WO2024251925A1 - Inducible gene expression system - Google Patents

Inducible gene expression system Download PDF

Info

Publication number
WO2024251925A1
WO2024251925A1 PCT/EP2024/065689 EP2024065689W WO2024251925A1 WO 2024251925 A1 WO2024251925 A1 WO 2024251925A1 EP 2024065689 W EP2024065689 W EP 2024065689W WO 2024251925 A1 WO2024251925 A1 WO 2024251925A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleotide sequence
seq
polynucleotide
sequence
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2024/065689
Other languages
French (fr)
Inventor
Martin Ebeling
Ravi Jagasia
Mateusz Piotr MENDEL
Filip ROUDNICKY
Lina SCHUKUR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Hoffmann La Roche Inc
Original Assignee
F Hoffmann La Roche AG
Hoffmann La Roche Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG, Hoffmann La Roche Inc filed Critical F Hoffmann La Roche AG
Priority to CN202480046722.2A priority Critical patent/CN121488042A/en
Priority to EP24732224.1A priority patent/EP4724585A1/en
Priority to AU2024286618A priority patent/AU2024286618A1/en
Priority to KR1020257043252A priority patent/KR20260022333A/en
Publication of WO2024251925A1 publication Critical patent/WO2024251925A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/002—Vectors comprising a special translation-regulating system controllable or inducible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/50—Vectors comprising a special translation-regulating system utilisation of non-ATG initiation codon

Definitions

  • RTT Rett Syndrome
  • X-linked neurological disorder associated with severe motor abnormalities and reduced lifespan that occurs in 1 in 1,000 females.
  • RTT is characterized by seemingly normal neurological and physical development during early postnatal period followed by a rapid regression with the loss of the purposeful motor skills and the onset of repetitive and autistic behaviors 1,2 .
  • the child loses purposeful hand skills and spoken language, experiences motor impairments, and develops breathing abnormalities and may also develop autistic-like features and seizures.
  • MeCP2 is highly expressed in neurons and functions as a ubiquitous transcriptional regulator by binding to methylated DNA and recruiting protein partners and regulatory complexes to control transcriptional activity. MeCP2 regulates neuronal physiology and maintenance and landmark studies have demonstrated that restoring levels of the MeCP2 protein dramatically reverses symptoms in mice 4,5 .
  • Inducible gene expression systems allow for reversibility and flexibility and enable the production of therapeutics upon demand, thereby circumventing overdose-associated side effects. Such regulation can be achieved using small molecules, with which transgene expression can be induced (generating ON- switches) or repressed (OFF-switch).
  • the tetracycline (Tet)-regulable system is the most widely exploited tool and can be used as an ON- or an OFF-switch.
  • Tet-inducible systems are derived from bacteria and require the expression of the regulator protein, e.g.
  • the ON-switch components include the tetracycline-controlled transactivator (tTA), which consists of the bacterial tetR with a C-terminal domain of VP16 (virion protein 16), derived from the herpes simplex virus.
  • tTA tetracycline-controlled transactivator
  • VP16 virion protein 16
  • riboswitches Another class of genetic switches originating from bacteria are riboswitches, RNA elements that are able to control gene expression in response to ligand binding, have a small genomic footprint and do not depend on other proteins for activity, which renders these types of regulated gene expression an attractive alternative to protein-based expression control systems 13 .
  • riboswitches suffer from a poor dynamic range as well as high basal activity 14 .
  • a new class of small molecules that regulate splicing of SMN2 exon 7 was recently identified, via screening using a reporter system based on the SMN2 transcript (described e.g. in WO 2009/151546 A2).
  • SMN2 reporter systems have since been proposed to be used as gene expression switches, where expression of a transgene is regulated through small molecule-controlled splicing of the expression cassette (Monteys et al. Nature (2021) 596: 291-295).
  • Known human SMN2 exon 6 to exon 8-derived transgene expression systems are described e.g. in Zhang, et al., Gene Ther. (2001) 8: 1532-1538, WO 2022/204471 A1, Monteys et al. Nature (2021) 596: 291-295 and WO 2021/163556 A1.
  • SMN2-based switch systems have very long nucleotide sequences, limiting the size of the transgene that can be employed under the control of the system. That is, the size of an insert comprising the switch and the coding sequence for a therapeutic polypeptide to be delivered as gene therapy would very often be much larger than the packaging limit for vectors routinely employed in the delivery of gene therapies, e.g. adeno-associated viral (AAV) vectors.
  • AAV adeno-associated viral
  • the present disclosure provides a polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:222, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least P37829 80% sequence identity to a nucleotide sequence according to SEQ ID NO:378 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:226; (iii) a
  • the present disclosure provides a polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:1, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26, wherein the third nucleotide sequence comprises ‘A’ at the position corresponding
  • the polynucleotide when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:222.
  • the first nucleotide sequence comprises, or consists of, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:106, SEQ ID NO:219 or SEQ ID NO:220.
  • the first nucleotide sequence comprises, or consists of, SEQ ID NO:3.
  • the second nucleotide sequence comprises SEQ ID NO:7 at its 5’ end, and comprises SEQ ID NO:8 or SEQ ID NO:377 at its 3’ end.
  • the second nucleotide sequence consists of fewer than 500 nucleotides.
  • the second nucleotide sequence comprises, or consists of, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:228.
  • the first nucleotide sequence comprises, or consists of, SEQ ID NO:10.
  • the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:226. In some embodiments, the third nucleotide sequence consists of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:223 or SEQ ID NO:224. In some embodiments, the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:26. In some embodiments, the third nucleotide sequence consists of SEQ ID NO:13 or SEQ ID NO:27. In some embodiments, the fourth nucleotide sequence comprises SEQ ID NO:15 or SEQ ID NO:379 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end.
  • the fourth nucleotide sequence consists of fewer than 500 nucleotides. In some embodiments, the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:227 or SEQ ID NO:340. In some embodiments, the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17. In some embodiments, the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’ or ‘TG’, or (b) comprises, or consists of, a nucleotide sequence according to SEQ ID NO:19.
  • the fifth nucleotide sequence (a) consists of the dinucleotide ‘GA’ or ‘TG’, or (b) comprises, or consists of, SEQ ID NO:20 or SEQ ID NO:21.
  • the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:164 and SEQ ID NO:171.
  • the polynucleotide comprises at least 80% sequence identity to SEQ ID NO:28 or SEQ ID NO:29. In some embodiments, the polynucleotide further comprises a promoter sequence 5’ to the start codon. In some embodiments, the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a polypeptide of interest. In some embodiments, the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end.
  • ITR inverted terminal repeat
  • the vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the present disclosure also provides a pharmaceutical composition comprising a polynucleotide or vector according to the present disclosure, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.
  • the present disclosure also provides a cell comprising a polynucleotide or vector according to the present disclosure.
  • the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion.
  • the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • the present disclosure also provides a method for modifying a cell to express a polypeptide of interest, comprising: (i) introducing into a cell a polynucleotide or vector according to the present disclosure; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion.
  • the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • the present disclosure also provides a method for expressing a polypeptide of interest in a cell, comprising contacting a cell according to the present disclosure with a splicing modifier that promotes SMN2 exon 7 inclusion.
  • the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • the present disclosure also provides a method for inhibiting expression of a polypeptide of interest in a cell, comprising contacting a cell according to the present disclosure with a splicing modifier that promotes SMN2 exon 7 inclusion.
  • the present disclosure also provides a method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell a polynucleotide or vector according to the present disclosure.
  • the present disclosure also provides a polynucleotide, vector or pharmaceutical composition according to the present disclosure, for use in a method of medical treatment or prophylaxis.
  • the present disclosure also provides a polynucleotide, vector or pharmaceutical composition according to the present disclosure, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest.
  • the present disclosure also provides the use of a polynucleotide, vector or pharmaceutical composition according to the present disclosure, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest.
  • the present disclosure also provides a method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject a polynucleotide, vector or pharmaceutical composition according to the present disclosure.
  • treating or preventing the disease or condition further comprises administering to the subject a splicing modifier that promotes SMN2 exon 7 inclusion.
  • the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • the disease or condition is a disease or condition characterised by deficiency of the polypeptide of interest.
  • the present disclosure also provides a kit, comprising: (i) a polynucleotide, vector or pharmaceutical composition according to the present disclosure; and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion.
  • the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • the present disclosure relates to inducible transgene expression systems using small molecule splicing modifiers (SMSM), and based on the described mechanism of action of the Survival of Motor Neuron 2 (SMN2) splicing modifier risdiplam (and variants thereof).
  • SMSM binding sites for SMN2 have previously been described to be two distinct sites within SMN2 exon 7 pre-mRNA: the ESE2 region and the 5’ splice donor site (5’ss) ( Figure 2a) 15 .
  • Interaction of the small molecules with the mRNA-protein complex is thought to be critical for the high selectivity of the compound.
  • This study suggested that both ESE2 and 5’ss regions are required for full activity of the SMSM compounds in a dose-dependent manner 15 .
  • RNA helix 16 Structural analysis using a compound from the same chemical class as risdiplam (SMN-C5) has demonstrated that the drug selectively promotes the recognition of the weak 5’ splice site of SMN2 exon 7 by U1 snRNP by stabilizing an unpaired adenine at the exon-intron junction in the RNA helix 16 .
  • the present disclosure provides novel SMN2 exon 6 to exon 8-derived constructs providing for the SMSM-inducible regulation of transgene expression, which are suitable to be employed for chemically- inducible regulation of the expression of gene therapies.
  • the present disclosure provides ON-switch constructs: (i) having a size permitting their application for the SMSM-inducible expression of polypeptides to be delivered as gene therapies, (ii) minimising undesirable expression of the polypeptide in the absence of the SMSM, and (iii) which are highly responsive to the SMSM, thus providing for strong induction of expression of the polypeptide in the presence of the SMSM, and (iv) that minimise the number of/completely remove extraneous amino acids P37829 at the N-terminus of the expressed polypeptide.
  • the present disclosure also provides OFF-switch constructs: (i) having a size permitting their application for the SMSM-inducible expression of polypeptides to be delivered as gene therapies, (ii) maximising expression of the polypeptide in the absence of the SMSM, and (iii) which are highly responsive to the SMSM, thus minimising expression of the polypeptide in the presence of the SMSM, and (iv) that minimise the number of/completely remove extraneous amino acids at the N-terminus of the expressed polypeptide.
  • Polynucleotides Aspects and embodiments of the present disclosure relate to polynucleotides.
  • a 'polynucleotide' refers to a polymer chain of a plurality of nucleotide monomers linked by bonds between the monomers, typically phosphodiester bonds (e.g. in the case of polynucleotides formed by naturally-occurring nucleotide monomers).
  • Polynucleotides include oligonucleotides, which generally comprise ⁇ 50 nucleotides.
  • a polynucleotide may be single-stranded, or may be double-stranded (i.e. may comprise a duplex formed by hydrogen-bonding between complementary nucleotides).
  • Polynucleotides according to the present disclosure may comprise or consist of: single-stranded DNA, double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single-stranded RNA, double- stranded RNA, RNA that is mixture of single- and double-stranded regions, single-stranded molecules comprising DNA and RNA, double-stranded molecules comprising DNA and RNA, and molecules comprising DNA and RNA having a mixture of single- and double-stranded regions.
  • a polynucleotide comprises or consists of DNA.
  • a polynucleotide is a polydeoxyribonucleotide.
  • a polynucleotide comprises or consists of RNA. In some embodiments, a polynucleotide is a polyribonucleotide. In aspects and embodiments wherein the polynucleotide of the present disclosure is defined by reference to a given nucleotide sequence, and wherein the given nucleotide sequence comprises or consists of RNA and/or is a polyribonucleotide, it will be appreciated that instances of ‘T’ for thymidine in such sequences are replaced with ‘U’, for uracil. The present disclosure also contemplates polynucleotides comprising modified nucleotides, e.g.
  • phosphonate and/or ribose and/or base of a deoxyribonucleotide or ribonucleotide is/are chemically modified.
  • Nucleotide modifications contemplated in accordance with the present disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), which is hereby incorporated by reference in its entirety.
  • Phosphonate modifications may be selected from phosphorothioate (e.g.
  • Rp isomer, Sp isomer), phosphorodithioate, methylphosphonate, methoxypropylphosphonate, 5'-(E)-vinylphosphonate, 5'- methylphosphonate, (S)-5'-C-methyl with phosphate, 5’-phosphorothioate, and peptide nucleic acid modifications.
  • Ribose modifications may be selected from 2'-O-methyl, 2'-O-methoxyethyl, 2’-fluoro, 2’- deoxy-2’-fluoro, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, 2'-arabino-fluoro, 2’-O-benzyl, 2’-O-methyl-4-pyridine, locked nucleic acid, (S)-cEt-BNA, tricyclo-DNA, PMO, unlocked P37829 nucleic acid, hexitol nucleic acid and glycol nucleic acid modifications.
  • Base modifications may be selected from pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5’-methylcytidine, 5’-fluoro-2’- deoxyuridine, N-ethylpiperidine 7'-EAA triazole-modified adenine, N-ethylpiperidine 6'-triazole-modified adenine, 6'-phenylpyrrolo-cytosine, 2',4'-difluorotoluyl ribonucleoside and 5'-nitroindole modifications.
  • a modified nucleotide may be selected from 2'-O-methyluridine-3'-phosphate, 2'- O-methyladenosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-O- methylguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphorothioate, 2'-fluorouridine-3'- phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphorot
  • Nucleotide sequences of the polynucleotides of the present disclosure are defined herein by reference to constituent nucleotide sequences. It will be appreciated that the constituent nucleotide sequences of polynucleotides according to the present disclosure are provided as subsequences of the complete sequence of the polynucleotide. In some aspects and embodiments, the constituent nucleotide sequences of polynucleotides according to the present disclosure are provided in a particular order in the sequence of the polynucleotide, e.g. from 5’ to 3’. By way of illustration, the first nucleotide sequence is provided 5’ to (i.e.
  • constituent nucleotide sequences of the polynucleotides of the present disclosure are non- overlapping.
  • constituent nucleotide sequences of the polynucleotides are provided in tandem in the context of the complete sequence of the polynucleotide.
  • constituent nucleotide sequences of the polynucleotide are immediately adjacent to one another (i.e.
  • positions 1 to 45 form the first nucleotide sequence
  • positions 46 to 309 form the second nucleotide sequence
  • positions 310 to 364 form the third nucleotide sequence
  • positions 356 to 616 form the fourth nucleotide sequence
  • positions 617 and 618 form the fifth nucleotide sequence.
  • the first nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 exon 6.
  • a ‘variant’ of a given reference nucleotide sequence comprises one or more differences relative to the reference nucleotide sequence.
  • a variant P37829 of a given reference nucleotide sequence may comprise insertion, deletion or substitution of one or more nucleotides relative to the reference nucleotide sequence.
  • the first nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:2.
  • the first nucleotide sequence comprises, or consists of, a nucleotide sequence having ⁇ 100% sequence identity to SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1.
  • a nucleotide sequence according to SEQ ID NO:1 refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:1.
  • Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:1 include SEQ ID NOs:3, 4, 5, 6 and 106.
  • the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:3.
  • the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:4.
  • the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:6.
  • the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221.
  • Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:1 include SEQ ID NOs:219 and 220.
  • the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • the first nucleotide sequence comprises ‘CAG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2 (cf. e.g. positions 109 to 111 of SEQ ID NO:6).
  • position(s) of a nucleotide sequence which ‘corresponds to’ specified position(s) of a reference nucleotide sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Söding, J.2005, P37829 Bioinformatics 21, 951-960).
  • sequence alignment software such as ClustalOmega (Söding, J.2005, P37829 Bioinformatics 21, 951-960).
  • sequence alignment software such as ClustalOmega (Söding, J.2005, P37829 Bioinformatics 21, 951-960).
  • the first nucleotide sequence comprises ‘CTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2 (cf. e.g. positions 109 to 111 of SEQ ID NO:5). In some embodiments, the first nucleotide sequence comprises ‘GGC’ at the positions corresponding to positions 64 to 66 of SEQ ID NO:2 (cf. e.g. positions 64 to 66 of SEQ ID NO:5, positions 64 to 66 of SEQ ID NO:6). In some embodiments, the first nucleotide sequence comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2 (cf. e.g.
  • the first nucleotide sequence comprises ‘GGC’ at the positions corresponding to positions 64 to 66 of SEQ ID NO:2, and comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2.
  • the first nucleotide sequence comprises ‘GTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2.
  • the first nucleotide sequence comprises ‘TTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2.
  • the first nucleotide sequence comprises ‘TAG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises ‘CAG’ at the positions corresponding to positions 39 to 41 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence consists of fewer than 111 nucleotides. In some embodiments the first nucleotide sequence consists of ⁇ 100 nucleotides, e.g.
  • the first nucleotide sequence consists of 45 nucleotides. In some embodiments, the first nucleotide sequence consists of 51 nucleotides. In some embodiments, the first nucleotide sequence consists of 81 nucleotides.
  • the first nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and (ii) comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) consists of ⁇ 45 nucleotides. Examples of such first nucleotide sequences include SEQ ID NOs:3, 4 and 106.
  • the first nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and (ii) comprises ‘GGC’ at the positions corresponding to positions 64 to 66 of SEQ ID NO:2; and (iii) comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iv) comprises ‘CTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2.
  • the first nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and (ii) comprises ‘GGC’ at the positions corresponding to positions 64 to 66 of SEQ ID NO:2; and (iii) comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iv) comprises ‘CAG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:3, 5, 6, 219, 220, and 230 to 237.
  • a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:2.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:3.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:5.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:6.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the second nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 intron 6. Accordingly, in some embodiments, the second nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:30.
  • the second nucleotide sequence comprises, or consists of, a nucleotide sequence having ⁇ 100% sequence identity to SEQ ID NO:30. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence according to SEQ ID NO:378 refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:378.
  • Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:378 include SEQ ID NO:8 and SEQ ID NO:377.
  • the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end.
  • the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g.
  • the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end, and comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end.
  • the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end, and comprises a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence refers to the region of the nucleotide sequence formed by nucleotides 5’ to (i.e. upstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence, or a subsequence of this region.
  • the region 5’ to the ‘midpoint’ includes the nucleotide immediately preceding the midpoint.
  • the region 5’ to the midpoint consists of positions 1 to 5.
  • the region 3’ to the ‘midpoint’ includes the nucleotide immediately after the midpoint.
  • the region 3’ to the midpoint consists of positions 6 to 10.
  • the region 5’ to the ‘midpoint’ includes the nucleotides 5’ to (i.e. upstream of) the nucleotide provided at the midpoint.
  • the region 5’ to the midpoint consists of positions 1 to 4.
  • the region 3’ to the ‘midpoint’ includes the nucleotides 3’ to (i.e. downstream of) the nucleotide provided at the midpoint.
  • the region 3’ to the midpoint consists of positions 6 to 9.
  • a sequence provided at the 5’ end of SEQ ID NO:9 refers to a nucleotide sequence formed by positions 1 to 207 of SEQ ID NO:9, or a subsequence thereof.
  • the ‘3’ end’ of a given nucleotide sequence refers to the region of the nucleotide sequence formed by nucleotides 3’ to (i.e.
  • a sequence provided at the 3’ end of SEQ ID NO:9 refers to a nucleotide sequence formed by positions 208 to 414 of SEQ ID NO:9, or a subsequence thereof.
  • a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes at least 5%, e.g.
  • a sequence provided at the 5’ end of SEQ ID NO:9 may comprise at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region formed by positions 1 to 207 of SEQ ID NO:9.
  • a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region of the nucleotide sequence formed by nucleotides 3’ to (i.e. downstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence.
  • a sequence provided at the 3’ end of SEQ ID NO:9 may comprise at least 5%, e.g.
  • a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes one or more nucleotides provided within 25 nucleotides, e.g. within one of 20, 15, 10 or 5 nucleotides, of the 5’ nucleotide of the given nucleotide sequence.
  • a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes the 5’ nucleotide of the given nucleotide sequence.
  • a sequence provided at the 5’ end of SEQ ID NO:9 may comprise one or more nucleotides provided within positions 1 to 25, e.g. within positions 1 to 20, 1 to 15, 1 to 10, or 1 to 5 of SEQ ID NO:9.
  • a sequence provided at the 5’ end of SEQ ID NO:9 may comprise position 1 of SEQ ID NO:9.
  • a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes one or more nucleotides provided within 25 nucleotides, e.g. within one of 20, 15, 10 or 5 nucleotides, of the 3’ nucleotide of the given nucleotide sequence.
  • a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes the 3’ nucleotide of the given nucleotide sequence.
  • a sequence provided at the 3’ end of SEQ ID NO:9 may comprise one or more nucleotides provided within positions 389 to 414, e.g. within positions 394 to 414, 399 to 414, 404 to 414, or 409 to 414 of SEQ ID NO:9.
  • a sequence provided at the 3’ end of SEQ ID NO:9 may comprise position 414 of SEQ ID NO:9.
  • the second nucleotide sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 in the region 5’ to its midpoint.
  • the second nucleotide sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:377 in the region 3’ to its midpoint.
  • the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 accounts for at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region 5’ to the midpoint of the second nucleotide sequence.
  • the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:377 accounts for at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region 3’ to the midpoint of the second nucleotide sequence.
  • the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the second nucleotide sequence.
  • nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 comprises the 5’ nucleotide of the second nucleotide sequence.
  • nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:377 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the second nucleotide sequence.
  • nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:377 comprises the 3’ nucleotide of the second nucleotide sequence.
  • nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end is SEQ ID NO:9. Positions 1 to 102 of SEQ ID NO:9 correspond to positions 1 to 102 of SEQ ID NO:7.
  • An exemplary sequence comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 at its 3’ end is SEQ ID NO:9. Positions 253 to 414 of SEQ ID NO:9 correspond to positions 1 to 162 of SEQ ID NO:8.
  • the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:10.
  • the second nucleotide sequence comprises ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30 (cf. e.g. position 411 of SEQ ID NO:9, position 261 of SEQ ID NO:10).
  • the second nucleotide sequence consists of fewer than 1044 nucleotides, e.g. one of ⁇ 1000 nucleotides, ⁇ 900 nucleotides, ⁇ 800 nucleotides, ⁇ 750 nucleotides, ⁇ 700 nucleotides, ⁇ 650 nucleotides, ⁇ 600 nucleotides, ⁇ 550 nucleotides or ⁇ 500 nucleotides.
  • the second nucleotide sequence consists of fewer than 500 nucleotides, e.g. one of ⁇ 450 nucleotides, ⁇ 400 nucleotides, ⁇ 350 nucleotides, ⁇ 300 nucleotides, ⁇ 250 nucleotides or ⁇ 200 nucleotides. In some embodiments, the second nucleotide sequence consists of 1044 nucleotides. In some embodiments, the second nucleotide sequence consists of 414 nucleotides. In some embodiments, the second nucleotide sequence consists of 264 nucleotides. In some embodiments, the second nucleotide sequence consists of 189 nucleotides.
  • the second nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) a nucleotide sequence having at least 80% (e.g.
  • the second nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g.
  • the second nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g.
  • a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:9, 10, 228, 238 to 255, 375 and 376.
  • a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:240.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a second nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:9, 10, 228, 238, 239, 241 to 255, 375 and 376.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a second nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:9.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g.
  • a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:10.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a second nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the third nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 exon 7. Accordingly, in some embodiments, the third nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:12.
  • the third nucleotide sequence comprises, or consists of, a nucleotide sequence having ⁇ 100% sequence identity to SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11.
  • nucleotide sequence according to SEQ ID NO:11 refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:11.
  • Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:11 include SEQ ID NOs:13 and 14.
  • the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence according to SEQ ID NO:26 refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:26.
  • Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:26 include SEQ ID NOs:13 and 27.
  • the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:225.
  • Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:225 include SEQ ID NOs:223 and 224.
  • the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:226.
  • the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:13.
  • the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:14.
  • the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • the third nucleotide sequence comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12 (cf. e.g. position 2 of SEQ ID NOs:11, 13, 14, 26 and 27). In some embodiments, the third nucleotide sequence comprises insertion of ‘GCCACC’ after the position corresponding to position 6 of SEQ ID NO:12 (cf. e.g. positions 7 to 12 of SEQ ID NO:14).
  • the third nucleotide sequence comprises ‘TG’ at the positions corresponding to positions 8 to 9 of SEQ ID NO:12 (cf. e.g. positions 14 and 15 of SEQ ID NO:14). In some embodiments, the third nucleotide sequence comprises insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12 (cf. e.g. position 49 of SEQ ID NO:13, position 55 of SEQ ID NO:14). In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 24 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the nucleotide at the position corresponding to position 20 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘T’ at the position corresponding to position 27 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘T’ at the position corresponding to position 28 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 21 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 29 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘T’ at the position corresponding to position 21 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘A’ at the position corresponding to position 31 of SEQ ID NO:12, and comprises ‘A’ at the position corresponding to position 34 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the positions corresponding to positions 9 to 20 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the positions corresponding to positions 33 to 41 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘CAC’ at the positions corresponding to positions 34 to 36 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘G’ after the position corresponding to position 39 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘GC’ at the positions corresponding to positions 47 to 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘CACCATG’ after the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘C’ at the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘AA’ after the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘CAT’ at the positions corresponding to positions 49 to 51 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘GCC’ at the positions corresponding to positions 46 to 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘CCA’ at the positions corresponding to positions 49 to 51 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘T’ after the position corresponding to position 51 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘GC’ at the positions corresponding to positions 39 to 40 of SEQ P37829 ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘CA’ after the position corresponding to position 40 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘ATG’ at the positions corresponding to positions 43 to 45 of SEQ ID NO:12, and deletion of the position corresponding to position 46 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘GCCACCATG’ after the position corresponding to position 9 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘AGCACCATG’ after the position corresponding to position 15 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 16 of SEQ ID NO:12.
  • the third nucleotide sequence comprises insertion of ‘CACCATG’ after the position corresponding to position 15 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘ATG’ after the position corresponding to position 15 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘ATG’ after the position corresponding to position 21 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘GG’ after the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘AT’ after the position corresponding to position 30 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘G’ at the position corresponding to position 31 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘G’ after the position corresponding to position 33 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘A’ after the position corresponding to position 43 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘GG’ at the positions corresponding to positions 45 and 46 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘AT’ after the position corresponding to position 52 of SEQ ID NO:12.
  • the third nucleotide sequence comprises deletion of the positions corresponding to positions 49 and 50 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 51 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 40 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 35 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘A’ after the position corresponding to position 39 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘G’ at the position corresponding to position 41 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘C’ at the position corresponding to position 43 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘ATG’ after the position corresponding to position 42 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘TG’ after the position corresponding to position 45 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘ATGGA’ after the position corresponding to position 44 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘A’ at the position corresponding to position 44 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 49 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the position corresponding to position 50 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding P37829 to position 50 of SEQ ID NO:12.
  • the third nucleotide sequence comprises ‘G’ at the position corresponding to position 44 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the position corresponding to position 49 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the positions corresponding to positions 42 to 44 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘G’ after the position corresponding to position 39 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the position corresponding to position 48 of SEQ ID NO:12.
  • the third nucleotide sequence comprises insertion of ‘GA’ after the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g.
  • sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprises insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; and (iv) comprises insertion of ‘GCCACC’ after the position corresponding to position 6 of SEQ ID NO:12; and (v) comprises ‘TG’ at the positions corresponding to positions 8 and 9 of SEQ ID NO:12.
  • the third nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:26; and (ii) comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprises deletion of the position corresponding to position 20 of SEQ ID NO:12; and (iv) comprises insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:13, 14, 27, 223, 224 and 256 to 329.
  • a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:13.
  • a polynucleotide according to the present P37829 disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the fourth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 intron 7. Accordingly, in some embodiments, the fourth nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having ⁇ 100% sequence identity to SEQ ID NO:18.
  • the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:380 at its 5’ end.
  • a nucleotide sequence according to SEQ ID NO:380 refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:380.
  • Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:380 include SEQ ID NO:15 and SEQ ID NO:379.
  • the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end.
  • the fourth nucleotide sequence P37829 comprises a nucleotide sequence having at least 80% (e.g.
  • the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:379 at its 5’ end.
  • the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end.
  • the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end, and comprises a nucleotide sequence having at least 80% (e.g.
  • the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:379 at its 5’ end, and comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end.
  • the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 or SEQ ID NO:379 in the region 5’ to its midpoint. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 in the region 3’ to its midpoint. In some embodiments, the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 or SEQ ID NO:379 accounts for at least 5%, e.g.
  • the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 accounts for at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region 3’ to the midpoint of the fourth nucleotide sequence.
  • nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 or SEQ ID NO:379 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 or SEQ ID NO:379 comprises the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 comprises one or more nucleotides provided within 25, e.g.
  • nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 comprises the 3’ nucleotide of the fourth nucleotide sequence.
  • An exemplary sequence comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 at its 5’ end is SEQ ID NO:17. Positions 1 to 102 of SEQ ID NO:17 correspond to positions 1 to 102 of SEQ ID NO:15.
  • P37829 An exemplary sequence comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end is SEQ ID NO:18.
  • the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:16.
  • the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:17.
  • the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:18.
  • the fourth nucleotide sequence consists of fewer than 444 nucleotides, e.g. one of ⁇ 425 nucleotides, ⁇ 400 nucleotides, ⁇ 350 nucleotides, ⁇ 300 nucleotides, ⁇ 250 nucleotides or ⁇ 200 nucleotides. In some embodiments, the fourth nucleotide sequence consists of fewer than 255 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 252 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 177 nucleotides.
  • the fourth nucleotide sequence consists of greater than 444 nucleotides, e.g. one of >450 nucleotides, >475 nucleotides or >500 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 508 nucleotides. In some embodiments, the fourth nucleotide sequence comprises ‘C’ at the position corresponding to position 441 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises ‘T’ at the position corresponding to position 441 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises ‘TT’ at the positions corresponding to positions 436 and 437 of SEQ ID NO:18.
  • the fourth nucleotide sequence comprises ‘TCCTC’ at the positions corresponding to positions 11 to 15 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises insertion of ‘TTT’ after the position corresponding to position 10 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises insertion of ‘CCC’ after the position corresponding to position 10 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence having at least 80% e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consists of fewer than 500 nucleotides.
  • the fourth nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or P37829 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fourth nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:337.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g.
  • a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:18, 227, 330 to 336, 338 to 370 and 381.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g.
  • a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:18.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fourth nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the fifth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 exon 8. Accordingly, in some embodiments, the fifth nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:32.
  • the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having ⁇ 100% sequence identity to SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘GA’, ‘TG’ or ‘TT’. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘GA’. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘TG’. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘TT’. In some embodiments, the fifth nucleotide sequence consists of the trinucleotide ‘GAG’.
  • the fifth nucleotide sequence consists of the nucleotide ‘A’. P37829
  • the fifth nucleotide sequence encodes a polypeptide of interest (e.g. as described hereinbelow).
  • the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘GA’, 'TG' or ‘TT’ at positions 1 and 2.
  • the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘GA’ at positions 1 and 2.
  • the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘TG’ at positions 1 and 2.
  • the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘TT’ at positions 1 and 2. In some embodiments, the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘GAG’ at positions 1 to 3. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:19.
  • nucleotide sequence according to SEQ ID NO:19 refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:19.
  • Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:19 include SEQ ID NOs:20 and 21.
  • the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:20.
  • the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:21.
  • the fifth nucleotide sequence comprises ‘GA’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:32 (cf. e.g. positions 1 and 2 of SEQ ID NO:20).
  • the fifth nucleotide sequence comprises ‘TG’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:32 (cf. e.g. positions 1 and 2 of SEQ ID NO:21). In some embodiments, the fifth nucleotide sequence comprises fewer than 577 nucleotides. In some embodiments the fifth nucleotide sequence consists of ⁇ 500 nucleotides, e.g. one of ⁇ 400 nucleotides, ⁇ 300 nucleotides, ⁇ 200 nucleotides, ⁇ 100 nucleotides or ⁇ 50 nucleotides. In some embodiments, the fifth nucleotide sequence comprises fewer than 25 nucleotides, e.g.
  • the fifth nucleotide sequence comprises 23 nucleotides. In some embodiments, the fifth nucleotide sequence comprises 2 nucleotides. P37829 In some embodiments, the fifth nucleotide sequence comprises 11 nucleotides. In some embodiments, the fifth nucleotide sequence comprises 8 nucleotides. In some embodiments, the fifth nucleotide sequence comprises 5 nucleotides. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.
  • the fifth nucleotide sequence comprises insertion of ‘G’ after the position corresponding to position 2 of SEQ ID NO:32.
  • the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. ⁇ 90% or 100%) sequence identity to positions 1 to 11 of SEQ ID NO:32.
  • the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.100%) sequence identity to positions 1 to 8 of SEQ ID NO:32.
  • the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.100%) sequence identity to positions 1 to 5 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises, or consists of, positions 1 and 2 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises ‘A’ at the position corresponding to position 1 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises insertion of ‘T’ after the position corresponding to position 1 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises ‘G’ at the position corresponding to position 3 of SEQ ID NO:32.
  • the fifth nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:19; and (ii) comprises ‘GA’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:32.
  • An example of such a fifth nucleotide sequence is SEQ ID NO:20.
  • the fifth nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:19; and (ii) comprises ‘TG’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:32.
  • An example of such a fifth nucleotide sequence is SEQ ID NO:21.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g.
  • a fifth nucleotide sequence comprising, or consisting of: the dinucleotide ‘TG’, the dinucleotide ‘GA’, the dinucleotide ‘TT’, the trinucleotide ‘GAG’, the nucleotide ‘A’, or a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:20, 21 and 371 to 374.
  • a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:20.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of: the dinucleotide ‘TG’, the dinucleotide ‘GA’, the dinucleotide ‘TT’, the trinucleotide ‘GAG’, the nucleotide ‘A’, or a nucleotide sequence having at least 80% (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of the dinucleotide ‘TG’.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of the dinucleotide ‘TT’.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of the trinucleotide ‘GAG’.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g.
  • a fifth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:20.
  • a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequences encoding a polypeptide of interest comprises, or consists of, a nucleotide sequence encoding a polypeptide of interest.
  • the sixth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a polypeptide of interest.
  • a polypeptide of interest may be any polypeptide.
  • a polypeptide of interest may be an antigen- binding polypeptide, an aptamer, an antigen-binding polypeptide complex, an antibody or an antigen- binding fragment or derivative thereof, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, a decoy receptor for a ligand, a decoy ligand for a receptor, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, a transcription factor, an epigenetic modifier, a constituent protein of a site-specific nuclease nucleic acid editing system (e.g.
  • a polypeptide of interest may be an antigen- binding polypeptide or an antigen-binding polypeptide complex.
  • a polypeptide of interest may be a chimeric antigen-receptor (CAR).
  • a polypeptide of interest is a polypeptide suitable for use in therapy or prophylaxis of a disease/condition.
  • a polypeptide of interest is a detectable polypeptide or a polypeptide having detectable activity.
  • a polypeptide suitable for use in therapy or prophylaxis of a disease/condition may be any polypeptide whose administration is useful for the treatment or prevention of a disease/condition.
  • a polypeptide suitable for use in therapy or prophylaxis of a disease/condition may be a polypeptide for which deficiency thereof is positively associated with, or implicated in the pathology of, a disease or condition.
  • the polypeptide of interest may be MeCP2. Deficiency of MeCP2 is associated with Rett syndrome.
  • a polypeptide suitable for use in therapy or prophylaxis of a disease/condition may be a polypeptide which inhibits the expression and/or activity of a factor whose expression or activity is positively associated with, or implicated in the pathology of, a disease or condition.
  • a detectable polypeptide may be or comprise a fluorescent polypeptide.
  • Fluorescent polypeptides include green fluorescent protein and variants thereof (e.g. enhanced green fluorescent protein), yellow fluorescent protein (e.g. citrine), red fluorescent protein and variants thereof (e.g.
  • a detectable polypeptide may be or comprise an epitope tag.
  • Epitope tags include e.g.
  • a polypeptide having detectable activity may be or comprise an enzymatic moiety.
  • Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g.
  • a polypeptide of interest expressed from a polynucleotide of the present disclosure may additionally comprises one or more extraneous amino acids added at the N-terminus of the polypeptide, i.e. immediately upstream of the amino acid sequence of the polypeptide of interest. Such extraneous amino acids may be characterised as forming an N-terminal tag on the polypeptide of interest.
  • the N-terminal tag consists of fewer than 50 amino acids, e.g. one of ⁇ 40 amino acids, ⁇ 30 amino acids, ⁇ 25 amino acids, ⁇ 20 amino acids, ⁇ 15 amino acids, ⁇ 10 amino acids, ⁇ 9 amino acids, ⁇ 8 amino acids, ⁇ 7 amino acids, ⁇ 6 amino acids, ⁇ 5 amino acids, ⁇ 4 amino acids, ⁇ 3 amino acids, ⁇ 2 amino acids or 1 amino acid.
  • the polypeptide of interest lacks an N-terminal tag.
  • the polypeptide of the present disclosure comprises one or more cleavage sites.
  • a cleavage site refers to a sequence of amino acids that acts as a substrate for an enzyme capable of cleaving peptide bonds. Many such cleavage sites are known to, and can be employed by, the person skilled in the art of molecular biology.
  • the cleavage sequence comprises an autocleavage site. Autocleavage sites include the 2A cleavage sequence from Picornavirus ‘NPGP’, which is cleaved at ’G/P’. Further autocleavage sites are described e.g.
  • T2A, E2A, P2A and F2A cleavage sites include e.g. T2A, E2A, P2A and F2A cleavage sites.
  • the amino acid sequences of T2A, E2A, P2A and F2A cleavage sites are shown in SEQ ID NOs: 107, 108, 109 and 110, respectively.
  • a cleavage site may be included in a polypeptide according to the present disclosure to provide for removal of extraneous amino acids added at the N-terminus of the polypeptide, i.e. immediately upstream of the amino acid sequence of the polypeptide of interest.
  • a cleavage site may be included for the removal of an N-terminal tag as described hereinabove.
  • a polypeptide according to the present disclosure comprises a cleavage site adjacent to (i.e. in the amino acid sequence of the polypeptide, e.g. immediately downstream of) extraneous amino acid(s), e.g. extraneous amino acid(s) forming an N-terminal tag.
  • a cleavage site according to the present disclosure is a 2A cleavage site, e.g. selected from a T2A, E2A, P2A and F2A cleavage site.
  • cleavage site is a T2A cleavage site.
  • a polypeptide according to the present disclosure comprises an amino acid sequence having at least 60%, preferably one of ⁇ 70%, ⁇ 75%, ⁇ 80%, ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% amino acid sequence identity to SEQ ID NO:107, 108, 109 or 110.
  • a nucleotide sequence encoding a polypeptide of interest comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:213 or 217.
  • nucleotide sequences of the polynucleotide Polynucleotides according to the present disclosure may comprise additional nucleotide sequences and/or sequence features in addition to the first, second, third, fourth, fifth and/or sixth nucleotide sequences as described hereinabove.
  • the polynucleotides of the present disclosure comprise a start codon 5’ to (i.e. upstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a polypeptide of interest.
  • the start codon is preferably the trinucleotide ‘ATG’.
  • a start codon is provided in the polynucleotide such that following splicing (e.g. where the polynucleotide is a polyribonucleotide, or splicing of a polyribonucleotide transcribed from the polynucleotide where the polynucleotide is a polydeoxyribonucleotide), the start codon is provided in the mature mRNA molecule in such a way as to serve as the initiator codon for translation of the polypeptide of interest (encoded by the fifth or sixth nucleotide sequence).
  • the splicing referred to in the preceding paragraph is splicing performed in the presence of a splicing modifier (e.g. as described herein).
  • a splicing modifier e.g. as described herein.
  • splicing of the polyribonucleotide having the nucleotide sequence of SEQ ID NO:22 in the presence of RG7800/RG76196 results in the production of a mature mRNA molecule comprising an initiator codon for translation of a polypeptide of interest.
  • the splicing referred to in the preceding paragraph is splicing performed in the absence of a splicing modifier (e.g. as described herein).
  • the polynucleotide further comprises a Kozak sequence.
  • the Kozak sequence is provided immediately upstream of the start codon for initiating translation of the polypeptide of interest.
  • the Kozak sequence consists of a nucleotide sequence conforming to the consensus of SEQ ID NO:33.
  • the Kozak sequence consists of SEQ ID NO:34.
  • the polynucleotide comprises a nucleotide sequence consisting of SEQ ID NO:35 5’ to the nucleotide sequence encoding a polypeptide of interest.
  • SEQ ID NO:35 is provided in the polynucleotide such that following splicing (e.g.
  • the start codon of SEQ ID NO:35 is provided in the mature mRNA molecule in such a way as to serve as the initiator codon for translation of the polypeptide of interest (encoded by the fifth or sixth nucleotide sequence).
  • the splicing referred to in the preceding paragraph is splicing performed in the presence of a splicing modifier (e.g. as described herein).
  • the splicing referred to in the preceding paragraph is splicing performed in the absence of a splicing modifier (e.g. as described herein).
  • the polynucleotide further comprises a promoter sequence.
  • the promoter sequence is preferably 5’ to the first nucleotide sequence.
  • the polynucleotide P37829 further comprises one or more enhancer sequences.
  • the one or more enhancer sequences are preferably 5’ to the first nucleotide sequence.
  • the polynucleotide further comprises a stop codon.
  • the stop codon is preferably provided immediately 3’ to (i.e.
  • the polynucleotide further comprises a polyadenylation signal sequence.
  • the polyadenylation signal sequence is provided 3’ to (i.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a polypeptide of interest.
  • the polynucleotide further comprises a terminator sequence.
  • the terminator sequence is preferably 3’ to the nucleotide sequence encoding a polypeptide of interest (and 3’ to the polyadenylation signal sequence, when present).
  • the constituent nucleotide sequences of the polynucleotides i.e. the first, second, third, fourth, fifth and/or sixth nucleotide sequences as described hereinabove
  • the polynucleotide further comprises one or more linker nucleotide sequences between one or more of the constituent nucleotide sequences of the polynucleotide.
  • Linker nucleotide sequences may comprise, or consist of, 1-10, e.g.
  • a polynucleotide according to the present disclosure comprises one more linker nucleotide sequences (i.e. provided between the first, second, third, fourth, fifth and/or sixth nucleotide sequences), the linker sequences are preferably selected such that they do not substantially affect post-transcriptional processing of the polynucleotide when the polynucleotide is a polyribonucleotide.
  • splicing of the polynucleotide when the polynucleotide is a polyribonucleotide is substantially the same as splicing of an equivalent polyribonucleotide lacking the linker nucleotide sequence(s).
  • the linker sequences are preferably selected such that they do not alter the amino acid sequence of a polypeptide encoded by the polynucleotide.
  • a polynucleotide according to the present disclosure comprises one more linker sequences
  • the polynucleotide encodes the same polypeptide as the equivalent polyribonucleotide lacking the linker nucleotide sequence(s).
  • the polynucleotide further comprises inverted terminal repeat (ITR) sequences.
  • ITR inverted terminal repeat
  • the polynucleotide comprises an ITR 5’ to the first nucleotide sequence (and 5’ to the P37829 promoter and/or enhancer sequences, when present).
  • the polynucleotide comprises an ITR 3’ to the nucleotide sequence encoding a polypeptide of interest (and 3’ to the stop codon, polyadenylation signal sequence and/or terminator sequence, when present).
  • the polynucleotide comprises an ITR sequence at its 5’ end, and an ITR sequences at its 3’ end.
  • the first nucleotide of the ITR sequence provided at the 5’ end of the polynucleotide is provided within 1 to 25 nucleotides, e.g. within one of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides of the first nucleotide of the polynucleotide (i.e.
  • the final nucleotide of the ITR sequence provided at the 3’ end of the polynucleotide is provided within 1 to 25 nucleotides, e.g. within one of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides of the final nucleotide of the polynucleotide (i.e. the terminal position of the nucleotide sequence of the polynucleotide).
  • the polynucleotide of the present disclosure i.e.
  • the polynucleotide consists of a nucleotide sequence having a size within the packaging limit of a vector for delivering the polynucleotide. In some embodiments, the polynucleotide has a size within the packaging limit of an AAV vector.
  • the polynucleotide has a size within the packaging limit of an AAV vector of one of the following serotypes: AAV9, AAV9.45, AAV-PHP.B, AAV1, AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV10 or AAVrh74. In some embodiments, the polynucleotide has a size within the packaging limit of an AAV vector of one of the following serotypes: AAV9, AAV9.45, AAV-PHP.B, AAV1, AAV2, AAV2.7m8, AAV2i8, AAV5, AAV6, AAV8, AAV10 or AAVrh74.
  • the polynucleotide of the present disclosure consists of fewer than 6,000 nucleotides, e.g. one of ⁇ 5,000, ⁇ 4,750, ⁇ 4,500, ⁇ 4,250, ⁇ 4,000, ⁇ 3,750, ⁇ 3,500, ⁇ 3,250, ⁇ 3,000, ⁇ 2,750, ⁇ 2,500, ⁇ 2,250, ⁇ 2,000, ⁇ 1,750, ⁇ 1,500, ⁇ 1,250 or ⁇ 1,000 nucleotides.
  • the polynucleotide of the present disclosure consists of fewer nucleotides than the number of nucleotides of a known SMN2 exon 6 to exon 8-derived transgene expression system (e.g. an SMN2 exon 6 to exon 8-derived transgene expression system described in Zhang, et al., Gene Ther. (2001) 8: 1532-1538, WO 2022/204471 A1, Monteys et al. Nature (2021) 596: 291-295 or WO 2021/163556 A1).
  • a known SMN2 exon 6 to exon 8-derived transgene expression system e.g. an SMN2 exon 6 to exon 8-derived transgene expression system described in Zhang, et al., Gene Ther. (2001) 8: 1532-1538, WO 2022/204471 A1, Monteys et al. Nature (2021) 596: 291-295 or WO 2021/163556 A1).
  • the polynucleotide of the present disclosure consists of fewer nucleotides than a polynucleotide comprising: (i) SEQ ID NO:36 and (ii) a nucleotide sequence encoding a polypeptide of interest (i.e. adjacent and 3’ to SEQ ID NO:36, the context of the complete sequence of the polynucleotide).
  • a polynucleotide comprising: (i) SEQ ID NO:36 and (ii) a nucleotide sequence encoding a polypeptide of interest i.e.
  • the polynucleotide of the present disclosure consists of a number of nucleotides that is less than 1 times, e.g.
  • the region of a polynucleotide of the present disclosure formed by the first, second, third, fourth and fifth nucleotide sequences consists of fewer nucleotides than the number of nucleotides in SEQ ID NO:36.
  • the region of a polynucleotide of the present disclosure formed by the first, second, third, fourth and fifth nucleotide sequences consists of a number of nucleotides that is less than 1 times, e.g. ⁇ 0.99 times, ⁇ 0.95 times, ⁇ 0.9 times, ⁇ 0.85 times, ⁇ 0.8 times, ⁇ 0.75 times, ⁇ 0.7 times, ⁇ 0.65 times, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times or ⁇ 0.25 times the number of nucleotides in SEQ ID NO:36.
  • the polynucleotide of the present disclosure may comprise 5’ cap, 5’ UTR, 3’ UTR and/or PolyA tail nucleotide sequences.
  • the polynucleotide comprises a 5’ UTR 5’ to (i.e. upstream of, in the context of the nucleotide sequence of the polynucleotide) a start codon.
  • the polynucleotide comprises a 3’ UTR 3’ to (i.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) a stop codon.
  • the polynucleotide comprises a 3’ UTR 5’ to a polyadenylation signal sequence.
  • the polynucleotide comprises a 3’ UTR 3’ to a stop codon and 5’ to a polyadenylation signal sequence.
  • the polynucleotide of the present disclosure comprises one or more nucleotide sequences encoding a selectable marker, to facilitate identification and/or selection of cells comprising/expressing the polynucleotide.
  • Selectable markers include proteins that confer resistance to antibiotics or other toxins, e.g., blasticidin, ampicillin, neomycin, methotrexate, or tetracycline, and proteins that complement auxotrophic deficiencies.
  • the polynucleotide of the present disclosure comprises a nucleotide sequence encoding an internal ribosome entry site (IRES).
  • the polynucleotide comprises a nucleotide sequence permitting two or more polypeptides to be translated separately from a single polyribonucleotide.
  • the polynucleotides of the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.
  • a polynucleotide according to the present disclosure comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from Column A of Table A; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence selected from Column B of Table A; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence selected from Column C of Table A; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence selected from Column D of Table A and a fifth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from Column D of Table A; and a fifth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence selected from Column E of Table A.
  • nucleotide sequence selected from Column A of Table A, and the nucleotide sequence selected from Column B of Table A, and the nucleotide sequence selected from Column C of Table A, and the nucleotide sequence selected from Column B of Table A, and the nucleotide sequence selected from Column D of Table A, and the nucleotide sequence selected from Column B of Table A, and the nucleotide sequence selected from Column E of Table A are all selected form the same row of Table A.
  • the nucleotide sequences selected from Columns A, B, C, D and E of Table A may be selected from row 3 of Table 1, and therefore may be SEQ ID NOs:5, 91418 and 20, respectively.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence according to SEQ ID NO:1; and comprising ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) consisting of ⁇ 45 nucleotides; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • P37829 sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:106; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:22.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:3; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and comprising ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) comprising ‘CTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; and (iv) comprising insertion of ‘GCCACC’ after the position corresponding to position 6 of SEQ ID NO:12; and (v) comprising ‘TG’ at the positions corresponding to positions 8 and 9 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:20.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:24.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and comprising ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) comprising ‘CAG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:20.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:25.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:28.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and comprising ‘GCC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) consisting of ⁇ 45 nucleotides;
  • the polynucleotide comprises: a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising deletion of the position corresponding to position 20 of SEQ ID NO:12; and (iv) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% P37829 (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:29.
  • the polynucleotide does not consist of, or does not comprise, SEQ ID NO:36.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to one of SEQ ID NOs:37 to 105.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence according to SEQ ID NO:221; and (ii) consisting of ⁇ 45 nucleotides; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • P37829 sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:111.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221; and (ii) consisting of ⁇ 45 nucleotides; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 300 nucleotides; P37829 a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence a nucleotide sequence according to SEQ ID NO:225; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:116.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221; and (ii) consisting of ⁇ 45 nucleotides; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 300 nucleotides; P37829 a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence a nucleotide sequence according to SEQ ID NO:225; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:220; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:164.
  • the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221; and (ii) consisting of ⁇ 45 nucleotides; a second nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence a nucleotide sequence according to SEQ ID NO:229; a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence a nucleotide sequence according to SEQ ID NO:225; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • sequence identity to SEQ ID NO:220; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to SEQ ID NO:171.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100%) sequence identity to one of SEQ ID NOs:111 to 200.
  • the polynucleotide comprises a nucleotide sequence having at least 80% (e.g.
  • Splicing modifiers are molecules that influence splicing of polyribonucleotides. Such molecules are reviewed e.g.
  • Splicing modifiers generally reduce/prevent association of factors required for normal post-transcriptional processing (e.g. components of the spliceosome) with RNA. Splicing modifiers typically bind to a nucleotide sequence of a polyribonucleotide and either inhibit or promote the association of RNA-binding proteins and/or non-coding RNAs which function as splicing activators or repressors.
  • splicing modifiers can alter the equilibrium of the splicing-regulatory RNA structures, and thus promote an increase or decrease in skipping or inclusion of an exon and/or retention or excision of an intron in the mature RNA molecule produced by splicing.
  • a splicing modifier is a small molecule or a splice-switching nucleic acid (e.g. a splice-switching oligonucleotide).
  • a ‘small molecule’ refers to a low molecular weight ( ⁇ 1000 daltons, typically about 300 to about 700 daltons) organic compound.
  • Splice-switching nucleic acids are reviewed e.g. in Haves and Hastings, Nucleic Acids Res. (2016) 44(14): 6549–6563, which is hereby incorporated by reference in its entirety.
  • Splice-switching nucleic acids include e.g. splice-switching oligonucleotides (SSOs). They disrupt the normal splicing of target RNA transcripts by blocking the RNA:RNA base-pairing and/or protein:RNA binding interactions that occur between components of the splicing machinery and pre-mRNA.
  • SSOs splice-switching oligonucleotides
  • Splice-switching nucleic acids may be designed to target a specific region of the target transcript, e.g. to promote skipping of exon(s) of interest and/or to promote inclusion of exon(s) of interest and/or to promote retention of introns of interest and/or to promote excision of introns of interest.
  • SSOs often comprise alterations to oligonucleotide sugar-phosphate backbones in order to reduce/prevent RNAseh degradation, such as e.g. phosphorothioate linkages, phosphorodiamidate linkages such as phosphorodiamidate morpholino (PMOs), and may comprise e.g.
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • methoxyethyl nucleotide modifications e.g.2′O-methyl (2′OMe) and 2′-O-methoxyethyl (MOE) ribose modifications and/or 5’-methylcytosine modifications.
  • Small molecule splicing modifiers contemplated in accordance with the present disclosure include RG- 7800 and RG-7916 (also known as risdiplam), and analogs thereof.
  • RG-7800 and RG-7916 bind to the 5’ splice site of intron 7 and exonic splicing enhancer 2 of exon 7 of human SMN2, thereby stabilising the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 snRNP complex, and promoting inclusion of exon 7 in the mature RNA molecule obtained following splicing of pre-mRNA transcribed from SMN2.
  • RG-7800 and RG-7916 are useful to restore functional SMN2 protein expression from SMN2 alleles comprising the spinal muscular atrophy (SMA)-associated polymorphism c.840C>T (which potentiates skipping of exon 7).
  • SMA spinal muscular atrophy
  • RG-7916 DrugBank Acc.
  • No. DB15305 is a close structural analog of RG-7800 having improved potency, pharmacokinetics and safety profile than RG-7800, and is approved by the FDA for the treatment of SMA. Further analogs of RG-7800 and RG-7916 having similar splicing modifier activity include SMN-C2, SMN-C3, SMN-C5 and TEC-1.
  • Branaplam (DrugBank Acc. No. DB14918; also known as LMI-070) is another small molecule splicing modifier that promotes inclusion of exon 7 in the mature RNA expressed from SMN2. Like RG-7800 and RG-7916, branaplam binds to the 5’ splice site of intron 7 of human SMN2.
  • PK4C9 (also known as homocarbonyltopsentin) is another small molecule splicing modifier that promotes inclusion of exon 7 in the mature RNA expressed from SMN2. It is thought to bind to the 5’ splice site of exon 7 and TSL2, and improve accessibility of the 5′ splice site via stabilising a triloop structure of TSL2. Small molecule splicing modifiers that promote inclusion of exon 7 in the mature RNA expressed from SMN2 are described e.g. in WO 2015/173181 A1 and WO 2009/151546 A2, which are hereby P37829 incorporated by reference in their entirety.
  • Nusinersen (DrugBank Acc. No. DB13161) is a splice-switching oligonucleotide that promotes exon 7 retention in mature RNA expressed from human SMN2.
  • Nusinersen is an 18-mer 2’-MOe phosphorothioate antisense oligonucleotide that hybridises to intronic splicing silencer site 1 of intron 7, occupying the site and thereby inhibiting the association of the splicing suppressor ribonucleoproteins hnRNPs A1/A2, thus promoting inclusion of exon 7 in the mature RNA molecule.
  • the splicing modifier according to the present disclosure preferably promotes inclusion of SMN2 exon 7 in RNA obtained following splicing of pre-mRNA transcribed from human SMN2.
  • RNA molecules comprising SMN2 exon 7 increase the proportion of RNA molecules comprising SMN2 exon 7 among RNA molecules obtained following splicing of pre-mRNA transcribed from human SMN2 (i.e. relative to the proportion obtained in the absence of the splicing modifier).
  • a splicing modifier that promotes inclusion of SMN2 exon 7 in RNA obtained following splicing of pre-mRNA transcribed from human SMN2 may be referred to herein simply as ‘a splicing modifier that promotes SMN2 exon 7 inclusion’
  • the splicing modifier promotes inclusion of exon 7 in RNA obtained following splicing of pre-mRNA transcribed from an allele of human SMN2 comprising c.840C>T.
  • the splicing modifier increases the proportion of RNA molecules comprising SMN2 exon 7 among RNA molecules obtained following splicing of pre-mRNA transcribed from an allele of human SMN2 comprising c.840C>T (i.e. relative to the proportion obtained in the absence of the splicing modifier).
  • the splicing modifier increases the level of human SMN2 protein comprising amino acids encoded by exon 7 of human SMN2 (i.e. relative to the level detected in the absence of the splicing modifier).
  • the splicing modifier increases the proportion of polypeptides comprising amino acids encoded by exon 7 of human SMN2 among polypeptides expressed from human SMN2 (i.e. relative to the proportion obtained in the absence of the splicing modifier). In some embodiments, the splicing modifier increases the proportion of polypeptides comprising amino acids encoded by exon 7 of human SMN2 among polypeptides expressed from an allele of human SMN2 comprising c.840C>T (i.e. relative to the proportion obtained in the absence of the splicing modifier).
  • the splicing modifier promotes inclusion of the third nucleotide sequence in the product of splicing of a polynucleotide according to the present disclosure (i.e. where the polynucleotide is a polyribonucleotide). In some embodiments, the splicing modifier increases the proportion of molecules comprising the third nucleotide sequence among molecules obtained following splicing of a polynucleotide according to the present disclosure (i.e. where the polynucleotide is a polyribonucleotide).
  • the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:13 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence P37829 of SEQ ID NO:22. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:13 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:22.
  • the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:13 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:23. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:13 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:23. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:14 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:24.
  • the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:14 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:24. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:13 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:25.
  • the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:13 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:25. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:13 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:28.
  • the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:13 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:28. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:27 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:29.
  • the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:27 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:29.
  • Splicing modifiers having such functional properties can be identified by analysis e.g. in suitable in vitro assays. Such assays may comprise culturing cells in vitro in the presence or absence of a candidate splicing modifier, and analysing the RNA and/or protein produced after an appropriate period of time for an effect of the candidate splicing modifier to be observed.
  • cells can be transfected with a vector comprising DNA having the sequence of SEQ ID NO:22 and cultured in the presence or absence of a candidate splicing modifier for a suitable period of time (e.g.24, 48, 72 hours).
  • RNA can subsequently be isolated from the cells, and analysed (e.g. by qRT-PCR) to determine the P37829 level/proportion of mature RNA molecules comprising the nucleotide sequence consisting of SEQ ID NO:13.
  • a splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound of formula (I) of WO 2015/173181 A1.
  • a splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound selected from those listed in claim 39 of WO 2015/173181 A1. In some embodiments, a splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound selected from those listed in claim 40 of WO 2015/173181 A1. In some embodiments, the splicing modifier according to the present disclosure is selected from: RG- 7916, RG-7800, SMN-C2, SMN-C3, SMN-C5, TEC-1, branaplam, PK4C9 and nusinersen.
  • the splicing modifier is selected from: RG-7916, RG-7800, SMN-C2, SMN-C3, SMN-C5 and TEC-1. In some embodiments, the splicing modifier is selected from RG-7916 and RG-7800. In preferred embodiments, the splicing modifier is risdiplam (RG-7916).
  • Functional properties of the polynucleotides In aspects and embodiments of the present disclosure, the polynucleotides of the present disclosure may be characterised by reference to one or more functional properties.
  • polynucleotide is a polyribonucleotide
  • splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence.
  • splicing of the polyribonucleotide in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides comprising the third nucleotide sequence.
  • inclusion of the third nucleotide sequence is promoted in splicing of the polyribonucleotide, favouring the production of mature RNA molecules encoding the polypeptide of interest, and thus expression of the polypeptide of interest at the protein level.
  • Such polynucleotides may be referred to herein as ‘ON-switch’ polynucleotides.
  • cells comprising an ON-switch polynucleotide of the present disclosure substantially do not express the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion.
  • cells comprising the ON-switch polynucleotide express the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion.
  • exemplary ON-switch polynucleotides according to the present disclosure include polynucleotides comprising SEQ ID NO:22, 23, 24 or 25.
  • Further exemplary ON-switch polynucleotides according to the present disclosure include polynucleotides comprising one of SEQ ID NOs:37 to 105.
  • P37829 Inclusion of the third nucleotide sequence as described herein in the products of splicing of a polyribonucleotide according to the present disclosure can be evaluated using methods that are well known to the person skilled in the art. Such methods include qRT-PCR based methods for the detection and/or quantification of RNA molecules comprising nucleotide sequences of interest.
  • cells may be transduced in vitro with a vector comprising a polynucleotide according to the present disclosure and subsequently cultured in vitro in the presence or absence of a splicing modifier that promotes SMN2 exon 7 inclusion for a period of time appropriate for an effect on splicing of a polyribonucleotide according to the present disclosure to be observed.
  • a splicing modifier that promotes SMN2 exon 7 inclusion for a period of time appropriate for an effect on splicing of a polyribonucleotide according to the present disclosure to be observed.
  • RNA may be isolated from the cells
  • cDNA may be prepared from the total RNA
  • the number/proportion of mature RNA molecules comprising the third nucleotide sequence may be evaluated by qPCR using oligonucleotides providing for the specific amplification and/or detection of the products of splicing of the polyribonucleotide (e.g. oligonucleotides hybridising to nucleotide sequences spanning exon:exon boundaries).
  • the qPCR analysis may employ oligonucleotides enabling distinction between RNA molecules comprising the third nucleotide sequence, and RNA molecules lacking the third nucleotide sequence.
  • qRT-PCR-based methods for analysing the products of splicing of a polyribonucleotide are described in the experimental examples of the present disclosure.
  • Expression of a polypeptide of interest can be evaluated using any suitable technique for the detection and/or quantification of the relevant polypeptide. Such techniques include e.g. antibody-based methods, (for example flow cytometry, immunocytochemistry, western blot, ELISA), fluorescence microscopy and flow cytometry.
  • expression of a polypeptide of interest can be evaluated as described in the experimental examples of the present disclosure.
  • expression of a polypeptide of interest may be evaluated by flow cytometry.
  • the third nucleotide sequence may be present in fewer than 15%, e.g. one of ⁇ 10%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2% or ⁇ 1% of the polyribonucleotides.
  • the third nucleotide sequence may be present in more than 80%, e.g.
  • the polypeptide may be expressed by fewer than 15%, e.g. one of ⁇ 10%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2% or ⁇ 1% of the cells.
  • the polypeptide may be expressed by more than 80%, e.g.
  • cells that ‘substantially do not express’ a polypeptide of interest may display a level of expression of the polypeptide of interest which is less than 0.2 times, e.g. one of ⁇ 0.1 times, ⁇ 0.09 times, ⁇ 0.08 times, ⁇ 0.07 times, ⁇ 0.06 times, ⁇ 0.05 times, ⁇ 0.04 times, ⁇ 0.03 times, ⁇ 0.02 times, or ⁇ 0.01 times the level of expression by cells that express the polypeptide of interest.
  • cells that ‘express’ a polypeptide of interest may display a level of expression of the polypeptide of interest which is greater than 5 times, e.g. one of ⁇ 10 times, ⁇ 20 times, ⁇ 50 times, ⁇ 100 times, ⁇ 1000 times, ⁇ 5000 times or ⁇ 10000 times level of expression by cells that ‘substantially do not express’ the polypeptide of interest.
  • the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 in cells comprising a polynucleotide according to the present disclosure following culture in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ⁇ 2 times, ⁇ 3 times, ⁇ 4 times, ⁇ 5 times, ⁇ 10 times, ⁇ 20 times, ⁇ 50 times, ⁇ 100 times, ⁇ 1000 times, ⁇ 5000 times or ⁇ 10000 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion.
  • the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 in cells comprising a polynucleotide according to the present disclosure following culture in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, e.g. one of ⁇ 50 times, ⁇ 20 times, ⁇ 10 times, ⁇ 5 times, ⁇ 4 times, ⁇ 3 times, ⁇ 2 times or ⁇ 1 times the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 in equivalent cells not comprising the polynucleotide.
  • the level of a polypeptide of interest in cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ⁇ 2 times, ⁇ 3 times, ⁇ 4 times, ⁇ 5 times, ⁇ 10 times, ⁇ 20 times, ⁇ 50 times, ⁇ 100 times, ⁇ 1000 times, ⁇ 5000 times or ⁇ 10000 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion.
  • the level of expression of a polypeptide of interest by cells comprising a polynucleotide according to the present disclosure is less than 100 times, e.g. one of ⁇ 50 times, ⁇ 20 times, ⁇ 10 times, ⁇ 5 times, ⁇ 4 times, ⁇ 3 times, ⁇ 2 times or ⁇ 1 times the level of the polypeptide of interest in equivalent cells not comprising the polynucleotide.
  • the proportion of cells expressing a polypeptide of interest within a population of cells comprising a polynucleotide according to the present disclosure is greater than 1 times, e.g. one of ⁇ 2 times, ⁇ 3 times, ⁇ 4 times, ⁇ 5 times, ⁇ 10 times, ⁇ 20 times, ⁇ 50 times, ⁇ 100 times, ⁇ 1000 times, ⁇ 5000 times or ⁇ 10000 times the proportion of such cells expressing the polypeptide of interest cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion.
  • the proportion of cells expressing a polypeptide of interest within a population of cells comprising a polynucleotide according to the present disclosure is less than 100 P37829 times, e.g. one of ⁇ 50 times, ⁇ 20 times, ⁇ 10 times, ⁇ 5 times, ⁇ 4 times, ⁇ 3 times, ⁇ 2 times or ⁇ 1 times the proportion of such cells expressing the polypeptide of interest cultured in the presence of the splicing modifier that promotes SMN2 exon 7 inclusion.
  • inclusion of the third nucleotide sequence is promoted in splicing of the polyribonucleotide, favouring the production of mature RNA molecules encoding a premature stop codon, and thus preventing expression of the polypeptide of interest at the protein level.
  • Such polynucleotides may be referred to herein as ‘OFF- switch’ polynucleotides.
  • cells comprising an OFF-switch polynucleotide of the present disclosure express the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion.
  • cells comprising the OFF-switch polynucleotide do not express the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion.
  • Exemplary OFF-switch polynucleotides according to the present disclosure include polynucleotides comprising SEQ ID NO:28 or 29.
  • the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26 in cells comprising a polynucleotide according to the present disclosure following culture in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g.
  • the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26 in cells comprising a polynucleotide according to the present disclosure following culture in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, e.g.
  • the level of a polypeptide of interest in cells comprising a polynucleotide according to the present disclosure is greater than 1 times, e.g.
  • the level of expression of a polypeptide of interest by cells comprising a polynucleotide according to the present disclosure is less than 100 times, e.g.
  • the proportion of cells expressing a polypeptide of interest within a population of cells comprising a polynucleotide according to the present disclosure is greater than 1 times, e.g.
  • the proportion of cells expressing a polypeptide of interest within a population of cells comprising a polynucleotide according to the present disclosure is less than 100 times, e.g.
  • polynucleotides of the present disclosure possess novel and/or improved properties relative to known transgene expression systems, e.g. known transgene expression systems comprising a SMSM-mediated switch derived from SMN2 exons 6 to 8 (e.g. SMN2 exon 6 to exon 8-derived transgene expression systems described in Zhang, et al., Gene Ther.
  • the polynucleotides of the present disclosure possess novel and/or improved properties relative to a SMN2ind minigene polynucleotide (which is defined hereinabove).
  • cells comprising a polynucleotide of the present disclosure cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion produce a population of RNA molecules having an increased proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26, compared to the population of RNA molecules produced by cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions.
  • the polynucleotide of the present disclosure is more effective at promoting inclusion of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to a SMN2ind minigene polynucleotide. That is, the polynucleotides of the present disclosure are more responsive to induction of SMN2 exon 7 variant inclusion in the products of splicing in response to a splicing modifier that promotes SMN2 exon 7 inclusion than SMN2ind minigene polynucleotides.
  • the proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 among RNA molecules obtained from cells comprising a polynucleotide of the present disclosure cultured in the P37829 presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g.
  • RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 among RNA molecules obtained from cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions.
  • the functional properties described herein are evaluated employing the same experimental conditions for the evaluation of cells comprising the different polynucleotides (i.e. the polynucleotide of the present disclosure, and the SMN2ind minigene polynucleotide).
  • the same cell type, the same splicing modifier that promotes SMN2 exon 7 inclusion, the same concentration of the splicing modifier that promotes SMN2 exon 7 inclusion, etc. are used, the same culture period is provided, and the cells are analysed in order to determine the proportion of RNA molecules comprising the relevant nucleotide sequence and/or the level of the polypeptide of interest in the same way.
  • cells comprising a polynucleotide of the present disclosure cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion produce a population of RNA molecules having a decreased proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26, compared to the population of RNA molecules produced by cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions.
  • the polynucleotide of the present disclosure is more effective at excluding a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to a SMN2ind minigene polynucleotide. That is, the polynucleotides of the present disclosure are less ‘leaky’ with respect to SMN2 exon 7 variant inclusion in the products of splicing in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to SMN2ind minigene polynucleotides.
  • RNA molecules comprising an SMN2 exon 7 variant may also be referred to herein as ‘background’ production of such RNA molecules.
  • the proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 among RNA molecules obtained from cells comprising a polynucleotide of the present disclosure cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 1 times, e.g.
  • RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or P37829 26 among RNA molecules obtained from cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions.
  • cells comprising a polynucleotide of the present disclosure e.g. an ON-switch polynucleotide
  • a splicing modifier that promotes SMN2 exon 7 inclusion display an increased level of protein expression of the polypeptide of interest, compared to the level of protein expression of the polypeptide of interest by cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions.
  • the polynucleotide of the present disclosure is more effective at promoting protein expression of the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to a SMN2ind minigene polynucleotide. That is, the polynucleotides of the present disclosure are more responsive to induction of expression of the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to SMN2ind minigene polynucleotides. It will be appreciated that for the purposes of such comparison, the different polynucleotides (i.e.
  • the polynucleotide of the present disclosure and the SMN2ind minigene polynucleotide preferably encode the same polypeptide of interest.
  • the level of protein expression of the polypeptide of interest from cells comprising a polynucleotide of the present disclosure e.g. an ON-switch polynucleotide
  • a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g.
  • cells comprising a polynucleotide of the present disclosure e.g.
  • an ON-switch polynucleotide) cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion display a decreased level of protein expression of the polypeptide of interest, compared to the level of protein expression of the polypeptide of interest by cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. That is, in some embodiments the polynucleotide of the present disclosure is more effective at preventing protein expression of the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to a SMN2ind minigene polynucleotide.
  • the polynucleotides of the present disclosure are less ‘leaky’ with respect to expression of the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to SMN2ind minigene polynucleotides.
  • Leaky expression of the polypeptide of interest i.e. in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion
  • the level of protein expression of the polypeptide of interest from cells comprising a polynucleotide of the present disclosure e.g.
  • an ON-switch polynucleotide) cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 1 times, e.g. ⁇ 0.99 times, ⁇ 0.95 times, P37829 ⁇ 0.9 times, ⁇ 0.85 times, ⁇ 0.8 times, ⁇ 0.75 times, ⁇ 0.7 times, ⁇ 0.65 times, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times, ⁇ 0.25 times, ⁇ 0.2 times, ⁇ 0.15 times, ⁇ 0.1 times, ⁇ 0.05 times, or ⁇ 0.01 times the level of protein expression of the polypeptide of interest from cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions.
  • a polypeptide of interest expressed from a polynucleotide of the present disclosure comprises fewer extraneous additional amino acids (i.e. amino acids additional to the amino acid sequence of the polypeptide of interest) compared to a polypeptide of interest expressed from SMN2ind minigene polynucleotide.
  • a polypeptide of interest expressed from a polynucleotide of the present disclosure comprises a smaller N-terminal tag (i.e. formed of extraneous, additional amino acids in addition to the amino acid sequence of the polypeptide of interest) than a polypeptide of interest expressed from SMN2ind minigene polynucleotide.
  • the polynucleotides of the present disclosure provide for the inducible expression of polypeptides of interest comprising fewer extraneous additional amino acids, relative to polypeptides of interest expressed from SMN2ind minigene polynucleotide.
  • Vectors The present disclosure provides vectors comprising the polynucleotides according to the present disclosure. It will be appreciated that a vector is also a polynucleotide, and so in some embodiments a polynucleotide according to the present disclosure may be a vector.
  • a ‘vector’ as used herein refers to a polynucleotide used as a vehicle to transfer exogenous nucleic acid into a cell.
  • the vector may be a vector for expression of the nucleic acid in the cell (i.e. the vector may be an expression vector).
  • Such vectors may include a promoter sequence operably linked to the nucleotide sequence to be expressed.
  • Vectors may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used in a vector according to the present disclosure.
  • the term ‘operably linked’ may include the situation where nucleic acid encoding a polypeptide of interest according to the present disclosure and regulatory nucleic acid sequence(s) (e.g.
  • a promoter and/or enhancers are covalently linked in such a way as to place the expression of the nucleic acid encoding a polypeptide under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette).
  • a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence.
  • the resulting transcript(s) may then be translated into the desired polypeptide(s).
  • Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g.
  • F plasmids F plasmids
  • non-conjugative plasmids R plasmids, col plasmids, episomes
  • viral vectors e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno- associated virus vectors, vaccinia virus vectors, baculoviral vectors and herpesvirus vectors), transposon- based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g.
  • retroviral vectors e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector)
  • lentiviral vectors e.g. murine Leukemia virus (MLV)
  • the vector may be a eukaryotic vector, i.e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell.
  • the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
  • the vector comprises a CMV (e.g. mCMV), SV40, RSV or PGK promoter.
  • the polynucleotide according to the present disclosure comprises a CMV promoter, a CAG promoter, a hEF1a promoter, a hUbiC promoter, an RSV promoter, a TK promoter, a PGK promoter, or a CAG minimal promoter.
  • the polynucleotide according to the present disclosure comprises a promoter having a nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g.
  • a vector is selected based on tropism for a cell type/tissue/organ to which it is desired to deliver the polynucleotide according to the present disclosure. In some embodiments, a vector is selected based on tropism for a cell type/tissue/organ in which it is desired to express the polypeptide of interest.
  • the polynucleotide may be desired to deliver the polynucleotide to, and/or express the polypeptide of interest in, a cell type/tissue/organ affected by a disease/condition to be treated/prevented in accordance with the present disclosure (e.g. a cell type/tissue/organ in which the symptoms of the disease/condition manifest).
  • a cell type/tissue/organ affected by a disease/condition to be treated/prevented in accordance with the present disclosure e.g. a cell type/tissue/organ in which the symptoms of the disease/condition manifest.
  • the vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • a vector may be an adeno-associated virus vector described in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378.
  • a vector may be an adeno-associated virus vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272.
  • the vector is a self-complementary adeno-associated virus (scAAV) vector.
  • scAAV self-complementary adeno-associated virus
  • Conventional AAV have a P37829 single-stranded DNA genome, and depend on the DNA replication machinery of a transduced cell to synthesise the complementary strand, delaying transgene expression.
  • scAAV contain complementary sequences that spontaneously anneal upon infection, eliminating the requirement for DNA synthesis in the transduced host cell.
  • a vector may be an adeno-associated viral vector of one of the following serotypes: AAV9 (including AAV9 variants AAV-PHP.B and AAV9.45), AAV1, AAV2 (including AAV2 variant AAV2i8), AAV5, AAV6, AAV8, AAV10 or AAVrh74.
  • AAV9 including AAV9 variants AAV-PHP.B and AAV9.45
  • AAV1 including AAV2 variant AAV2i8
  • AAV5 AAV6, AAV8, AAV10 or AAVrh74.
  • a vector may be an adeno-associated viral vector of one of the following serotypes: AAV9 (including AAV9 variants AAV- PHP.B and AAV9.45), AAV1, AAV2 (including AAV2 variants AAV2.7m8 and AAV2i8), AAV5, AAV6, AAV8, AAV10 or AAVrh74.
  • the vector is an AAV9 vector.
  • a vector comprises modification to increase binding to and/or transduction of a cell-type of interest (i.e. as compared to the level of binding/transduction by the unmodified vector).
  • modification is to a capsid protein.
  • a vector comprises a capsid protein comprising a cell-targeting peptide.
  • the cell-targeting peptide is a cell-targeting peptide described in Büning and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12: 248-265, which is hereby incorporated by reference in its entirety, e.g. a cell-targeting peptide shown in Table 1, 2, 3 or 4 thereof.
  • a vector comprises a capsid protein comprising substitution to one or more tyrosine residues, e.g. one or more surface-exposed tyrosine residues.
  • a vector comprises a capsid protein in which one or more tyrosine residues are substituted with another amino acid as described in Iida et al., Biomed Res Int. (2013) 2013: 974819, which is hereby incorporated by reference in its entirety.
  • a vector may be an adeno-associated virus vector described in Büning and Srivastava, supra.
  • a vector may be an adeno-associated virus vector described in Iida et al., supra.
  • the vector comprises a control element for inducible expression of the polynucleotide of the disclosure.
  • a sequence for controlling expression of the polynucleotide may provide for expression of the polynucleotide by cells of a particular type or tissue.
  • expression may be under the control of a cell type- or tissue-specific promoter.
  • Promoters for cell type- or tissue-specific expression of a polynucleotide in accordance with the present disclosure can be selected in accordance with a disease/condition to be treated/prevented.
  • the promoter may drive expression in a cell type/tissue/an organ affected by the disease/condition (e.g. a cell type/tissue/an organ in which the symptoms of the disease/condition manifest).
  • a promoter may provide for expression of the polynucleotide in neuronal cells/tissue.
  • a promoter may be a neuron-specific promoter (e.g. a CaMKII, NSE or SynI-miniCMV promoter)
  • a promoter may provide for expression of the polynucleotide in muscle cells/tissue (e.g. cardiac and/or skeletal muscle cells/tissue).
  • a promoter may be a cardiac or cardiomyocte-specific promoter (e.g. a cTNT, ⁇ -MHC or MLC2v promoter).
  • a promoter may be a skeletal muscle/striated muscle cell- specific promoter (e.g. a MCK, MHCK7 or desmin promoter).
  • a promoter may be a vascular endothelial cell-specific promoter (e.g. a Tie2 promoter).
  • a promoter may be a vascular smooth muscle cell-specific promoter (e.g. a SM22a promoter).
  • a promoter may be a monocyte/macrophage-specific promoter (e.g. a LysM promoter).
  • a sequence for controlling expression of the polynucleotide may provide for expression of the polynucleotide in response to e.g.
  • expression may be under the control of inducible promoter.
  • the agent may provide for inducible expression of the polynucleotide in vivo by administration of the agent to a subject having been administered with a modified cell according to the disclosure, or ex vivo/in vitro by administration of the agent to cells in culture ex vivo or in vitro.
  • a polynucleotide or vector according to the present disclosure may employ a conditional expression system for controlling expression of the polynucleotide by cells comprising the polynucleotide/vector.
  • ‘Conditional expression’ may also be referred to herein as ‘inducible expression’, and refers to expression contingent on certain conditions, e.g.
  • Cells The present disclosure also provides a cell comprising or expressing a polynucleotide according to the present disclosure. Also provided is a cell comprising or expressing vector according to the present disclosure.
  • a polynucleotide according to the present disclosure e.g. a polyribonucleotide
  • the cell may be a eukaryotic cell, e.g. a mammalian cell.
  • the mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).
  • the cell may be a human cell.
  • P37829 The cell may be an immune cell.
  • the cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte.
  • the lymphocyte may be e.g. a T cell, B cell, NK cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof.
  • the cell may express e.g. CD3 polypeptides (e.g. CD3 ⁇ CD3 ⁇ CD3 ⁇ or CD3 ⁇ ), TCR polypeptides (TCR ⁇ or TCR ⁇ ), CD27, CD28, CD4 or CD8.
  • the cell is a T cell.
  • the T cell is a CD3+ T cell.
  • the T cell is a CD3+, CD8+ T cell.
  • the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).
  • CTL cytotoxic T lymphocyte
  • the present disclosure also provides a method for producing a cell comprising or expressing a polynucleotide/vector according to the present disclosure, the method comprising introducing a polynucleotide/vector of the present disclosure into a cell.
  • introducing a polynucleotide/vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. adeno-associated viral transduction).
  • the polynucleotide/vector is introduced to the cell in vivo, e.g. by administration of a vector according to the present disclosure (e.g. a viral vector, e.g. an adeno-associated viral vector) to a subject.
  • a vector according to the present disclosure e.g. a viral vector, e.g. an adeno-associated viral vector
  • the polynucleotide/vector is introduced into cells in culture ex vivo or in vitro. Any suitable method may be employed to produce a cell according to the present disclosure. Such methods may comprise nucleic acid transfer for permanent (i.e. stable) or transient expression of the polynucleotide of the present disclosure.
  • the polynucleotide following introduction into a cell, may be integrated into or form part of the genomic DNA of the cell.
  • the polynucleotide may be maintained extrachromosomally.
  • Any suitable genetic engineering platform may be used, and include gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol. (2014) 32:189-225, hereby incorporated by reference in its entirety. Methods also include those described e.g. in Wang and Rivière Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety.
  • Suitable methods for introducing nucleic acid(s)/vector(s) into cells include transduction, transfection and electroporation. In some embodiments, the methods additionally comprise maintaining the cell under conditions suitable for expression of the polynucleotide/vector by the cell.
  • the present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure.
  • Compositions The present disclosure also provides compositions comprising the polynucleotides, vectors and cells described herein. In particular, the present disclosure provides pharmaceutical compositions and medicaments comprising the polynucleotides, vectors and cells of the present disclosure. P37829 Such compositions may comprise the relevant article (i.e. the polynucleotide/vector/cell) in a formulation suitable for clinical use.
  • compositions/medicaments comprising polynucleotides and vectors according to the present disclosure.
  • the compositions of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents/excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g.
  • vitamin A vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben
  • anti-oxidants e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium
  • lubricants e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin
  • binders e.g.
  • PEG polyethylene glycol
  • PVP polyvinylpyrrolidone
  • xylitol sorbitol, mannitol
  • stabilisers solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).
  • solubilisers e.g., surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).
  • surfactants e.g., wetting agents
  • masking agents or colouring agents e.g. titanium oxide
  • each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation.
  • Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed.(A)Adejare), 23rd Edition (2020), Academic Press.
  • the pharmaceutical compositions/medicaments according to the present disclosure may be formulated for administration to a subject, e.g. administration via a route of administration as appropriate for the nature of the therapeutic agent and the disease to be treated/prevented.
  • a pharmaceutical composition/medicament may be formulated for parenteral, systemic, topical, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal,, oral or transdermal administration.
  • a pharmaceutical composition/medicament may be formulated for administration by injection or infusion, or administration by ingestion.
  • Medicaments and pharmaceutical compositions may be formulated for administration to a blood vessel, or to a tissue/organ of interest (e.g. a tissue/organ affected by a disease/condition, e.g. a tissue/organ in which symptoms of the disease/condition manifest).
  • the pharmaceutical compositions/medicaments may comprise the polynucleotide/vector/cell in a sterile or isotonic medium.
  • the pharmaceutical compositions/medicaments may be provided in fluid, including gel, P37829 form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via cannula) to a blood vessel, or a selected region of the human or animal body.
  • the pharmaceutical compositions/medicaments may be provided in solid form, e.g. in lyophilised form.
  • the present disclosure also provides methods for producing pharmaceutical compositions/medicaments according to the present disclosure.
  • Such methods may comprise mixing a polynucleotide/vector/cell described herein with a pharmaceutically-acceptable carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent.
  • a pharmaceutically-acceptable carrier diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent.
  • Such methods generally include the step of bringing into association the polynucleotide/vector/cell with a carrier which constitutes one or more accessory ingredients.
  • the compositions are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping
  • Polynucleotides, vectors, cells and compositions according to the present disclosure may be modified and/or formulated to facilitate delivery to, and/or uptake by, a cell type/tissue/organ of interest (e.g. a cell type/tissue/organ in which symptoms of a disease/condition manifest).
  • a cell type/tissue/organ of interest e.g. a cell type/tissue/organ in which symptoms of a disease/condition manifest.
  • Strategies for targeted delivery of polynucleotides are reviewed e.g. in Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated by reference in their entirety.
  • articles of the present disclosure may be encapsulated in a nanoparticle or a liposome.
  • articles of the present disclosure may be (covalently or non-covalently) associated with a cell-penetrating peptide (e.g. a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide), a cationic polymer, a cationic lipid or a viral carrier.
  • a cell-penetrating peptide e.g. a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide
  • Nanoparticles may be organic, e.g. micelles, liposomes, proteins, solid-lipid particles, solid polymer particles, dendrimers, and polymer therapeutics.
  • Nanoparticles may be inorganic, e.g. such as nanotubes or metal particles, optionally with organic molecules added.
  • a nanoparticle is a nanoparticle described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, which is hereby incorporated by reference in its entirety.
  • a nanoparticle is a PLGA, polypeptide, poly( ⁇ -amino ester), DOPE, ⁇ -cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan or polyphosophoester nanoparticle.
  • polynucleotides and vectors according to the present disclosure comprise modification to incorporate one or more moieties facilitating delivery to, and/or uptake by, a cell type, organ or tissue of interest (e.g.
  • polynucleotides or vectors according to the present disclosure are linked (e.g. chemically conjugated to) one or more moieties facilitating delivery to, and/or uptake by, a cell type, tissue or organ of interest.
  • moieties facilitating delivery to, and/or uptake by, cell types, tissues or organs of interest are described e.g. in Benizri et al., Bioconjug Chem. (2019) 30(2): 366–383, which is hereby incorporated by reference in its entirety.
  • moieties include e.g.
  • N-acetylgalactosamine (GalNAc), ⁇ -tocopherol, cell-penetrating peptides, nucleic acid aptamers, antibodies and antigen-binding fragments/derivatives thereof, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g. palmitic acid) and nucleolipid moieties.
  • Articles of the present disclosure may be formulated in a sustained release delivery system, in order to release the polynucleotide, vector, cell or composition at a predetermined rate. Sustained release delivery systems may maintain a constant drug/therapeutic/prophylactic concentration for a specified period of time.
  • articles of the present disclosure are formulated in a liposome, gel, implant, device, or drug-polymer conjugate e.g. hydrogel.
  • a composition according to the present disclosure may further comprise a splicing modifier that promotes SMN2 exon 7 inclusion (e.g. as described herein).
  • Therapeutic/prophylactic applications The polynucleotides, vectors, cells and compositions of the present disclosure find use in therapy and prophylaxis. Accordingly, the present disclosure provides a polynucleotide, vector, cell or composition described herein for use in a method of medical treatment or prophylaxis.
  • a polynucleotide, vector, cell or composition described herein for use in a method of treating or preventing a disease/condition described herein. Also provided is the use of a polynucleotide, vector, cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically- or prophylactically- effective amount of a polynucleotide, vector, cell or composition described herein.
  • the intervention described in the preceding paragraph may be effective to reduce the development or progression of a disease/condition, alleviate the symptoms of a disease/condition or reduce the pathology of a disease/condition.
  • the intervention may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition.
  • the intervention may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition.
  • the intervention may prevent progression/development of the disease/condition a later stage (e.g. a chronic stage).
  • the polynucleotides, vectors, cells and compositions described herein may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from an increase in the level of the polypeptide of interest (i.e. the polypeptide of interest encoded by the polynucleotide).
  • the disease/condition may be a disease/condition associated with and/or characterised by deficiency/insufficiency of the polypeptide of interest. Deficiency/insufficiency of the polypeptide of interest may be positively associated with the onset, development or progression of the disease/condition, and/or positively associated with the severity of one or more symptoms of the disease/condition.
  • Deficiency/insufficiency of the polypeptide of interest may be a risk factor for the onset, development or progression of the disease/condition.
  • the disease/condition may be characterised by a decreased level of expression or activity of the polypeptide of interest, e.g. as compared to the level of expression/activity in the absence of the disease/condition.
  • the disease/condition may be characterised by a decrease in the number/proportion/activity of cells expressing the polypeptide of interest, e.g. as compared to the level/number/proportion/activity in the absence of the disease/condition (e.g. in a healthy subject, or in equivalent non-diseased tissue).
  • the polypeptide of interest may be MeCP2, and the disease/condition to be treated/prevented in accordance with the present disclosure may be a disease/condition caused by deficiency/insufficiency of MeCP2, e.g. Rett syndrome.
  • the disease/condition may be a disease/condition in which the target antigen, or cells comprising/expressing the target antigen are pathologically-implicated, e.g.
  • a disease/condition in which an increased level/activity of the target antigen, or an increase in the number/proportion/activity of cells comprising/expressing the target antigen is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition.
  • an increased level/activity of the target antigen, or an increase in the number/proportion/activity of cells comprising/expressing the target antigen may be a risk factor for the onset, development or progression of the disease/condition.
  • the disease/condition may be characterised by an increase in the level of expression or activity of the target antigen, e.g. as compared to the level of expression/activity in the absence of the disease/condition.
  • the disease/condition may be characterised by an increase in the number/proportion/activity of cells expressing the target antigen, e.g. as compared to the level/number/proportion/activity in the absence of the disease/condition (e.g. in a healthy subject, or in equivalent non-diseased tissue).
  • Therapeutic/prophylactic intervention in accordance with the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of the target antigen; a reduction in the activity of the target antigen; and/or a reduction in the number/proportion/activity of cells comprising/expressing the target antigen.
  • the disease/condition may be a disease/condition to be treated by nucleic acid editing
  • the polypeptide of interest may be a constituent protein of an appropriate site-specific nuclease nucleic acid editing system.
  • the present disclosure provides the articles of the present disclosure for use, uses of articles of the present disclosure, and methods comprising administering polynucleotides, vectors, cells and P37829 compositions according to the present disclosure to a subject (e.g. a subject in need of treatment).
  • the methods comprise administering to the subject a splicing modifier that promotes SMN2 exon 7 inclusion (e.g. a splicing modifier that promotes SMN2 exon 7 inclusion as described herein).
  • Administration of the articles of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of the disease/condition and the particular article administered.
  • Prescription of treatment e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed.(A)Adejare), 23rd Edition (2020), Academic Press.
  • Administration of the articles of the present disclosure may be parenteral, systemic, intravenous, intra- arterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral. In some cases, the articles of the present disclosure may be formulated for targeted delivery to specific cells, a tissue, an organ and/or a tumor. Multiple doses of an article of the present disclosure may be provided.
  • the present disclosure provides the articles of the present disclosure for use, uses of articles of the present disclosure, and methods comprising administering: (i) a polynucleotide, vector, cell or composition according to the present disclosure, and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, to a subject (e.g. a subject in need of treatment).
  • provision of (i) and (ii) may be as a combination therapy.
  • (i) and (ii) may be provided simultaneously or sequentially.
  • Simultaneous administration refers to administration of the two or more agents (e.g. a polynucleotide, vector, cell or composition according to the present disclosure, and a splicing modifier that promotes SMN2 exon 7 inclusion) together, for example as a pharmaceutical composition containing both agents (i.e. as a combined preparation), or immediately after each other (e.g. within 1, 4, 6, 8 or 12 hours), and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel.
  • agents e.g. a polynucleotide, vector, cell or composition according to the present disclosure, and a splicing modifier that promotes SMN2 exon 7 inclusion
  • Sequential administration refers to administration of one of the agents followed after a given time interval P37829 by separate administration of another agent. It is not required that the agents are administered by the same route, although this is the case in some embodiments.
  • the time interval may be any time interval.
  • a splicing modifier that promotes SMN2 exon 7 inclusion is administered to a subject after administration of a polynucleotide, vector, cell or composition according to the present disclosure.
  • a polynucleotide, vector, cell or composition according to the present disclosure is administered to a subject, and a splicing modifier that promotes SMN2 exon 7 inclusion is administered continuously to the subject thereafter.
  • the splicing modifier that promotes SMN2 exon 7 inclusion is administered in a quantity, and/or with a periodicity, selected to achieve a desired level of expression of the polypeptide of interest. That is, in some embodiments the quantity of, and/or periodicity with which, a splicing modifier that promotes SMN2 exon 7 inclusion administered to a subject in accordance with the present disclosure is selected to achieve a desired level of expression of the polypeptide of interest. In some embodiments, the quantity of, and/or periodicity with which, a splicing modifier that promotes SMN2 exon 7 inclusion is administered to a subject is adjusted through the course of its administration to change (i.e.
  • the quantity and/or periodicity of administration of a splicing modifier that promotes SMN2 exon 7 inclusion may be increased to increase the level of expression of the polypeptide of interest.
  • the quantity and/or periodicity of administration of a splicing modifier that promotes SMN2 exon 7 inclusion may be decreased to decrease the level of expression of the polypeptide of interest.
  • the quantity and/or periodicity of administration of a splicing modifier that promotes SMN2 exon 7 inclusion may be decreased to increase the level of expression of the polypeptide of interest.
  • the quantity and/or periodicity of administration of a splicing modifier that promotes SMN2 exon 7 inclusion may be increased to decrease the level of expression of the polypeptide of interest.
  • the present disclosure also provides a method for modifying a cell to comprise or express a polynucleotide according to the present disclosure, comprising introducing into a cell a polynucleotide or vector according to the present disclosure.
  • introducing a polynucleotide or vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).
  • Transfection relates to the process of introducing nucleic acid into cells using means other than viral infection and is hence a non-viral method. Transfection may be performed by physical/mechanical methods (including electroporation, sonoporation, magnetofection, gene microinjection and laser irradiation) or chemical methods (liposomal-based or non-liposomal based).
  • Liposomal-based transfection P37829 reagents are chemicals which enable the formation of positively charged lipid aggregates, which can then merge with the phospholipid bilayer of the cell to facilitate the entry of foreign genetic material.
  • liposomal-based transfection reagents include, but are not limited to Oligofectamine®, Lipofectamine® and DharmaFECT®.
  • Non-liposomal transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendrimers and non-liposomal lipids.
  • a non- liposomal transfection reagent is polyethylenimine (PEI). Electroporation may be performed e.g.
  • Transduction is a process by which nucleic acids may be introduced into a cell by a virus or a viral vector.
  • the polynucleotide is, or is comprised in, a viral vector, or the vector is a viral vector.
  • Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola- Ila, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety.
  • Agents may be employed in the methods of the present disclosure to enhance the efficiency of transduction.
  • Hexadimethrine bromide is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface.
  • Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).
  • the methods comprise centrifuging the cells into which it is desired to introduce polynucleotide or vector according to the present disclosure in the presence of cell culture medium comprising viral vector(s) comprising the polynucleotide (referred to in the art as ‘spinfection’).
  • the methods comprise culturing the cell under conditions suitable for expression of the polynucleotide or vector by the cell.
  • the methods comprise culturing the cell under conditions suitable for transcription of a polydeoxyribonucleotide.
  • the methods comprise culturing the cell under conditions suitable for post-transcriptional processing (e.g. splicing) of a polyribonucleotide.
  • the methods comprise culturing the cell under conditions suitable for translation of a polypeptide from a polyribonucleotide.
  • Methods for culturing (including generating and/or expanding) populations of cells in vitro/ex vivo – including suitable culture conditions (i.e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), cell numbers, culture periods etc. – are well known to the skilled person.
  • suitable culture conditions i.e. cell culture media, additives, stimulations, temperature, gaseous atmosphere
  • cell numbers, culture periods etc. – are well known to the skilled person.
  • cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2.
  • the present disclosure also provides a method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell a polynucleotide or vector according to the present disclosure.
  • the cell may express the polypeptide of interest following introduction of the polynucleotide/vector into the cell.
  • the method may further comprise contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion as described herein.
  • the method comprises (i) introducing into a cell a polynucleotide or vector according to the present disclosure; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion.
  • Contacting’ a cell with a splicing modifier may comprise bringing a cell into contact with a splicing modifier in a cell culture, and may be achieved by applying the splicing modifier to the cells in culture.
  • the present disclosure also provides a method for inhibiting expression of a polypeptide of interest in a cell. The method comprises contacting a cell comprising a polynucleotide or vector according to the present disclosure comprising/encoding an OFF-switch with a splicing modifier that promotes SMN2 exon 7 inclusion as described herein. Any suitable quantity/concentration of a splicing modifier may be employed in the methods of the present disclosure.
  • the quantity/concentration of the splicing modifier is preferably selected such as to achieve the desired effect, i.e. increased inclusion of the SMN2 exon 7 variant nucleotide sequence in the products of splicing of the relevant polyribonucleotide.
  • Subjects A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use).
  • the subject to be administered with an article of the present disclosure e.g. in accordance with therapeutic or prophylactic intervention
  • the subject is preferably mammalian, more preferably human.
  • the subject may be a non-human mammal, but is more preferably human.
  • the subject may be male or female.
  • kits of parts The present disclosure also provides kits of parts.
  • a kit of parts according to the present disclosure comprises (i) a polynucleotide, vector, or a pharmaceutical composition according to the present disclosure, and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion.
  • Kits of parts according to the present disclosure may comprise a predetermined quantity of articles according to (i) and/or (ii), as described in the preceding paragraph.
  • articles according to (i) and/or (ii) are provided in containers (e.g. in vials or bottles).
  • the kit may provide articles according to (i) and/or (ii) together with instructions (e.g. a protocol) as to how to employ them in accordance with a therapeutic or prophylactic intervention as described herein.
  • the kit of parts may comprise a polynucleotide or vector according to the present disclosure, and optionally materials for introducing the polynucleotide/vector into a cell.
  • the kit of parts may comprise a system for producing a cell according to the present disclosure.
  • the kit of parts may comprise a (closed) bag cell incubation system in which a polynucleotide or vector can be introduced into a cell.
  • the kit of parts may comprise materials for formulating a polynucleotide or vector according to the present disclosure to a pharmaceutical composition, e.g. a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.
  • the kit of parts further comprises reagents, buffers and/or standards required for execution of a method according to the present disclosure. Kits according to the present disclosure may include instructions for use, e.g.
  • kits of parts in the form of an instruction booklet or leaflet.
  • the instructions may include a protocol for performing any one or more of the methods described herein.
  • the manufacture of kits of parts according to the present disclosure preferably follows standard procedures which are known to the person skilled in the art. Sequence identity
  • sequence identity between a given nucleotide sequence (e.g. of a polynucleotide) and a reference nucleotide sequence is calculated by determining the percentage of the nucleotides in the given nucleotide sequence that are identical to those of the reference nucleotide sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percentage sequence identity between the two sequences.
  • sequence identity between a given amino acid sequence (e.g. of a polypeptide) and a reference amino acid sequence is calculated by determining the percentage of the amino acids in the given amino acid sequence that are identical to those of the reference amino acid sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percentage sequence identity between the two sequences. Pairwise and multiple sequence alignment for the purposes of evaluating sequence identity between two or more nucleotide or amino acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Söding, J.
  • P37829 2. The polynucleotide according to para 1, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. 3. The polynucleotide according to para 1 or para 2, wherein the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:222. 4.
  • the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:226.
  • the polynucleotide according to any one of paras 1 to 11, wherein the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:227 or SEQ ID NO:340. 13.
  • ITR inverted terminal repeat
  • a vector comprising a polynucleotide according to any one of paras 1 to 18.
  • AAV adeno-associated virus
  • a pharmaceutical composition comprising a polynucleotide according to any one of paras 1 to 18, or a vector according to para 19 or para 20, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.
  • a cell comprising a polynucleotide according to any one of paras 1 to 18, or a vector according to para 19 or para 20.
  • the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 24.
  • a method for modifying a cell to express a polypeptide of interest comprising: (i) introducing into a cell a polynucleotide according to any one of paras 1 to 18, or a vector according to para 19 or para 20; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 25.
  • a method for expressing a polypeptide of interest in a cell comprising contacting a cell according to para 22 with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • P37829 26 A polynucleotide according to any one of paras 1 to 18, a vector according to para 19 or para 20, or a pharmaceutical composition according to para 21, for use in a method of medical treatment or prophylaxis. 27.
  • a method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest comprising administering to a subject a polynucleotide according to any one of paras 1 to 18, a vector according to para 19 or para 20, or a pharmaceutical composition according to para 21.
  • a kit comprising: (i) a polynucleotide according to any one of paras 1 to 18, a vector according to para 19 or para 20, or a pharmaceutical composition according to para 21; and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 33.
  • polynucleotide according to para 33 wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:1.
  • the first nucleotide sequence comprises, or consists of, SEQ ID NO:3. 37.
  • 39. The polynucleotide according to any one of paras 33 to 38, wherein the second nucleotide sequence comprises, or consists of, SEQ ID NO:10. 40.
  • the polynucleotide according to any one of paras 33 to 41, wherein the fourth nucleotide sequence comprises SEQ ID NO:15 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end. P37829 43.
  • ITR inverted terminal repeat
  • a vector comprising a polynucleotide according to any one of paras 33 to 48.
  • 50. The vector according to para 49, wherein the vector is an adeno-associated virus (AAV) vector. 51.
  • AAV adeno-associated virus
  • a pharmaceutical composition comprising a polynucleotide according to any one of paras 33 to 48, or a vector according to para 49 or para 50, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.
  • 52. A cell comprising a polynucleotide according to any one of paras 33 to 48, or a vector according to para 49 or para 50.
  • 53. The cell according to para 52, wherein the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 54.
  • a method for modifying a cell to express a polypeptide of interest comprising introducing into a cell a polynucleotide according to any one of paras 33 to 48, or a vector according to para 49 or para 50.
  • a method for inhibiting expression of a polypeptide of interest in a cell comprising contacting a cell according to para 52 with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. P37829 56.
  • a polynucleotide according to any one of paras 33 to 48, a vector according to para 49 or para 50, or a pharmaceutical composition according to para 51 in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest.
  • a method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest comprising administering to a subject a polynucleotide according to any one of paras 33 to 48, a vector according to para 49 or para 50, or a pharmaceutical composition according to para 51. 60.
  • a kit comprising: (i) a polynucleotide according to any one of paras 33 to 48, a vector according to para 49 or para 50, or a pharmaceutical composition according to para 51; and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 1A.
  • 9A The polynucleotide according to any one of paras 1A to 8A, wherein the third nucleotide sequence consists of SEQ ID NO:13 or SEQ ID NO:14. 10A.
  • polynucleotide according to any one of paras 1A to 14A wherein the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25. 16A.
  • ITR inverted terminal repeat
  • AAV adeno-associated virus
  • a pharmaceutical composition comprising a polynucleotide according to any one of paras 1A to 18A, or a vector according to para 19A or para 20A, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.
  • 22A A cell comprising a polynucleotide according to any one of paras 1A to 18A, or a vector according to para 19A or para 20A.
  • 23A The cell according to para 22A, wherein the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 24A.
  • a method for modifying a cell to express a polypeptide of interest comprising: P37829 (i) introducing into a cell a polynucleotide according to any one of paras 1A to 18A, or a vector according to para 19A or para 20A; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 25A.
  • a method for expressing a polypeptide of interest in a cell comprising contacting a cell according to para 22A with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • 26A A polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A, for use in a method of medical treatment or prophylaxis.
  • 28A Use of a polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest.
  • a method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest comprising administering to a subject a polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A. 30A.
  • 31A The polynucleotide, vector, or pharmaceutical composition for use according to para 27A or para 30A, the use according to para 28A or para 30A, or the method according to para 29A or para 30A, wherein the disease or condition is a disease or condition characterised by deficiency of the polypeptide of interest. 32A.
  • a kit comprising: (i) a polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A; and P37829 (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 33A.
  • polynucleotide according to para 33A wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence.
  • 35A The polynucleotide according to para 33A or para 34A, wherein the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:1.
  • 36A The polynucleotide according to any one of paras 33A to 35A, wherein the first nucleotide sequence comprises, or consists of, SEQ ID NO:3.
  • 48A The polynucleotide according to any one of paras 33A to 47A, wherein the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. 49A.
  • ITR inverted terminal repeat
  • 50A The vector according to para 49A, wherein the vector is an adeno-associated virus (AAV) vector.
  • a pharmaceutical composition comprising a polynucleotide according to any one of paras 33A to 48A, or a vector according to para 49A or para 50A, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.
  • 52A. A cell comprising a polynucleotide according to any one of paras 33A to 48A, or a vector according to para 49A or para 50A.
  • P37829 54A A method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell a polynucleotide according to any one of paras 33A to 48A, or a vector according to para 49A or para 50A. 5 55A.
  • a method for inhibiting expression of a polypeptide of interest in a cell comprising contacting a cell according to para 52A with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • 56A A polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 10 50A, or a pharmaceutical composition according to para 51A, for use in a method of medical treatment or prophylaxis.
  • 57A A polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 10 50A, or a pharmaceutical composition according to para 51A, for use in a method of medical treatment or prophylaxis.
  • 58A. Use of a polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 50A, or a pharmaceutical composition according to para 51A, in the manufacture of a medicament 20 for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest.
  • a method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest comprising 25 administering to a subject a polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 50A, or a pharmaceutical composition according to para 51A.
  • 61A The polynucleotide, vector, or pharmaceutical composition for use according to para 57A, the use according to para 58A, or the method according to para 59A, wherein the disease or condition is a 30 disease or condition characterised by deficiency of the polypeptide of interest.
  • a kit comprising: (i) a polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 50A, or a pharmaceutical composition according to para 51A; and 35 (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
  • a ‘peptide’ refers to a chain of two or more amino acid monomers linked by peptide bonds.
  • a peptide typically has a length in the region of about 2 to 50 amino acids.
  • a ‘polypeptide’ is a 20 polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.
  • Reference herein to peptides, polypeptides and proteins also includes P37829 glycopeptides/glycopolypeptides/glycoproteins, lipopeptides/lipopolypeptides/lipoproteins, nucleopeptides/nucleopolypeptides/nucleoproteins, etc.
  • an amino acid sequence, or a region of a polypeptide, which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60%, e.g. one of at least ⁇ 65%, ⁇ 70%, ⁇ 75%, ⁇ 80%, ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity to the amino acid sequence of the amino acid sequence/polypeptide/region.
  • amino acid sequence/region/position of a polypeptide/amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence/region/position of a polypeptide/amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Söding, J.2005, Bioinformatics 21, 951-960).
  • sequence alignment software such as ClustalOmega (Söding, J.2005, Bioinformatics 21, 951-960).
  • a nucleotide sequence, or a region of a polynucleotide, which ‘corresponds’ to a specified reference nucleotide sequence or region of a polynucleotide has at least 60%, e.g.
  • a polynucleotide/region/position of a polynucleotide/nucleotide sequence which ‘corresponds’ to a specified reference nucleotide sequence/region/position of a polynucleotide/nucleotide sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g.
  • an amino acid sequence (e.g. the amino acid sequence of a peptide/polypeptide/domain/region) which is ‘derived from’ a reference amino acid sequence (e.g. the amino acid sequence of a reference peptide/polypeptide/domain/region) comprises, or consists of, an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference amino acid sequence.
  • nucleotide sequence (e.g. a nucleotide sequence of a polynucleotide) which is ‘derived from’ a reference nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference nucleotide sequence.
  • the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise.
  • Ranges may be expressed herein as from ‘about’ one particular value, and/or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.
  • a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.
  • P37829 Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with/on intact multi-cellular organisms.
  • Figure 1 Schematic representation of the design of the In-frame ON-switch.
  • the cassette comprising the in-frame switch is constructed using the exon 6-intron 6-exon 7-intron 7-exon 8 region of human SMN2 followed by the coding sequence of the gene of interest (GOI) downstream of exon 8.
  • the translation start site is at the 5’ end of exon 6.
  • Intron 6 is modified to reduce its length and exon 7 contains an insertion of the nucleotide A after position 48.
  • the sequence of exon 8 is only the initial 23 nucleotides.
  • the graph shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • Figure 3. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pLS41 and pLS76 ON-switches.
  • B-D FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS41 or pLS76 constructs.
  • B Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the data of Figure 3B, as the total median GFP signal without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT) , or in cells expressing the indicated constructs, in the absence of risdiplam.
  • (F) Shows the results of analysis of the products of splicing by RT-PCR performed on HEK293 stably expressing pLS76, and treated with low (100nM) or high (1 ⁇ M) concentrations of risdiplam, or DMSO.
  • Figure 4. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e.
  • FIG. 1 Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the data of Figure 4B, as the total median GFP signal without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 5 Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e.
  • FIG. 1 Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the data of Figure 5B, as the total median GFP signal without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 6. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e.
  • (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (C) Shows the data of Figure 6B, as the total median GFP signal without normalization to DMSO control.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • FIG. 7 Shows the results of analysis of the products of splicing by RT-PCR performed on HEK293 stably expressing pLS179, and treated with low (100nM) or high (1 ⁇ M) concentrations of risdiplam, or DMSO.
  • Figure 7. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM70, pMM71, pMM72, pMM73 and pMM112 ON-switches, which have reduced exon 8 lengths relative to the pLS179 ON-switch.
  • FIG. 1 Schematic representation of the differences between the pLS179, pMM70, pMM71, pMM72, pMM73 and pMM112 ON-switches.
  • B-D FACS-based measurements of GFP expression upon induction with increasing concentrations of P37829 risdiplam in HEK293 cells stably expressing the pLS179, pMM70, pMM71, pMM72, pMM73 or pMM112 constructs.
  • B Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • FIG. 7B Shows the data of Figure 7B, as the total median GFP signal without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • FIG. 1 Schematic representation of the differences between the pMM112 and pMM130 ON-switches.
  • B-D FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS179, pMM112 or pMM130 constructs.
  • B Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the data of Figure 8B, as the total median GFP signal without normalization to DMSO control.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 9. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM143, pMM144, pMM145, pMM146 and pMM147 ON-switches, in which the ISS-N1 region of intron 7 is mutated to reduced leakiness.
  • FIG. 1 Schematic representation of the differences between the pLS179, pMM143, pMM144, pMM145, pMM146 and pMM147 ON-switches.
  • B- D FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS179, pMM143, pMM144, pMM145, pMM146 or pMM147 constructs.
  • B Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • FIG. 9B Shows the data of Figure 9B, as the total median GFP signal without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 11. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM151 and pMM152 ON-switches, which comprise deletions in exon 7.
  • (A) Schematic representation of the differences between the pMM112, pMM151 and pMM152 ON- switches. (B-D).
  • FIG. 1 Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 12. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM56, pMM59, pMM60, pMM61, pMM62 and pMM63 ON-switches, in which ATG codons in exon 6 are removed, and in which comprise modifications in exon 7.
  • FIG. 1 Schematic representation of the differences between (Ai) pLS76 and pLS174, and (Aii) pLS174, pMM56, pMM59, pMM60, pMM61, pMM62 and pMM63 ON-switches.
  • B-D FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS76, pMM56, pMM59, pMM60, pMM61, pMM62 and pMM63 constructs.
  • FIG. 12B Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the data of Figure 12B, as the total median GFP signal without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 13 Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e.
  • FIG. 14 Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 14 Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM242, pMM243, pMM244, pMM245, pMM246, and pMM247 ON- switches, in which ATG codons are introduced into exon 7. Insertions relative to the sequence of pLS174 are shown in black and in bold, and mutations relative to the sequence of pLS174 are shown in black, italics and bold.
  • FIG. 14B Shows the data of Figure 14B, as the total median GFP signal without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 15. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 16. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM201, pMM202, pMM203, pMM204, pMM205 and pMM206 ON- switches, in which ATG codons are introduced into exon 7 downstream of ESE2.
  • FIG. 16B Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the data of Figure 16B, as the total median GFP signal without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 17. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e.
  • FIG. 1 Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 18. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM252, pMM254, pMM255, pMM256 and pMM257 ON-switches, in which ATG codons are introduced into exon 7 downstream of ESE2.
  • A Schematic representation of the differences between the pMM130, pMM252, pMM254, pMM255, pMM256 and pMM257 ON-switches.
  • B- D Schematic representation of the differences between the pMM130, pMM252, pMM254, pMM255, pMM256 and pMM257 ON-switches.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 19 Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM198, pMM199 and pMM200 ON-switches, in which an ATG codon is introduced, spanning exons 7 and 8.
  • C-E FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM112, pMM198, pMM199 or pMM200 constructs.
  • FIG. 19C Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • D Shows the data of Figure 19C, as the total median GFP signal without normalization to DMSO control.
  • E Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • F Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 20 Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e.
  • FIG. 10 Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 21 Analysis of constitutive reporter expression (i.e. in the absence of risdiplam), and risdiplam- induced suppression of reporter expression for the pMM193 and pMM194 OFF-switches.
  • A Schematic representation of splicing of SMN2 exon 6 to exon 8-derived ON and OFF-switches.
  • FIG. 1 Schematic representation of the differences between the pMM193 and pMM194 OFF-switches.
  • D-F FACS-based measurements of GFP expression upon treatment of HEK293 cells stably expressing the pMM130, pMM193, or pMM194 constructs with increasing concentrations of risdiplam.
  • D Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO- treated control cells, 48h after risdiplam treatment.
  • E Shows the data of Figure 21D, as the total median GFP signal without normalization to DMSO control.
  • (F) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells, at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (G) Shows the level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • Figure 22 Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e.
  • (E) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • (G) Shows a fragment of Fig.22C with induction rate at 125 nM risdiplam concentration calculated as median GFP expression normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment, GFP-positive cells.
  • Figure 23. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e.
  • (C) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (D) Shows the total median GFP signal of GFP positive cells without normalization to DMSO control.
  • (E) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • WT wildtype HEK293 cells
  • (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (C) Shows the total median GFP signal of GFP positive cells without normalization to DMSO control.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or P37829 in cells expressing the indicated constructs, in the absence of risdiplam.
  • FIG. 25 Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • Figure 25 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273, pMM358, pMM359, pMM360, and pMM361.
  • A Schematic representation of the differences between the pMM273, pMM358, pMM359, pMM360, and pMM361 ON-switches.
  • B-F Schematic representation of the differences between the pMM273, pMM358, pMM359, pMM360, and pMM361 ON-switches.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • F Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • FIG. 26 Shows the results of analysis of the products of splicing by PCR performed on HEK293 stably expressing pMM273, pMM358, pMM359 and pMM360 and treated with increasing concentrations of risdiplam, or DMSO.
  • Figure 26 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273 and pMM365 - pMM375.
  • A Schematic representation of the differences between the pMM273 and pMM365 - pMM375 (B-G).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • Figure 27 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e.
  • (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • FIG. 1 Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • Figure 28 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273, pMM436, pMM437, and pMM438.
  • A Schematic representation of the differences between the pMM273, pMM436, pMM437, and pMM438 (B- F).
  • (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • F Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • Figure 29 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e.
  • (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • FIG. 30 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273 and pMM464 - pMM474
  • A Schematic representation of the differences between the pMM273 and pMM464 - pMM474 as well as a table describing intron modifications in every construct. Intron sequences added to the constructs were taken from the SMN2 transcript (B-F). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273 and pMM464 - pMM474 constructs.
  • (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • FIG. 1 Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • Figure 31 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM466, pMM470, pMM472, pMM474 and pMM567 - pMM570
  • A Schematic representation of the differences between the pMM466, pMM470, pMM472, pMM474 and pMM567 - pMM570.
  • B-F FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM466, pMM470, pMM472, pMM474 and pMM567 - pMM570 constructs.
  • B Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • D Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • (F) Shows induction of GFP expression calculated as median GFP expression at 62.5nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • Figure 32 Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • D) Shows the percentage of GFP-positive (% P37829 GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • FIG. 1 Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • G Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • Figure 33 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM642 - pMM651, and pMM709.
  • FIG. 1 Schematic representation of the intron 6 deletion screen performed using pMM569 construct.5 nt step by step deletions spanning the whole intron 6 were introduced and tested.
  • B Selected intron 6 mutations tested in constructs pMM642 - pMM651, and pMM709.
  • C-H FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM642 - pMM651, and pMM709.
  • (C) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (D) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • (E) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • G Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • H Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • Figure 34 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM634 - pMM641, pMM711 and pMM712.
  • FIG. 1 Schematic representation of the intron 7 deletion screen performed using pMM569 construct.5 nt step by step deletions spanning the whole intron 7 were introduced and tested.
  • B Selected intron 7 mutations tested in constructs pMM634 - pMM641, pMM711 and pMM712.
  • C-H FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM634 - pMM641, pMM711 and pMM712 constructs.
  • (C) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (D) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • (E) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • G Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control P37829 cells, 48h after risdiplam treatment.
  • H Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • Figure 35 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM579, pMM595, and pMM596.
  • (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • FIG. 1 Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • G Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • Figure 36 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM618, and pMM619.
  • pMM618 has 13 nt deletion at the 5’ side of exon 6, while pMM619 has 30 nt deletion of exon 65’ side.
  • B-G FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM618, and pMM619 constructs.
  • (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • (F) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • FIG. 37 Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • Figure 37 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569 switch with different promoters (pMM569, pMM581 - pMM584, pMM589, pMM590, and pMM562, and pMM619).
  • A Schematic representation of the pMM569 switch and the list of all tested promoters.
  • B-F FACS-based measurements of GFP P37829 expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM581 - pMM584, pMM589, pMM590, and pMM562, and pMM619 constructs.
  • B Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • F Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO- treated control cells, 48h after risdiplam treatment.
  • FIG. 1 Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM).
  • Figure 38 Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM263, pMM274, pMM273, pMM359, and pMM569.
  • FIG. 1 Schematic representation of the differences between the pMM263, pMM274, pMM273, pMM359, and pMM569. These constructs represent various iterations of the switch design.
  • B-G FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM263, pMM274, pMM273, pMM359, and pMM569 constructs.
  • B Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • E Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • F Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO- treated control cells, 48h after risdiplam treatment.
  • FIG. 39 Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • Figure 39 Analysis of risdiplam-induced NanoLuc (NLuc) luciferase reporter expression, and background NLuc reporter expression (i.e. in the absence of risdiplam) for pMM568, pMM569, pMM570 and pMM579.
  • A Schematic representation of the differences between the pMM568, pMM569, pMM570 and pMM579.
  • B-G Luminescence-based measurements of NLuc luciferase expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM568, pMM569, pMM570 and pMM579 constructs.
  • B Shows induction of NLuc expression calculated as NLuc signal expression at each concentration of risdiplam, normalized to NLuc signal in DMSO-treated control cells, 48h after risdiplam treatment.
  • C Shows the total NLuc signal without normalization to DMSO control.
  • D Shows the total NLuc signal for WT HEK293 cells as well as cells expressing indicated constructs, in the absence of risdiplam (DMSO treated).
  • P37829 Figure 40 Risdiplam-mediated expression of Cas9 in HEK293T stable cell lines assessed by CD81 staining.
  • A Schematic representation of the differences between the constructs pMM263, pMM274, pMM273, pMM359, and pMM569, representing various iterations of the switch design
  • B Experimental setup. HEK293T with stably incorporated pMM263, pMM274, pMM273, pMM359, and pMM569 switches controlling Cas9 expression were nucleoporated with 3 different sgRNAs targeting CD81 locus, each at 150 nM concentration, seeded at 96-well plates and right after treated with various concentrations of risdiplam.
  • C Percentage of CD81 positive cells in cells expressing pMM263 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam.
  • D Percentage of CD81 positive cells in cells expressing pMM274 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam.
  • E Percentage of CD81 positive cells in cells expressing pMM273 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam
  • F Percentage of CD81 positive cells in cells expressing pMM359 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam.
  • G Percentage of CD81 positive cells in cells expressing pMM569 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam.
  • H Percentage of CD81 positive cells in cells expressing pMM596 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam.
  • Figure 41 Percentage of CD81 positive cells in cells expressing pMM596 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam.
  • (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment.
  • (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control.
  • D) Shows the percentage of GFP- positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 ⁇ M).
  • (E) Shows the background level of total median GFP expression in wildtype Jurkat cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam.
  • hESC-derived neurons were differentiated from the neuronal stem cell (NSC)- state for 28 days, transduced with AAV7.m8 containing the sequence illustrated in A. After three days of AAV-transduction, Risdiplam was added to the cell culture at varying concentrations as indicated. Medium was exchanged every 3 days with fresh addition of risdiplam and presented images were acquired 7 days after risdiplam treatment.
  • Figure 44. Optimized in-frame switch pMM569 allows for controlled expression of MeCP2.
  • A Schematic representation of the in-frame switch based on the sequence from pMM569.
  • H4 cells were used as wild type as a control for native MeCP2 expression in contrast to MeCP2-knockout (KO) H4 cells, which were generated as a single cell clone using CRISPR/Cas9-mediated knockout of MeCP2.
  • H4-KO cells were transfected with pMM569, encoding for MeCP2 as a therapeutic protein.
  • C Transfected H4-KO cells were treated with indicated concentrations of risdiplam for 24h and imaging was conducted using anti-MeCP2 antibody (cell signaling technology, cat#3456).
  • Example 1 Design and construction of the in-frame ON-switch
  • the in-frame ON-switch comprises exon 6, exon 7, and a partial sequence of exon 8 of SMN2 (survival of motor neuron 2), along with intervening intronic regions (intron 6 and intron 7).
  • GFP is used as a reporter gene.
  • Exon 7 contains a single nucleotide insertion (A at position 48). This allows in-frame translation of GFP upon inclusion of exon 7 (in the presence of risdiplam) by inactivation of the translation termination codon at the 3’ end of exon 7 17 .
  • exon 6 The entirety of exon 6 was used, while the downstream intronic region was reduced to 414 nucleotides (from its original length of 5769 nucleotides) comprising the first 202 and the last 212 nucleotides of intron 6.
  • the entirety of intron 7 was used, followed by the first 23 nucleotides of exon 8 ( Figure 1).
  • a Kozak sequence (GCCACC) as well as an ATG codon were placed upstream of exon 6 for the initiation of translation.
  • the gene expression was driven by the human cytomegalovirus (CMV) promoter.
  • CMV human cytomegalovirus
  • Example 3 Optimization of the in-frame switch: Reduction in leakiness (background expression).
  • An improved version of the in-frame switch was generated by introducing two point mutations within the 3’ splice site of exon 7: acagGGT (WT) to ccagGAT (mutant), which has been shown in a previous study to promote skipping of exon 7 18 (Figure 3A). This construct was termed pLS76 and was used as a starting construct for further modifications as described below.
  • HEK293 cells were transfected with WT (pLS41) and mutated (pLS76) constructs and treated with increasing amounts of risdiplam compound.
  • the nucleotide sequences of the pLS41 and pLS76 ON-switches are shown in SEQ ID NOs:37 and 38, respectively.
  • Fluorescence activated cell sorting (FACS)-based readouts revealed reduced background expression in the modified sequence (ccagGAT) compared to WT (acagGGT), with a comparable induction rate (Figure 3B-3D).
  • Figure 3B shows relative fold change in median GFP expression level normalized to DMSO control 48h after risdiplam treatment at the indicated concentrations.
  • the total GFP signal is shown in Figure 3C, whereas Figure 3D represents the % of GFP- positive cells normalized to the condition with the highest risdiplam treatment.
  • Untransfected, wildtype HEK293 cells serve thereby for gating out GFP-negative cells.
  • This type of data presentation allows the demonstration of the increase in the number of GFP-positive cells rather than the GFP intensity upon induction with risdiplam.
  • Figure 3E we assessed the leakiness of the cells by comparing total GFP expression in cells treated with DMSO only (Figure 3E) and found lower background GFP expression level in the construct that contains the acagGGT> ccagGAT mutation.
  • P37829 In a first step, the central region of intron 6 was removed, leaving 102 nucleotides of the 5’ end and 162 nucleotides of the 3’ end of intron 6. Similarly, the central region of intron 7 was removed, leaving 101 nucleotides at the 5’ end and 151 nucleotides at the 3’ end. In both cases, only the central intronic regions were removed, in order to minimise the chances of disrupting splicing regulatory sequences, which are typically located in close proximity to 5’ and 3’ splice sites (E adjacent to their upstream and downstream flanking exons).
  • This construct termed pLS159, reduced the total length of the in-frame switch by 314 nucleotides.
  • intron 6 and intron 7 were then further reduced to: intron 6 (72 nucleotides at 5’ end and 112 nucleotides at 3’ end); intron 7 (77 nucleotides at 5’ end and 100 nucleotides at 3’ end).
  • This construct termed pLS160, led to a total reduction of 492 nucleotides of the in-frame switch length.
  • the nucleotide sequences of the pLS159 and pLS160 ON-switches are shown in SEQ ID NOs:39 and 40, respectively.
  • Example 5 Optimization of the in-frame switch: Reduction in exon 6 length.
  • exonic splicing regulatory sequences are usually localized close to the 5’SS
  • the inventors investigated whether it was possible to further reduce the size of the in-frame switch, by gradually reducing its length from the 5’ end of exon 6.
  • two variants of the switch were generated that were lacking 30 (pLS167) or 60 (pLS168) nucleotides of the 5’ end of exon 6 (illustrated in Figure 5A). Results showed increased overall expression of the reporter gene (GFP) as measured by FACS-based analysis.
  • the nucleotide sequences of the pLS167 and pLS168 ON-switches are shown in SEQ ID NOs:41 and 42, respectively.
  • HEK293 cells were stably transfected, followed by treatment with risdiplam at the indicated concentrations for 48(H)Original in-frame switch construct pLS76 served as a control.
  • pLS167 partial truncation of exon 6 by 30 bp
  • Figure 5B partial truncation of exon 6 by 30 bp
  • Figure 5C total increase in median GFP signal
  • the pLS168 construct also showed increased GFP expression in comparison to pLS76 ( Figure 5B-5C).
  • Increased GFP expression from pLS167 and pLS168 constructs relative to pLS76 may be a consequence of the polypeptide added to GFP encoded by the pLS167 and pLS168 constructs. Increased GFP expression from pLS167 relative to pLS168 may be a consequence of pLS167 comprising a ‘G’ immediately after the Kozak and start codon sequences, instead of ‘A’ (as in the pLS168 construct).
  • P37829 The number of GFP-positive cells in cultures expressing the original construct, pLS76, increased proportionally with increasing concentration of risdiplam. Similar observations were made with cells expressing pLS168 ( Figure 5D).
  • Example 6 Combined modifications of reduced intron length and reduced exon length in one construct. Next, the modifications of pLS159 and pLS168 were combined into one construct. The minimal required intron length for optimal induction (pLS159) was combined with the minimal required length of exon 6 (pLS168).
  • Example 7 Reduction in nucleotide sequence of exon 8.
  • the final amino acid sequence expressed using the in-frame switch is composed of the transgene of interest linked to a fragment of SMN2 exon 6, exon 7, and exon 8 due to inclusion of these exons after P37829 RNA splicing.
  • the length of the nucleotide sequence of exon 8 was reduced in a stepwise manner.
  • the nucleotide sequences of the pLS179, pMM70, pMM71, pMM72, pMM73 and pMM112 ON-switches are shown in SEQ ID NOs:43, 44, 45, 46, 47 and 22, respectively.
  • HEK293 cells were stably transfected with these constructs, and treated with increasing amounts of risdiplam. FACS-based analysis showed that the reduction in exon 8 nucleotide sequence did not lead to significant changes in the relative ( Figure 7B) or in the total ( Figure 7C) GFP expression level in comparison to untruncated exon 8 (pLS179).
  • a successful translation driven by an ATG in closer proximity to the transgene sequence will reduce the length of any amino acids that will be attached to the final transgene output due to exon inclusion.
  • a new construct (pMM130) was generated based on pMM112, with the Kozak sequence/start site removed from the 5’ end of exon 6. The design of the constructs and their respective sequences are illustrated in Figure 8A. The nucleotide sequence of the pMM130 ON-switch is shown in SEQ ID NO:23.
  • HEK293 cells were stably transfected with the constructs and analysed by FACS to assess relative (Figure 8B) and total (Figure 8C) GFP expression levels.
  • the induction rate of the in-frame switch in response to varying concentrations of risdiplam could be maintained by the pMM130 construct that lacks a canonical Kozak (i.e. conforming to SEQ ID NO:33) and relies on the ATG site at the 3’ end of exon 6.
  • a canonical Kozak i.e. conforming to SEQ ID NO:33
  • no changes were observed in the number of GFP-positive cells as well as the background expression of GFP in DMSO-treated cells when comparing pMM130 to starting construct pMM112 or pLS179.
  • the length of the tag added to the final transgene output could be reduced by 15 amino acids.
  • ISS-N1 Intronic Splicing Silencer-N1
  • ISS-N1 is a 15-nucleotide long motif located immediately downstream of the 5’ splice site of exon 7 of the SMN2 gene ((5’..GA
  • ISS- N1 was described as a major inhibitory element and a master checkpoint of SMN2 exon 7 splicing because its deletion fully restored exon 7 inclusion (Singh et al. RNA (2010) 16(6):1167-81). Modifications within the inhibitory region could help reduce background splicing events (leading to inclusion of exon 7 in the absence of the SMSM) driven by the native SMN2 sequence. Therefore, mutations were introduced within the ISS-N1 region that were found to lead to increased inhibition of exon 7 inclusion 19 .
  • the nucleotide sequences of the pMM143, pMM144, pMM145, pMM146 and pMM147 ON-switches are shown in SEQ ID NOs:48, 49, 50, 51 and 52, respectively.
  • the modified constructs were stably transfected into HEK293 cells.
  • HEK293 cells were stably transfected with constructs carrying the respective mutations (pMM136-pMM142) and treated with increasing concentrations of risdiplam.
  • the nucleotide sequences of the pMM136, pMM137, pMM138, pMM139, pMM140, pMM141 and pMM142 ON-switches are shown in SEQ ID NOs:53, 54, 55, 56, 58 and 59, respectively. No increase in the relative (Figure 10B) or total (Figure 10C) GFP expression level was observed.
  • Example 12 Introducing translation start site within exon 7
  • the current construct design leads to the generation of a residual amino acid chain that is fused to the N- terminus of the gene of interest due to the positioning of the translation start site (Kozak and ATG) at the 5’ end of exon 6.
  • the Kozak and ATG start site at exon 6 were removed and all alternative ATGs within the exon 6 sequence were mutated as described in Figure 12A(i) (ATG>GGC).
  • This construct was named pLS174 and served as the starting construct to introduce Kozak and ATG start sites within the exon 7 sequence.
  • the nucleotide sequence of the pLS174 ON-switch is shown in SEQ ID NO:62.
  • the first construct contained mutations downstream of the ESE2 region to recreate a Kozak and ATG start site (GCTCACAT- - >GCCACCATG)
  • construct pMM59 contained an insertion of Kozak sequence and mutation of the sequence to resemble ATG upstream of the ESE2 region (------AGA > GCCACCATG)
  • pMM60 was modified by 2 point mutations and an insertion (AT------- > GCCACCATG)
  • pMM61 contained mutations and a single nucleotide insertion to create a Kozak-like sequence that has previously been shown to enhance translation initiation site 20 , (T-ATAAG > CAACATG)
  • pMM62 contained mutations and a single nucleotide insertion (AATATAA-G > GCCACCATG)
  • pMM63 contained mutations and 2 nucleotide insertion
  • the background expression of GFP in the absence of risdiplam was only slightly higher in pMM59 in comparison to pLS76, however, significantly lower than in pMM56 and pMM61-63.
  • the Kozak/ATG translation start site is provided upstream of ESE2
  • the Kozak/ATG translation start site is provided downstream of ESE2.
  • the presented data suggest that pMM59 with a Kozak sequence/start site introduced upstream of the ESE2 region provides very high induction rate as well as low leakiness and can be used to reduce the size of the tag on the final transgene output.
  • pMM59 represents a new in-frame switch design, where the translation start site is inside the inducible exon itself.
  • Example 13 Modifications in the 5’ splice site of exon 6 in order to remove the ATG start site.
  • Alternative ATG translation start sites within exon 6 were mutated in pLS176 as indicated in Figure 13A and highlighted in bold.
  • the previous construct pMM59 carried a modification at the 5’ splice site of exon 6 from ATG to CTG.
  • Example 14 Introduction of translation start site upstream of the ESE2 sequence in exon 7. Based on the previous results using pMM59, a construct in which Kozak and ATG start site were positioned upstream of the ESE2 sequence by point mutations and insertions, various constructs that contain Kozak and ATG at different positions upstream of the ESE2 region were generated, as illustrated in Figure 14A.
  • Constructs were generated containing: Kozak-ATG (GCCACCATG, in pMM59 and P37829 pMM242); Kozak-like/ATG (GCACCATG in pMM243 and pMM244); another Kozak-like/ATG sequence (CACCATG, pMM245); only ATG insertion (pMM246); and ATG insertion within the ESE2 region (pMM247).
  • pMM244, pMM245, and pMM246 contained a C>G mutation that was placed downstream of the inserted ATG.
  • Example 15 Introduction of translation start site within exon 7 and downstream of the ESE2 sequence. Different outcomes have been seen from different modifications and positioning of the translation start site in relation to the ESE2 region. Therefore, it was decided to further evaluate the effect of positioning the ATG start site downstream of the ESE2 sequence.
  • pLS175 was used as the starting construct that contained mutations of ATG start site (indicated in bold in Figure 15A) as well as ATG>CAG mutation in the 5’ end of exon 6.
  • Example 16 Additional modifications to pMM126 in order to improve performance. Following up on the successful insertion of an ATG site downstream of the ESE2 sequence in pMM126, pMM126 was further modified in order to increase the induction rate by generating a stronger translation start site.
  • the nucleotide sequences of the pMM201, pMM202, pMM203, pMM204, pMM205 and pMM206 ON- switches are shown in SEQ ID NOs:86, 87, 88, 89, 90 and 91, respectively.
  • the described constructs were transfected into HEK293 cells for stable expression and treated at increasing risdiplam concentrations for 48(H)Although FACS-based measurements showed that introducing the modifications described in Figure 16A did not result in improved GFP induction rate (Figure 16B) or total GFP median expression level (Figure 16C), constructs pMM204 and pMM205 showed reduced leakiness compared to pMM126 (lower median GFP signal in DMSO) with a comparable induction rate.
  • results showed an increased number of GFP-positive cells in the DMSO-treated cells, stably expressing pMM201, pMM202 and pMM206 (Figure 16C-16E).
  • single or double nucleotide mutations in constructs pMM201 and pMM202 lead to constitutive GFP expression, while bigger changes as in pMM203 had much smaller impact, suggesting again that the region downstream of ESE2 is very sensitive to modifications.
  • Example 17 Engineering of a stronger translation start site in pMM129 As described in Figure 15, pMM129 did show improved performance in terms of low leakiness and still contained acceptable induction rate, although to a significantly lower extent than pMM126.
  • the nucleotide sequences of the pMM248, pMM249, pMM250, and pMM251 ON-switches are shown in SEQ ID NOs:92, 93, 94 and 95, respectively.
  • the above-described constructs (illustrated in Figure 17A) were transfected into HEK293 cells for stable expression and treated at increasing risdiplam concentrations for 48h. No improved performance was observed through the introduction of these modifications, i.e. no increase in GFP induction rate (Figure 17B), increase in total level of median GFP including the DMSO-treated control ( Figure 17C-17E), suggesting that the introduced modifications led to increased leakiness of the constructs and no improvements in risdiplam-controlled GFP expression.
  • Example 18 Implementing learnings on translation start site (Kozak/ATG) within exon 7 on the short in-frame switch pMM130
  • Kozak/ATG translation start site
  • Figure 18A the ATG site in construct pMM130 was firstly removed to create pMM252, with all ATG sites mutated.
  • HEK293 cells were transfected with the indicated constructs and selected for stable expression under blasticidin selection marker. Cells were treated with varying concentrations of risdiplam for 48h before GFP expression was measured by FACS. Results showed a general increase in leakiness from each of the modifications that were introduced into the shorter in-frame switch pMM130.
  • pMM254 short in-frame switch with translation start site in exon 7
  • control construct pMM126 long in- frame switch with translation start site in exon 7
  • Similar observations were made with pMM255 and its control construct pMM128, as well as pMM256 and its control construct pMM129.
  • pLS175 contains 23 nucleotide long exon 8 sequence, an 8 amino acid tag still remains.
  • exon 8 can be truncated to just 2 nucleotides, thus completely removing the tag.
  • 3 constructs were generated with different mutations: pMM198 contained P37829 a mutation GA>TG in the 5’ end of exon 8, pMM199 and pMM200 contained the same mutation as pMM198 but were additionally modified in the intron 7 region in order to increase the strength of polypyrimidine tract with the aim of increasing splicing efficiency between exon 7 and exon 8 and consequently compensating for the generation of a weak splice site by mutating the 5’ end of exon 8 from GA (consensus) to TG (non-optimal splice site).
  • the described constructs were transfected into HEK293 cells for stable expression and treated with risdiplam at increasing concentrations for 48h before they were assessed for GFP expression by FACS-based measurements.
  • pMM112 the short improved in- frame switch was used as a control construct to compare the performance of the split-ATG constructs.
  • the nucleotide sequences of the pMM198, pMM199 and pMM200 ON-switches are shown in SEQ ID NOs:25, 101 and 102, respectively.
  • the in-frame switch can be successfully engineered to form an ATG translation start site dependent on exon 7 inclusion without compromising the risdiplam-mediated regulation of GFP expression.
  • Example 20 Introducing ATG start site in exon 8
  • pLS76 was used as a starting construct. The Kozak and ATG sites were removed from the 5’ end of exon 6 and alternative ATG start sites within exon 6 were mutated ( Figure 20A).
  • exon 6 was mutated in each of the modified constructs as follows: pMM236 (ATG>CAG), pMM237 (ATG>TAG), pMM238 (ATG>TTG).
  • the mutations in combination with the upstream sequence result in a suboptimal Kozak-like sequence.
  • the exon 7 sequence remained as described for pLS76.
  • a mutation was introduced at the 5’ end of exon 8 in pMM236-pMM238 (G-A>ATG) as illustrated in Figure 20A.
  • the risdiplam-mediated gene expression control is retained by the presence of a Kozak-like sequence within the native sequence of exon 7 (highlighted in bold in Figure 20B).
  • the nucleotide sequences of the pMM236, pMM237 and pMM238 ON switches are shown in SEQ ID NOs:103, 104 and 105, respectively.
  • the presented concept relies on the presence of risdiplam to allow inclusion of exon 7 and hence the introduction of an efficient Kozak-like sequence contained within exon 7 (TAAGGA) to enhance translation driven by the downstream ATG start codon present in exon 8 (at the 5’ end of pMM236- pMM238).
  • Example 21 Engineering a SMN2-based OFF-switch Inducible gene expression systems could not only be developed as an ON-switch to turn on gene expression but also as an OFF-switch to inhibit gene expression.
  • a risdiplam-inducible OFF-switch was developed that would respond to various concentrations of risdiplam by suppressing transgene translation.
  • the design strategy of the presented ON-switch was used and a construct was engineered to allow inhibition of translation by introducing a single nucleotide change (Figure 21A).
  • the design was based on the pMM130 construct having reduced intron length (intron 6 and 7), reduced exon length (exon 6 and 8), as well as reduced transgene tag size (by using the ATG codon positioned at the 3’ end of exon 6 as the translation start site) (Figure 21B). Presence of ATG at the 3’ end of exon 6 allows for complete tag removal in the OFF-switch.
  • pMM130 contains only 2 nucleotides of exon 8 (GA); by adding an additional nucleotide (G) downstream of the sequence ( Figure 21B), a frame-shift in the transgene sequence (positioned downstream of exon 8) can be introduced upon inclusion of exon 7 by risdiplam, giving rise to an OFF-switch. In contrast, in the absence of risdiplam (exclusion of exon 7), the transgene can be correctly translated. In order to reduce the length of a peptide produced in the OFF state and drive transcript degradation by nonsense mediated decay mechanism, the presented OFF-switch (pMM193, Figure 21B) was further modified by introducing premature termination codon in exon 7.
  • HEK293 cells were used, stably expressing the constructs pMM193, pMM194 and pMM130 as a control, cells were treated for 48h with increasing amounts of risdiplam and FACS used to measure changes in GFP expression as a response to risdiplam treatment.
  • GFP expression was significantly reduced in pMM193-, and pMM194-transfected cells with increasing concentrations of risdiplam, while cells transduced with pMM130 (the ON-switch) showed increased GFP expression level.
  • the number of GFP-positive cells in the cells expressing the OFF- P37829 (pMM193, pMM194) or the ON-switch (pMM130) was also measured. A complete reduction in GFP- positive cells in the population that carries the OFF-switch was not observed ( Figure 21F). It is possible that incubation of risdiplam for 48h is not sufficient to measure complete reduction of the GFP due to the enhanced GFP version used in these assays. Total GFP expression level in untreated cells was also measured. As expected, GFP expression is very high in pMM193 and pMM194-transfected cells in the absence of risdiplam (Figure 21G).
  • Example 22 Constructs lacking selection antibiotic resistance gene All previously tested constructs had IRES-blasticidin-SV40 sequence added after the GFP encoding sequence leading to expression of antibiotic resistance gene (blasticidin), which was used for stable cell line selection. However, as the in-frame switch is intended to be used without antibiotic selection in AAV viral vectors, the inventors decided to replace the whole IRES-blasticidin-SV40 fragment with BGH polyA ( Figure 22A).
  • the described constructs were transfected into HEK293 cells for stable expression and treated with risdiplam at increasing concentrations for 48 h before they were assessed for GFP expression by FACS-based measurements.
  • the nucleotide sequences of the pMM263, pMM262, pMM259, pMM260, pMM272, pMM274 switches are shown in SEQ ID NOs: 38, 42, 22, 23, 25 and 107, respectively.
  • Example 23 Generation of short split ATG switch [pMM273] Despite significantly smaller size and better induction rate compared to pMM263 and pMM274 switches, pMM259 still has a short peptide ‘tag’ added to the expressed transgene. In order to remove the tag entirely, we employed a split-ATG approach, as in Example 19, wherein the 5’ end of exon 8 was modified to contain ‘TG’ instead of ‘GA’, to create pMM273 ( Figure 23A).
  • the described constructs were transfected into HEK293 cells for stable expression and treated with risdiplam at increasing concentrations for 48h before they were assessed for GFP expression by FACS-based measurements.
  • pMM274, pMM263 and pMM259 were used as control constructs to compare the performance of the split-ATG constructs.
  • the nucleotide sequence of the pMM273 ON-switch is shown in SEQ ID NO:111.
  • the results show that split-ATG-based construct pMM273 showed sensitivity to risdiplam treatment as seen by induced GFP expression relative to DMSO control (Figure 23C), as well as the total level of median GFP expression (Figure 23D), although to a lesser extent than pMM259.
  • Example 24 Modifications of exon 75’splice site sequences in pMM273 Based on the results obtained with pMM273, we investigated the effect of modifications within the 5’ splice site (5’ss) within the exon 7 sequence.
  • Three new constructs were generated with the modifications indicated in Figure 24: pMM362 (G>A at position -3 in exon 7, a deletion of nucleotide C at position +7 of intron 7; and a mutation T>G at position +8 of intron 7), pMM363 (A>G and G>T at positions -5 and -3, respectively, and TC>AT mutation within intron 7), and pMM364 (C>G mutation at position +7 within intron 7).
  • HEK293 cells were stably transfected with each one the constructs or pMM273 (as a control), and treated with the indicated risdiplam concentrations for 48h.
  • the modification of pMM362 disrupted splicing regulation and rendered the switch constitutively active without dependency on the presence or absence of risdiplam ( Figure 24 A-C).
  • pMM363 and pMM364 clearly showed reduced leakiness in comparison to pMM273 ( Figure 24E).
  • the level of expression of the transgene was reduced from these constructs compared to pMM273 ( Figure 24C).
  • Example 25 ESE2 region deletions in pMM273 to reduce leakiness in DMSO
  • deletion of the ESE2 region and adjacent sequence within the exon 7 region would lead to reduced risdiplam-independent inclusion of exon 7.
  • Example 26 Testing ESE2 region deletions in pMM273 Based on the results obtained by removing the ESE2 sequence (Example 25), we hypothesized that a gradual reduction of the ESE2 region and adjacent sequences could result in a construct with reduced leakiness levels that maintains high expression and induction rate.
  • pMM365 has almost complete ESE2 region deletion (- AAAAAGAAGGAAG) and pMM366 has complete ESE2 region deletion (-AAAAAGAAGGAAGG).
  • pMM367 For pMM367, pMM372, pMM373, pMM374, the ESE2 region and downstream nucleotides were also deleted: pMM367 (-AAAAAGAAGGAAGGT), pMM372 (-AAAAAGAAGGAAGGTGC), pMM373 (- AAAAAGAAGGAAGGTGCTC), pMM374 (-AAAAAGAAGGAAGGTGCTCAC).
  • pMM367 (-AAAAAGAAGGAAGGT)
  • pMM372 -AAAAAGAAGGAAGGTGC
  • pMM373 - AAAAAGAAGGAAGGTGCTC
  • pMM374 -AAAAAGAAGGAAGGTGCTCAC
  • pMM375 we introduced the same deletion as in pMM374 (-AAAAAGAAGGAAGGTGCTCAC), but additionally mutated 4 downstream nucleotides from ATTC to GGTG. Finally, we also tested only partial ESE2 region deletions in pMM368 (-AAAAAGAAGG), pMM369 (-AAAAAGAAG), pMM370 (-AAAAAGAA) and pMM371 (-AAAAA).
  • HEK293 cells stably transfected with one of the various different constructs or pMM273 (as a control), and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels.
  • Results showed increased GFP signal in response to risdiplam for most of the constructs (Figure 26B-C, 26F), with the exception of the constructs having large deletions (pMM372, pMM373, pMM374), where induction was low and further decreased with increasing size of the deletion.
  • pMM375 – which has the same deletion as inactive pMM374, but with an additional ATTC> GGTG mutation downstream of the ESE2 region – had completely rescued risdiplam induction of transgene expression, which was higher than pMM273 ( Figure 26F).
  • Example 27 Testing ESE2 region deletions in pMM273 Based on the results obtained in the gradual ESE2 region deletions (Example 26), we tested several other deletions, where the ESE2 (AAAAAGAAGGAAGG, underlined hereafter) region was either partially or completely removed, while keeping the GGTG sequence intact and instead focusing on deletions of the region upstream of the ESE2 region (Figure 27A).
  • pMM377 and pMM378 we tested complete ESE2 region deletion combined with additional upstream deletions: pMM376 (- TCAAAAAGAAGGAA), pMM377 (-AAAATCAAAAAGAAGGAA) and pMM378 (- CAAAATCAAAAAGAAGGAA).
  • pMM379 the ESE2 region was only partially deleted (- ACAAAATCAAAA).
  • pMM380 tested deletion in a downstream region (-CTCACATTC), while pMM381 combined pMM379 (-ACAAAATCAAAA) and pMM380 deletions (-CTCACATTC).
  • pMM382 deleted the majority of SMN2 exon 7 (-ACAAAATCAAAAAGAAGGAAGGTGCTCACATTC), while pMM386 (-AAA) and pMM387 (-A) tested small ESE2 region deletions not tested in Example 26.
  • Results showed increased GFP expression in response to risdiplam for most of the constructs ( Figure 27B-C, 27F), except pMM382 (which has a large deletion, leading to no splicing) and pMM386 (where (AAA) deletion led to constitutive splicing of the construct).
  • Constructs pMM376, pMM377, pMM379, pMM380 and pMM381 showed increased induction rate compared to pMM273 at 250 nM concentration of risdiplam ( Figure 27F). However these constructs displayed reduced median GFP signal compared to pMM273 ( Figure 27C).
  • Example 28 Testing exon 7 deletions in pMM273 Based on the results obtained after GGTG deletion combined with the ESE2 region deletion (Examples 25 and 26), the GGTG sequence appears to have a regulatory role in exon 7 splicing.
  • pMM436 harboring partial ESE2 P37829 (AGAAGG) deletion and partial GGTG (TGCT) deletion, pMM437 where GGTG was deleted and pMM438 where GGTGCT sequence was deleted ( Figure 28A).
  • HEK293 cells stably transfected with one of the various different constructs or pMM273 (as a control), and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP signal in response to risdiplam for all the constructs, except pMM436 which was inducible only at higher risdiplam concentrations and showed very low median GFP expression ( Figure 28B-C, 28F).
  • HEK293 cells stably transfected with one of the various different constructs or pMM273 (as a control), and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP expression in response to risdiplam, although to various extents, for all the constructs ( Figure 29B-C, 29F).
  • pMM485 yielded very high GFP expression levels in DMSO ( Figure 29E) and displayed a low induction rate (Figure29B).
  • Results showed increased GFP signal in response to risdiplam for all the constructs ( Figure 30B-C, 30F). Although the percentage of GFP+ cells was comparable for all the constructs ( Figure 30D), the median GFP signal was significantly reduced for all the tested constructs compared to pMM273, with the highest GFP signal reduction in pMM466, pMM470, pMM472 and pMM474 ( Figure 30E). Similarly, the induction rate was increased relative to pMM273 for all the tested constructs at all risdiplam concentrations ( Figure 30B).
  • constructs pMM466, pMM470, pMM472 and pMM474 showed the highest induction (Figure 30F) and were selected for further evaluation.
  • Example 31 Exon 6 and 7 mutations in the improved constructs We further modified constructs selected in the Example 30 (pMM466, pMM470, pMM472, pMM474) by mutating a remaining ATG site in exon 6 (ATG>CTG) and deleting a TAA sequence in exon 7 ( Figure 31A).
  • the TAA sequence was identified to increase induction rate in a screen where every single, two, three, four or five nucleotides of exon 7 were deleted in construct pMM273 and analyzed by FACS (results not shown).
  • Example 33 Intron 6 deletion screen in pMM569 While Example 30 suggests that increasing intron length significantly reduces leakiness, we wanted to identify any remaining intronic sequences regulating SMN2 exon 7 splicing and to this end we performed a whole intron 6 deletion screen, with 5-nt long non-overlapping deletions ( Figure 33A).
  • Example 34 Intron 7 deletion screen in pMM569
  • Results showed increased GFP signal in response to risdiplam for all the constructs ( Figure 35B-C, 35F). Although none of the constructs displayed reduced median GFP expression compared to pMM569 (Figure 35E), pMM579 and pMM596 significantly reduced the percentage of GFP+ cells compared to pMM569 ( Figure 35G). While the leakiness of pMM579 and pMM596 was similar, the induction rate and median GFP expression was significantly better for pMM596 ( Figure 35B-C), with approximately 5-fold induction at 62.5 nM of risdiplam for pMM596, compared to 2-fold for pMM579 ( Figure 35F).
  • pMM569 design with 7 other promoters: pMM581 (full length (FL) CAG promoter), pMM582 (hEF1a promoter), pMM583 (hUBiC promoter), pMM584 (RSV promoter), pMM589 (TK promoter), pMM590 (PGK promoter) and pMM562 (minimal CAG promoter) (Figure 37A).
  • pMM581 full length (FL) CAG promoter
  • pMM582 hEF1a promoter
  • pMM583 hUBiC promoter
  • pMM584 RSV promoter
  • TK promoter pMM589
  • PGK promoter pMM590
  • pMM562 minimal CAG promoter
  • results showed increased GFP signal in response to risdiplam for all the constructs, except pMM589 (TK promoter) and pMM590 (PGK promoter), which showed a very modest increase only at the highest risdiplam concentrations investigated (Figure 37B-C).
  • constructs pMM582 (hEF1a) and pMM652 (minimal CAG) had comparable induction rate to pMM569 (CMV), while pMM581 (FL-CAG), pMM583 (hUbiC), pMM584 (RSV), although still inducible, were 2-3-fold lower P37829 (Figure 37F).
  • Construct pMM581 (FL-CAG promoter) showed very high leakiness measured both by median GFP expression and % of GFP+ cells (Figure39E, G), while minimal CAG promoter (pMM652) and hEF1a (pMM582) leakiness was comparable to CMV.
  • the percentage of GFP positive cells was lowest for the least inducible constructs (pMM263 and pMM274) and highest for pMM273, while pMM359 and pMM569 had approximately 50% GFP-positive cells in DMSO ( Figure 38D, 38G). While the pMM569 switch achieved almost 100% GFP-positive cells at 15 nM risdiplam, the starting construct (pMM263) as well as the published (pMM274) required much higher risdiplam concentrations. In conclusion, we identified pMM569 as an ON-switch construct that outperformed other variations in terms of induction rate, and having background GFP expression levels comparable to the least leaky constructs tested.
  • Example 39 Risdiplam-regulated NanoLuciferase (NLuc) expression
  • NLuc Risdiplam-regulated NanoLuciferase
  • HEK293 cells stably transfected with one of the various different constructs were treated with indicated risdiplam concentrations for 48h and cells were lysed and analyzed for NLuc expression (Figure 39B-D).
  • the deletion of the ESE2 region sequence in pMM612 switch pMM579 with NLuc as a reporter
  • Example 40 Risdiplam-regulated Cas9 expression for gene editing applications
  • Cas9 as a protein of interest under the control of risdiplam.
  • HEK293 cells In order to test the performance of the various versions of the in-frame switch, we stably transfected HEK293 cells with the above-mentioned constructs using piggybac method. Having established stable HEK293T cell lines, we have used nucleofection-mediated delivery of 3 different sgRNA (UUGGCUUCCUGGGCUGCUA, GCAGCCCUCCACUCCCAUGG, GGCGCUGUCAUGAUGUUCGU) that target the CD81 gene, encoding for a cell-surface protein.
  • sgRNA UUGGCUUCCUGGGCUGCUA, GCAGCCCUCCACUCCCAUGG, GGCGCUGUCAUGAUGUUCGU
  • Risdiplam was added at the indicated concentrations and cells were incubated for 72h before they were stained with anti-CD81 antibody to assess reduction of expression of the protein upon Cas9 induction with risdiplam using FACS-based analysis (Figure 40B). Cells that were not transfected with sgRNA mix served as a negative control.
  • Figure 40A we observed a risdiplam dose-dependent reduction in CD81 expression (Figure 40B-H), with a maximum decrease of 50% for the previously published construct (pMM274) ( Figure 40D) and up to 12% for pMM596 (Figure 40H).
  • Example 41 Risdiplam-regulated CAR expression in CAR-T therapy
  • CARs chimeric antigen receptors
  • An option for regulated expression of CARs in T cells could translate into huge benefits for patients, as regulated CAR expression could increase safety of the therapy, allowing some of the side effects observed in the CAR-T therapy space to be overcome, e.g. strong immune reaction and the well-established cytokine storm effect. Furthemore, it could allow for using more aggressive constructs, or using second inducible CAR-expressing constructs to prevent development of cancer resistance during CAR-T therapy.
  • the in-frame switch constructs pLS76 and its optimized counterpart pMM359 were incorporated into an adeno-associated virus (AAV) vector genome, subsequently packaged into AAV vectors with the AAV2.7m8 capsid, and employed to transduce human embryonic stem cell (hESC)- derived neurons at a multiplicity of infection (MOI) of 20,000 genome-containing vector particles per cell.
  • AAV adeno-associated virus
  • hESC human embryonic stem cell
  • MOI multiplicity of infection
  • neurons Prior to AAV infection, neurons underwent a differentiation period of 21 days.
  • varying concentrations of risdiplam were added to the cultures, with DMSO serving as the negative control.
  • the incucyte live cell imaging analysis system was utilized to assess the expression of the reporter gene GFP in neurons transduced with AAV vectors containing the different in-frame switches, providing insights into both transduction efficiency and the functionality of the in-frame switches within the AAV context. Consistent with our findings with cell lines transfected with the ON-switch constructs, induction of GFP expression was observed with the AAV genome containing the pLS76 switch as depicted in Figure 43B. Notably, the GFP expression mediated by the optimized pMM359 sequence was significantly enhanced at lower concentrations of risdiplam.
  • Example 43 Risdiplam-regulated expression of MeCP2 protein
  • the GFP reporter in the pMM569 switch was substituted with the MeCP2 coding sequence (as depicted in Figure 44A).
  • MeCP2-knockout H4 cells were transfected with the piggyBac system to introduce the MeCP2-expressing plasmid controlled by the in-frame switch.
  • the Operetta imaging system was employed to detect MeCP2 expression, with P37829 cells stained using an anti-MeCP2 antibody and DAPI to visualize nuclei. Following transfection, cells were selected with puromycin to isolate cells stably-expressing the constructs, which were then exposed to various concentrations of risdiplam for 24 hours (illustrated in Figures 44C-D).
  • Example 44 Materials and Methods Cell culture: HEK293 [American Type Culture Collection (ATCC): CRL-1573] and H4 [ATCC: HTB-148] cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; cat. no. #97068-085, lot no. #065K19, VWR) and 1% (v/v) penicillin/streptomycin solution (Sigma-Aldrich) later referred to as DMEM Complete Medium (DMEM).
  • DMEM Dulbecco’s modified Eagle’s medium
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • VWR fetal bovine serum
  • 1% penicillin/streptomycin solution Sigma-Aldrich
  • Jurkat cells (#BA131204, Hoffmann-La Roche, Nutley, NJ) were cultured in RPMI 1640 Medium with GlutaMAXTM Supplement (#61870-036, Gibco) supplemented with 10% FBS (#A3160402, Gibco) and 1% Penicillin-Streptomycin (10,000 U/mL) (#15140122, Gibco). Cells were maintained at 0.5-1.5 x 10 6 cells/ml. In order to select the antibiotic resistant cells, 0.8 - 1 ⁇ g/ml of puromycin (#A1113803, Gibco) or 20 ⁇ g/mL blasticidin (Invitrogen, cat. no. #A1113902) was added to the media.
  • hESC Human embryonic stem cell
  • hESC Human embryonic stem cell
  • MSPPLN521-05, VWR Maintenance medium: DMEM/F12 medium (Cat#31331028, ThermoFischer), mixed at a 1:1 ratio with Neurobasalmedium (Cat# 321103049, ThermoFischer), supplemented with B27 (Cat#12587010), M2 (Cat317502048, ThermoFischer), b- Mercaptoethanol (Cat#31350010, ThermoFischer), and Penicillin/Streptomycin (Cat#15140122, ThermoFischer).
  • BGAA differentiation medium
  • BDNF BDNF
  • GDNF GDNF
  • Aa2-P Cat#A8960, SigmaAldrich
  • cAMP Cat# D009, BioLog
  • Laminin Cat#11243217001, SigmaAldrich
  • HEK293 and H4 cells were transfected using the piggyBac transposase system and Lipofectamine 2000 (Invitrogen, cat. no. #11668019) used according to manufacturer’s instructions.0.35 ⁇ 10 6 cells were seeded per well in a 6-well plate 24 hours (h) before transfection. Cells were then co-transfected with a P37829 PiggyBac helper plasmid encoding for the transposase enzyme and the donor plasmid that contains the gene of interest under the control of the various splice switch versions at a 3:1 ratio (donor:helper plasmid).24h post-transfection, cells underwent blasticidin (Invitrogen, cat. no.
  • Nucleofection of HEK293 and Jurkat cells was performed using 4D-Nucleofector® X Unit (#AAF-1003X, Lonza) according to the manufacturer's protocol. In order to nucleofect Jurkat cells ,1 million cells per reaction was collected, washed once with DPBS, no calcium, no magnesium (#14190144, Gibco), spun down and resuspended in 100 ⁇ L of SE Cell Line NucleofectorTM Solution (#V4XC-1024, Lonza).
  • Resuspended cells were mixed with 2 ⁇ g of the donor plasmid that contains the gene of interest under the control of the various splice switch versions and 0.65 ⁇ g of PiggyBac encoding plasmid (3:1 ratio of donor:helper plasmids) and transferred to NucleocuvetteTM Vessel. Vessels were transferred to 4D- Nucleofector® X Unit and nucleoporated using the CL-120 program. Subsequently, 400 ⁇ L of culture media (RPMI 1640) was added to the vessels and cells were incubated for 5 min. at RT. Next, the whole volume was transferred to 12-well plates and an additional 0.5 mL of fresh media was added.
  • Cells were incubated for 48h after which they were selected with puromycin (0.8 ⁇ g/ml) for 5 days. After the selection, cells were washed with DPBS, resuspended in culture media, seeded on 96-well plates (60000 cells/well), and treated with varying concentrations of risdiplam or DMSO as a negative control.
  • HEK293 nucleoporation with sgRNAs 1 million HEK293 cells stably expressing splice-switch inducible Cas9 enzyme was collected, washed with DPBS, spun down and resuspended in 100 ⁇ L of SF Cell Line NucleofectorTM Solution (#V4XC-2032, Lonza) containing three sgRNAs at 150 nM concentration (sgRNA1: UUGGCUUCCUGGGCUGCUA, sgRNA2: GCAGCCCUCCACUCCCAUGG, sgRNA3: GGCGCUGUCAUGAUGUUCGU).
  • AAV transduction For transducing neuronal cells with AAVs vectoring GFP transgene under the control of a risdiplam- inducible ON-switch (Example 42), AAVs were produced using standard production protocols. HEK293T cells were cultured in DMEM supplemented with 10% FBS until they reached 50 to 70% confluency. The cells were then triple transfected with pHelper, pAAV ITR-expression, and pAAV Rep-Cap plasmids using PEI reagent in 1x cellFactory5. After 72 hours post-transfection, AAV particles were harvested from the cells.
  • the crude lysate was prepared by lysing the cell pellet with 0.1% Triton and digesting with Benzonase. Primary capture of AAV particles was performed using Poros CaptureSelect AAV-X P37829 (ThermoFisher). A secondary purification step was conducted using iodixanol gradient centrifugation to enrich for full capsids. For quality control, vector genome titer was determined by performing qPCR with ITR-specific primers (ITR_qPCR_F: CGGCCTCAGTGAGCGA, qPCR_R: ggaacccctagtgatggagtt). The purity of the final product was assessed via 10% SDS-PAGE using Stain technology (Bio-Rad).
  • GFP expression was monitored using Incucyte Live Imaging device and images were acquired for 7 days. Every 3 days, fresh BGAA medium was exchanged alongside risdiplam. Cloning of the constructs All the tested constructs were ordered and cloned using Gibson cloning at GenScript.
  • RNA isolation HEK293 cells with stably incorporated splice constructs were seeded on 6-well plates (0.5 x 10 6 cells/well) and treated with DMSO or risdiplam for 24(H)After 24h cells were washed with PBS (-/-), trypsynised, resuspended in DMEM CM and transferred to 1.5 mL Eppendorf tubes. Next, cells were centrifuged at 300 x g for 5 min at room temperature, washed once with PBS (-/-) and cell pellets were frozen at -80°C. RNA was isolated using RNeasy Mini Kit (Qiagen, cat. no. #74104) according to manufacturer’s protocol.
  • RNA concentration was measured and all samples were diluted with UltraPureTM DNase/RNase-Free Distilled Water (Invitrogen, cat. no. #10977015) to the same P37829 concentration.
  • the isolated RNA was stored at -80°C and subsequently used for cDNA synthesis by reverse transcriptase (RT)-PCR.
  • RT reverse transcriptase
  • cDNA synthesis 2 ⁇ g of the isolated total RNA were used for the cDNA synthesis using SuperScript IV VILO (Invitrogen, cat. no. #18090050) according to manufacturer’s protocol.
  • RNA was either mixed with SuperScript VILO reverse transcriptase (RT) (+RT) or without RT (–RT), 4x master mix and water in 0.2 mL 8-Strip Non-Flex PCR Tubes (Starlab, cat. no. #I1402-3700) to the final volume of 20 ⁇ L. Tubes were incubated at 25°C for 10 min, followed by 50°C for 30 min and 80°C for 5 min. Samples were diluted to 100 ⁇ L with UltraPureTM DNase/RNase-Free Distilled Water (Invitrogen, cat. no. #10977015) to the final cDNA concentration of 20 ng/ ⁇ L and stored at -20°C.
  • PCR Synthetized cDNA was used for PCRs to analyze splicing efficiency of the tested constructs.
  • PCRs were set up using 10 ⁇ L of Q5 Hot Start HF 2x Master Mix (NEB, cat. No. #M0494L), 1 ⁇ L forward (MM16, TTGACCTCCATAGAAGACACC) and 1 ⁇ L reverse primer (MM40, GCAGATGAACTTCAGGGTCAG) to the final concentration of 0.5 ⁇ M each, 6 ⁇ L H2O and 2 ⁇ L cDNA to the total volume of 20 ⁇ l in 0.2 mL 8- Strip Non-Flex PCR Tubes.
  • NEB Hot Start HF 2x Master Mix
  • PCR thermocycling conditions were as follows: 98°C for 1 min; 15 cycles of (98°C for 15 seconds, 64°C for 15 seconds, 72°C for 30 seconds); 72°C for 2 min; 4°C hold. PCR products were resolved for 20 min on 2% E-GelTM 48-well Agarose Gels (Invitrogen, cat. no. #G820802) with E-Gel 50 bp DNA Ladder (Invitrogen, cat. no. #10488099) and imaged using E-Gel Power Snap Electrophoresis System (G8300).
  • H4 cells were transfected with a knockout in MeCP2 with plasmid containing the in-frame switch according to sequence pMM569 and the MeCP2 sequence (plasmid pMM586) using piggybac system according to described protocol. Cells were selected for stable expressers using puromycin. The stable cell pool was cultured in DMEM, supplemented with FBS and P/S and induced with varying concentrations of risdiplam as indicated or DMSO as a negative control.
  • a secondary antibody (Goat anti-Rabbit, Cat# A32733, ThermoFischer) was then used at a 1:1000 dilution and incubated for 1 hour at RT, followed by 3 washing steps with PBS.
  • DAPI To visualize the nucleus, cells were incubated with DAPI at a 1:1000 for 5min, washed with PBS and images were acquired using the Operetta CLS High-Content Analysis System.
  • NanoLuc (NLuc) measurement NLuc measurements (Example 39) were performed using Nano-Glo® Luciferase Assay System (Promega, #N1120) according to the manufacturer protocol.
  • Nano-Glo reagent was prepared at P37829 room temperature by combining one volume of Nano-Glo® Luciferase Assay Substrate with 50 volumes of Nano-Glo® Luciferase Assay Buffer. Next, the reagent was added to each measured well using a volume of reagent equal to that of the cell culture media in each well (100 ⁇ L per one well of 96-well plate). Plates were incubated for 3 minutes and measured using PHERAstar FSX Microplate Reader (BMG Labtech).
  • HEK293 cells were nucleoporated with CD81-targeting sgRNAs as described in the nucleofection section hereinabove, seeded on 96-well plates and treated with varying concentrations of risdiplam or DMSO as a negative control for 3 days.
  • CD81+ on cell surface cells were first detached with 40 ⁇ L of Trypsin-EDTA (0.05%), phenol red (#25300054, Gibco) and next resuspended in the additional volume of 100 ⁇ L DBPS to a total volume of 140 ⁇ L.
  • Cells were transferred to fresh 96-well Microplates, PP, U-bottom, 0,3 ml (#701330, Brand), spun down and washed once with DPBS. Next, cells were resuspended in 50 ⁇ L of 1:10 mix of APC-conjugated CD81 (#561958, BD) and DBPS and incubated for 15 min. at 4 ⁇ C. Cells were spun down (180xg, 3 min.), the staining solution was removed, and cells were washed three times with DPBS. Finally, cells were resuspended in DBPS and analyzed by flow cytometry using CytoFLEX LX Flow Cytometer (BeckmanCoulter).
  • HLA-A*0201-restricted cytolytic responses to the rtTA transactivator dominant and cryptic epitopes compromise transgene expression induced by the tetracycline on system. Mol Ther 10, 279-289, doi:10.1016/j.ymthe.2004.05.012 (2004). 12. Hoyng, S.(A)et al. Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve. Gene Ther 21, 549-557, doi:10.1038/gt.2014.22 (2014). 13. Tickner, Z. J. & Farzan, M.
  • Splice site sequences of spinal muscular atrophy related SMN2 pre-mRNA include enhancers for nearby exons. ScientificWorldJournal 2014, 617842, doi:10.1155/2014/617842 (2014). 19. Gao, Y. et al. Systematic characterization of short intronic splicing-regulatory elements in SMN2 pre-mRNA. Nucleic Acids Res 50, 731-749, doi:10.1093/nar/gkab1280 (2022). 20. Noderer, W. L. et al. Quantitative analysis of mammalian translation initiation sites by FACS-seq. Mol Syst Biol 10, 748, doi:10.15252/msb.20145136 (2014).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present disclosure relates to the fields of molecular biology and nucleic acid technology. The present disclosure also relates to therapy and prophylaxis of disease.

Description

P37829 Inducible Gene Expression System This application claims priority from EP23178358.0 filed 9 June 2023, the contents and elements of which are herein incorporated by reference for all purposes. Technical Field The present disclosure relates to the fields of molecular biology and nucleic acid technology. The present disclosure also relates to therapy and prophylaxis of disease. Background Gene therapy as a modality aims at ameliorating disease-related phenotypes by using both viral and non- viral delivery systems. Significant efforts have been focusing on optimizing this therapy to be more efficient, non-immunogenic and less toxic while enabling a long-term expression of the gene of interest. One of the key challenges to achieve this goal is to regulate the expression of the transgene to meet the required level and to be expressed upon demand. For certain targets, where constitutive and unlimited expression leads to adverse events, regulated gene expression is necessary to render gene therapy a viable option as a treatment modality. One example is Rett Syndrome (RTT), an X-linked neurological disorder associated with severe motor abnormalities and reduced lifespan that occurs in 1 in 1,000 females. RTT is characterized by seemingly normal neurological and physical development during early postnatal period followed by a rapid regression with the loss of the purposeful motor skills and the onset of repetitive and autistic behaviors1,2. During the rapid progression stage, the child loses purposeful hand skills and spoken language, experiences motor impairments, and develops breathing abnormalities and may also develop autistic-like features and seizures. At later stages, further motor deterioration begins, typified by severe physical disability, which leads many patients to become dependent on the use of wheelchairs. Current treatment options are limited to symptom control. Mutations in the MECP2 gene on the X chromosome, encoding for methyl- CpG-binding protein 2 account for 95% of RTT cases3. MeCP2 is highly expressed in neurons and functions as a ubiquitous transcriptional regulator by binding to methylated DNA and recruiting protein partners and regulatory complexes to control transcriptional activity. MeCP2 regulates neuronal physiology and maintenance and landmark studies have demonstrated that restoring levels of the MeCP2 protein dramatically reverses symptoms in mice4,5. Given that it is a monogenetic disorder driven by the lack of MeCP2, a protein with multiple functions, gene therapy is one potential avenue to treat RTT. Studies in RTT mice have provided encouraging data showing attenuated neurological dysfunctions as well as extended lifespan by intravenous administration of an Adeno-associated virus serotype 9 (AAV9) expressing the wild-type (WT) MeCP2 gene5-8. Despite the promising potential of gene replacement therapy, high doses of MeCP2 by overexpression systems – which rely on the use of ubiquitous promoters resulting in uncontrolled level of the transgene – can lead to toxicity with phenotypes similar to those observed in duplication syndrome, a condition mainly affecting males, characterized by moderate to severe intellectual impairment, and caused by a duplication of the MeCP2 gene on the X-chromosome9,10. The requirement of keeping MeCP2 expression level within a window for normal function is one of the most challenging aspects that has yet to be solved. One solution P37829 would be to develop controlled transgene expression systems in order to overcome this limitation and make gene therapy a viable treatment option. Inducible gene expression systems allow for reversibility and flexibility and enable the production of therapeutics upon demand, thereby circumventing overdose-associated side effects. Such regulation can be achieved using small molecules, with which transgene expression can be induced (generating ON- switches) or repressed (OFF-switch). Among the existing inducible transcriptional gene regulatory systems, the tetracycline (Tet)-regulable system is the most widely exploited tool and can be used as an ON- or an OFF-switch. Tet-inducible systems, however, are derived from bacteria and require the expression of the regulator protein, e.g. the ON-switch components include the tetracycline-controlled transactivator (tTA), which consists of the bacterial tetR with a C-terminal domain of VP16 (virion protein 16), derived from the herpes simplex virus. The required co-expression of these components further increase the limitations in the size of the transgene for AAV packaging, and can cause both silencing and potential immunogenicity effects when introduced into human tissue, which has hampered further translation into the clinics11,12. Another class of genetic switches originating from bacteria are riboswitches, RNA elements that are able to control gene expression in response to ligand binding, have a small genomic footprint and do not depend on other proteins for activity, which renders these types of regulated gene expression an attractive alternative to protein-based expression control systems13. However, riboswitches suffer from a poor dynamic range as well as high basal activity14. A new class of small molecules that regulate splicing of SMN2 exon 7 was recently identified, via screening using a reporter system based on the SMN2 transcript (described e.g. in WO 2009/151546 A2). SMN2 reporter systems have since been proposed to be used as gene expression switches, where expression of a transgene is regulated through small molecule-controlled splicing of the expression cassette (Monteys et al. Nature (2021) 596: 291-295). Known human SMN2 exon 6 to exon 8-derived transgene expression systems are described e.g. in Zhang, et al., Gene Ther. (2001) 8: 1532-1538, WO 2022/204471 A1, Monteys et al. Nature (2021) 596: 291-295 and WO 2021/163556 A1. However, these known SMN2-based switch systems have very long nucleotide sequences, limiting the size of the transgene that can be employed under the control of the system. That is, the size of an insert comprising the switch and the coding sequence for a therapeutic polypeptide to be delivered as gene therapy would very often be much larger than the packaging limit for vectors routinely employed in the delivery of gene therapies, e.g. adeno-associated viral (AAV) vectors. Summary In a first aspect, the present disclosure provides a polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:222, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least P37829 80% sequence identity to a nucleotide sequence according to SEQ ID NO:378 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:226; (iv) a fourth nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:15 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end; (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘GA’, ‘TG’ or ‘TT’, or (b) comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:19, wherein the nucleotide sequence comprises ‘GA’, ‘TG’ or ‘TT’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:19, or (c) encoding a polypeptide of interest, and comprising ‘GA’, ‘TG’ or ‘TT’ at positions 1 and 2; and (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a) or (v)(b), a sixth nucleotide sequence encoding a polypeptide of interest; wherein the polynucleotide comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. In a second aspect, the present disclosure provides a polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:1, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26, wherein the third nucleotide sequence comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; (iv) a fourth nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end; (v) a fifth nucleotide sequence: (a) consisting of the trinucleotide ‘GAG’ or (b) encoding a polypeptide of interest, and comprising ‘GAG’ at positions 1 to 3; and (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a), a sixth nucleotide sequence encoding a polypeptide of interest; wherein the polynucleotide comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. In some embodiments in accordance with the various aspects of the present disclosure, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. P37829 In some embodiments, the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:222. In some embodiments, the first nucleotide sequence comprises, or consists of, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:106, SEQ ID NO:219 or SEQ ID NO:220. In some embodiments, the first nucleotide sequence comprises, or consists of, SEQ ID NO:3. In some embodiments, the second nucleotide sequence comprises SEQ ID NO:7 at its 5’ end, and comprises SEQ ID NO:8 or SEQ ID NO:377 at its 3’ end. In some embodiments, the second nucleotide sequence consists of fewer than 500 nucleotides. In some embodiments, the second nucleotide sequence comprises, or consists of, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:228. In some embodiments, the first nucleotide sequence comprises, or consists of, SEQ ID NO:10. In some embodiments, the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:226. In some embodiments, the third nucleotide sequence consists of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:223 or SEQ ID NO:224. In some embodiments, the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:26. In some embodiments, the third nucleotide sequence consists of SEQ ID NO:13 or SEQ ID NO:27. In some embodiments, the fourth nucleotide sequence comprises SEQ ID NO:15 or SEQ ID NO:379 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end. In some embodiments, the fourth nucleotide sequence consists of fewer than 500 nucleotides. In some embodiments, the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:227 or SEQ ID NO:340. In some embodiments, the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17. In some embodiments, the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’ or ‘TG’, or (b) comprises, or consists of, a nucleotide sequence according to SEQ ID NO:19. In some embodiments, the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’ or ‘TG’, or (b) comprises, or consists of, SEQ ID NO:20 or SEQ ID NO:21. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:164 and SEQ ID NO:171. In some embodiments, the polynucleotide comprises at least 80% sequence identity to SEQ ID NO:28 or SEQ ID NO:29. In some embodiments, the polynucleotide further comprises a promoter sequence 5’ to the start codon. In some embodiments, the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a polypeptide of interest. In some embodiments, the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. P37829 The present disclosure also provides a vector comprising a polynucleotide according to the present disclosure. In some embodiments, the vector is an adeno-associated virus (AAV) vector. The present disclosure also provides a pharmaceutical composition comprising a polynucleotide or vector according to the present disclosure, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. The present disclosure also provides a cell comprising a polynucleotide or vector according to the present disclosure. In some embodiments, the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. The present disclosure also provides a method for modifying a cell to express a polypeptide of interest, comprising: (i) introducing into a cell a polynucleotide or vector according to the present disclosure; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. The present disclosure also provides a method for expressing a polypeptide of interest in a cell, comprising contacting a cell according to the present disclosure with a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. The present disclosure also provides a method for inhibiting expression of a polypeptide of interest in a cell, comprising contacting a cell according to the present disclosure with a splicing modifier that promotes SMN2 exon 7 inclusion. The present disclosure also provides a method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell a polynucleotide or vector according to the present disclosure. The present disclosure also provides a polynucleotide, vector or pharmaceutical composition according to the present disclosure, for use in a method of medical treatment or prophylaxis. The present disclosure also provides a polynucleotide, vector or pharmaceutical composition according to the present disclosure, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. The present disclosure also provides the use of a polynucleotide, vector or pharmaceutical composition according to the present disclosure, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. P37829 The present disclosure also provides a method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject a polynucleotide, vector or pharmaceutical composition according to the present disclosure. In some embodiments, treating or preventing the disease or condition further comprises administering to the subject a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. In some embodiments, the disease or condition is a disease or condition characterised by deficiency of the polypeptide of interest. The present disclosure also provides a kit, comprising: (i) a polynucleotide, vector or pharmaceutical composition according to the present disclosure; and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. Description The present disclosure relates to inducible transgene expression systems using small molecule splicing modifiers (SMSM), and based on the described mechanism of action of the Survival of Motor Neuron 2 (SMN2) splicing modifier risdiplam (and variants thereof). The SMSM binding sites for SMN2 have previously been described to be two distinct sites within SMN2 exon 7 pre-mRNA: the ESE2 region and the 5’ splice donor site (5’ss) (Figure 2a)15. Interaction of the small molecules with the mRNA-protein complex is thought to be critical for the high selectivity of the compound. This study suggested that both ESE2 and 5’ss regions are required for full activity of the SMSM compounds in a dose-dependent manner15. Structural analysis using a compound from the same chemical class as risdiplam (SMN-C5) has demonstrated that the drug selectively promotes the recognition of the weak 5’ splice site of SMN2 exon 7 by U1 snRNP by stabilizing an unpaired adenine at the exon-intron junction in the RNA helix16. The present disclosure provides novel SMN2 exon 6 to exon 8-derived constructs providing for the SMSM-inducible regulation of transgene expression, which are suitable to be employed for chemically- inducible regulation of the expression of gene therapies. In particular, the present disclosure provides ON-switch constructs: (i) having a size permitting their application for the SMSM-inducible expression of polypeptides to be delivered as gene therapies, (ii) minimising undesirable expression of the polypeptide in the absence of the SMSM, and (iii) which are highly responsive to the SMSM, thus providing for strong induction of expression of the polypeptide in the presence of the SMSM, and (iv) that minimise the number of/completely remove extraneous amino acids P37829 at the N-terminus of the expressed polypeptide. The present disclosure also provides OFF-switch constructs: (i) having a size permitting their application for the SMSM-inducible expression of polypeptides to be delivered as gene therapies, (ii) maximising expression of the polypeptide in the absence of the SMSM, and (iii) which are highly responsive to the SMSM, thus minimising expression of the polypeptide in the presence of the SMSM, and (iv) that minimise the number of/completely remove extraneous amino acids at the N-terminus of the expressed polypeptide. Polynucleotides Aspects and embodiments of the present disclosure relate to polynucleotides. A 'polynucleotide' refers to a polymer chain of a plurality of nucleotide monomers linked by bonds between the monomers, typically phosphodiester bonds (e.g. in the case of polynucleotides formed by naturally-occurring nucleotide monomers). Polynucleotides include oligonucleotides, which generally comprise ≤50 nucleotides. A polynucleotide may be single-stranded, or may be double-stranded (i.e. may comprise a duplex formed by hydrogen-bonding between complementary nucleotides). Polynucleotides according to the present disclosure may comprise or consist of: single-stranded DNA, double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single-stranded RNA, double- stranded RNA, RNA that is mixture of single- and double-stranded regions, single-stranded molecules comprising DNA and RNA, double-stranded molecules comprising DNA and RNA, and molecules comprising DNA and RNA having a mixture of single- and double-stranded regions. In some embodiments, a polynucleotide comprises or consists of DNA. In some embodiments, a polynucleotide is a polydeoxyribonucleotide. In some embodiments, a polynucleotide comprises or consists of RNA. In some embodiments, a polynucleotide is a polyribonucleotide. In aspects and embodiments wherein the polynucleotide of the present disclosure is defined by reference to a given nucleotide sequence, and wherein the given nucleotide sequence comprises or consists of RNA and/or is a polyribonucleotide, it will be appreciated that instances of ‘T’ for thymidine in such sequences are replaced with ‘U’, for uracil. The present disclosure also contemplates polynucleotides comprising modified nucleotides, e.g. in which the phosphonate and/or ribose and/or base of a deoxyribonucleotide or ribonucleotide is/are chemically modified. Nucleotide modifications contemplated in accordance with the present disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), which is hereby incorporated by reference in its entirety. Phosphonate modifications may be selected from phosphorothioate (e.g. Rp isomer, Sp isomer), phosphorodithioate, methylphosphonate, methoxypropylphosphonate, 5'-(E)-vinylphosphonate, 5'- methylphosphonate, (S)-5'-C-methyl with phosphate, 5’-phosphorothioate, and peptide nucleic acid modifications. Ribose modifications may be selected from 2'-O-methyl, 2'-O-methoxyethyl, 2’-fluoro, 2’- deoxy-2’-fluoro, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, 2'-arabino-fluoro, 2’-O-benzyl, 2’-O-methyl-4-pyridine, locked nucleic acid, (S)-cEt-BNA, tricyclo-DNA, PMO, unlocked P37829 nucleic acid, hexitol nucleic acid and glycol nucleic acid modifications. Base modifications may be selected from pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5’-methylcytidine, 5’-fluoro-2’- deoxyuridine, N-ethylpiperidine 7'-EAA triazole-modified adenine, N-ethylpiperidine 6'-triazole-modified adenine, 6'-phenylpyrrolo-cytosine, 2',4'-difluorotoluyl ribonucleoside and 5'-nitroindole modifications. In some embodiments, a modified nucleotide may be selected from 2'-O-methyluridine-3'-phosphate, 2'- O-methyladenosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-O- methylguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphorothioate, 2'-fluorouridine-3'- phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'- phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphorothioate, 2'- fluoroadenosine-3'-phosphorothioate, and 2'-fluorouridine-3'-phosphorothioate. Nucleotide sequences of the polynucleotides of the present disclosure Polynucleotides of the present disclosure are defined herein by reference to constituent nucleotide sequences. It will be appreciated that the constituent nucleotide sequences of polynucleotides according to the present disclosure are provided as subsequences of the complete sequence of the polynucleotide. In some aspects and embodiments, the constituent nucleotide sequences of polynucleotides according to the present disclosure are provided in a particular order in the sequence of the polynucleotide, e.g. from 5’ to 3’. By way of illustration, the first nucleotide sequence is provided 5’ to (i.e. upstream of) the second nucleotide sequence, in the context of the sequence of the polynucleotide. Similarly, the second nucleotide sequence is 5’ to the third nucleotide sequence, etc. The constituent nucleotide sequences of the polynucleotides of the present disclosure are non- overlapping. In some embodiments, constituent nucleotide sequences of the polynucleotides are provided in tandem in the context of the complete sequence of the polynucleotide. In some embodiments, constituent nucleotide sequences of the polynucleotide are immediately adjacent to one another (i.e. the 3’ nucleotide of a given nucleotide sequence is followed immediately by the 5’ nucleotide of another given nucleotide sequence, in the context of the complete sequence of the polynucleotide). By way of illustration, in the polynucleotide of SEQ ID NO:22, positions 1 to 45 form the first nucleotide sequence, positions 46 to 309 form the second nucleotide sequence, positions 310 to 364 form the third nucleotide sequence, positions 356 to 616 form the fourth nucleotide sequence, and positions 617 and 618 form the fifth nucleotide sequence. First nucleotide sequence The first nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 exon 6. A ‘variant’ of a given reference nucleotide sequence comprises one or more differences relative to the reference nucleotide sequence. For example, a variant P37829 of a given reference nucleotide sequence may comprise insertion, deletion or substitution of one or more nucleotides relative to the reference nucleotide sequence. Accordingly, in some embodiments, the first nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having <100% sequence identity to SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1. It will be appreciated that ‘a nucleotide sequence according to SEQ ID NO:1’ refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:1. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:1 include SEQ ID NOs:3, 4, 5, 6 and 106. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:3. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:4. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:5. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:6. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:1 include SEQ ID NOs:219 and 220. In some embodiments, the first nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:222. In some embodiments, the first nucleotide sequence comprises ‘CAG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2 (cf. e.g. positions 109 to 111 of SEQ ID NO:6). As explained herein, position(s) of a nucleotide sequence which ‘corresponds to’ specified position(s) of a reference nucleotide sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Söding, J.2005, P37829 Bioinformatics 21, 951-960). By way of illustration, it will be appreciated that positions 43 to 45 of SEQ ID NO:3 correspond to positions 109 to 111 of SEQ ID NO:2. Similarly, positions 45 to 47 of SEQ ID NO:4 correspond to positions 109 to 111 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises ‘CTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2 (cf. e.g. positions 109 to 111 of SEQ ID NO:5). In some embodiments, the first nucleotide sequence comprises ‘GGC’ at the positions corresponding to positions 64 to 66 of SEQ ID NO:2 (cf. e.g. positions 64 to 66 of SEQ ID NO:5, positions 64 to 66 of SEQ ID NO:6). In some embodiments, the first nucleotide sequence comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2 (cf. e.g. positions 16 to 18 of SEQ ID NO:3, positions 18 to 20 of SEQ ID NO:4, positions 82 to 84 of SEQ ID NO:5). In some embodiments, the first nucleotide sequence comprises ‘GGC’ at the positions corresponding to positions 64 to 66 of SEQ ID NO:2, and comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises ‘GTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises ‘TTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises ‘TAG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises ‘CAG’ at the positions corresponding to positions 39 to 41 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2. In some embodiments, the first nucleotide sequence consists of fewer than 111 nucleotides. In some embodiments the first nucleotide sequence consists of <100 nucleotides, e.g. one of <85 nucleotides, <80 nucleotides, <75 nucleotides, <70 nucleotides, <65 nucleotides, <60 nucleotides, <55 nucleotides, <50 nucleotides or ≤45 nucleotides. In some embodiments, the first nucleotide sequence consists of 45 nucleotides. In some embodiments, the first nucleotide sequence consists of 51 nucleotides. In some embodiments, the first nucleotide sequence consists of 81 nucleotides. In some embodiments, the first nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and (ii) comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) consists of ≤45 nucleotides. Examples of such first nucleotide sequences include SEQ ID NOs:3, 4 and 106. P37829 In some embodiments, the first nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and (ii) comprises ‘GGC’ at the positions corresponding to positions 64 to 66 of SEQ ID NO:2; and (iii) comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iv) comprises ‘CTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2. An example of such a first nucleotide sequence is SEQ ID NO:5. In some embodiments, the first nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and (ii) comprises ‘GGC’ at the positions corresponding to positions 64 to 66 of SEQ ID NO:2; and (iii) comprises ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iv) comprises ‘CAG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2. An example of such a first nucleotide sequence is SEQ ID NO:6. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:3, 5, 6, 219, 220, and 230 to 237. In some embodiments, a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:2. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:3. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:5. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:6. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:219. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a first nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one P37829 of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:220. Second nucleotide sequence The second nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 intron 6. Accordingly, in some embodiments, the second nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:30. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having <100% sequence identity to SEQ ID NO:30. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:378 at its 3’ end. It will be appreciated that ‘a nucleotide sequence according to SEQ ID NO:378’ refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:378. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:378 include SEQ ID NO:8 and SEQ ID NO:377. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:377 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end, and comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end, and comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:377 at its 3’ end. As used herein, the ‘5’ end’ of a given nucleotide sequence refers to the region of the nucleotide sequence formed by nucleotides 5’ to (i.e. upstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence, or a subsequence of this region. P37829 In a nucleotide sequence having an even number of nucleotides, the region 5’ to the ‘midpoint’ includes the nucleotide immediately preceding the midpoint. For example, in a nucleotide sequence consisting of 10 nucleotides, the region 5’ to the midpoint consists of positions 1 to 5. Similarly, in a nucleotide sequence having an even number of nucleotides, the region 3’ to the ‘midpoint’ includes the nucleotide immediately after the midpoint. For example, in a nucleotide sequence consisting of 10 nucleotides, the region 3’ to the midpoint consists of positions 6 to 10. In a nucleotide sequence having an odd number of nucleotides, the region 5’ to the ‘midpoint’ includes the nucleotides 5’ to (i.e. upstream of) the nucleotide provided at the midpoint. For example, in a nucleotide sequence consisting of 9 nucleotides, the region 5’ to the midpoint consists of positions 1 to 4. Similarly, in a nucleotide sequence having an odd number of nucleotides, the region 3’ to the ‘midpoint’ includes the nucleotides 3’ to (i.e. downstream of) the nucleotide provided at the midpoint. For example, in a nucleotide sequence consisting of 9 nucleotides, the region 3’ to the midpoint consists of positions 6 to 9. By way of illustration, in SEQ ID NO:9, which consists of 414 nucleotides, a sequence provided at the 5’ end of SEQ ID NO:9 refers to a nucleotide sequence formed by positions 1 to 207 of SEQ ID NO:9, or a subsequence thereof. Conversely, the ‘3’ end’ of a given nucleotide sequence refers to the region of the nucleotide sequence formed by nucleotides 3’ to (i.e. downstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence, or a subsequence of this region. By way of illustration, in SEQ ID NO:9, a sequence provided at the 3’ end of SEQ ID NO:9 refers to a nucleotide sequence formed by positions 208 to 414 of SEQ ID NO:9, or a subsequence thereof. In some embodiments, a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region of the nucleotide sequence formed by nucleotides 5’ to (i.e. upstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence. By way of illustration, a sequence provided at the 5’ end of SEQ ID NO:9 may comprise at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region formed by positions 1 to 207 of SEQ ID NO:9. In some embodiments, a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region of the nucleotide sequence formed by nucleotides 3’ to (i.e. downstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence. By way of illustration, a sequence provided at the 3’ end of SEQ ID NO:9 may comprise at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region formed by positions 208 to 414 of SEQ ID NO:9. P37829 In some embodiments, a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes one or more nucleotides provided within 25 nucleotides, e.g. within one of 20, 15, 10 or 5 nucleotides, of the 5’ nucleotide of the given nucleotide sequence. In some embodiments, a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes the 5’ nucleotide of the given nucleotide sequence. By way of illustration, a sequence provided at the 5’ end of SEQ ID NO:9 may comprise one or more nucleotides provided within positions 1 to 25, e.g. within positions 1 to 20, 1 to 15, 1 to 10, or 1 to 5 of SEQ ID NO:9. By way of illustration, a sequence provided at the 5’ end of SEQ ID NO:9 may comprise position 1 of SEQ ID NO:9. In some embodiments, a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes one or more nucleotides provided within 25 nucleotides, e.g. within one of 20, 15, 10 or 5 nucleotides, of the 3’ nucleotide of the given nucleotide sequence. In some embodiments, a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes the 3’ nucleotide of the given nucleotide sequence. By way of illustration, a sequence provided at the 3’ end of SEQ ID NO:9 may comprise one or more nucleotides provided within positions 389 to 414, e.g. within positions 394 to 414, 399 to 414, 404 to 414, or 409 to 414 of SEQ ID NO:9. By way of illustration, a sequence provided at the 3’ end of SEQ ID NO:9 may comprise position 414 of SEQ ID NO:9. In some embodiments the second nucleotide sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 in the region 5’ to its midpoint. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:377 in the region 3’ to its midpoint. In some embodiments, the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 accounts for at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region 5’ to the midpoint of the second nucleotide sequence. In some embodiments, the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:377 accounts for at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region 3’ to the midpoint of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 comprises the 5’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:377 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:377 comprises the 3’ nucleotide of the second nucleotide sequence. P37829 An exemplary sequence comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end is SEQ ID NO:9. Positions 1 to 102 of SEQ ID NO:9 correspond to positions 1 to 102 of SEQ ID NO:7. An exemplary sequence comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 at its 3’ end is SEQ ID NO:9. Positions 253 to 414 of SEQ ID NO:9 correspond to positions 1 to 162 of SEQ ID NO:8. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:229. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:229 include SEQ ID NOs:10 and 228. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:9. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10. In some embodiments, the second nucleotide sequence comprises ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30 (cf. e.g. position 411 of SEQ ID NO:9, position 261 of SEQ ID NO:10). In some embodiments, the second nucleotide sequence consists of fewer than 1044 nucleotides, e.g. one of <1000 nucleotides, <900 nucleotides, <800 nucleotides, <750 nucleotides, <700 nucleotides, <650 nucleotides, <600 nucleotides, <550 nucleotides or <500 nucleotides. In some embodiments, the second nucleotide sequence consists of fewer than 500 nucleotides, e.g. one of <450 nucleotides, <400 nucleotides, <350 nucleotides, <300 nucleotides, <250 nucleotides or <200 nucleotides. In some embodiments, the second nucleotide sequence consists of 1044 nucleotides. In some embodiments, the second nucleotide sequence consists of 414 nucleotides. In some embodiments, the second nucleotide sequence consists of 264 nucleotides. In some embodiments, the second nucleotide sequence consists of 189 nucleotides. In some embodiments, the second nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprises ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consists of fewer than 1044 nucleotides. Examples of such second nucleotide sequences include SEQ ID NOs:9 and 10. P37829 In some embodiments, the second nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprises ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consists of fewer than 500 nucleotides. An example of such a second nucleotide sequence is SEQ ID NO:9. In some embodiments, the second nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprises ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consists of fewer than 300 nucleotides. An example of such a second nucleotide sequence is SEQ ID NO:10. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a second nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:9, 10, 228, 238 to 255, 375 and 376. In some embodiments, a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:240. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a second nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:9, 10, 228, 238, 239, 241 to 255, 375 and 376. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a second nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:9. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a second nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a second nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:228. P37829 Third nucleotide sequence The third nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 exon 7. Accordingly, in some embodiments, the third nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having <100% sequence identity to SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11. It will be appreciated that ‘a nucleotide sequence according to SEQ ID NO:11’ refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:11. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:11 include SEQ ID NOs:13 and 14. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:26. It will be appreciated that ‘a nucleotide sequence according to SEQ ID NO:26’ refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:26. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:26 include SEQ ID NOs:13 and 27. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:225. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:225 include SEQ ID NOs:223 and 224. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:226. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:13. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:14. In some embodiments, the third nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:27. P37829 In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12 (cf. e.g. position 2 of SEQ ID NOs:11, 13, 14, 26 and 27). In some embodiments, the third nucleotide sequence comprises insertion of ‘GCCACC’ after the position corresponding to position 6 of SEQ ID NO:12 (cf. e.g. positions 7 to 12 of SEQ ID NO:14). In some embodiments, the third nucleotide sequence comprises ‘TG’ at the positions corresponding to positions 8 to 9 of SEQ ID NO:12 (cf. e.g. positions 14 and 15 of SEQ ID NO:14). In some embodiments, the third nucleotide sequence comprises insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12 (cf. e.g. position 49 of SEQ ID NO:13, position 55 of SEQ ID NO:14). In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 24 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the nucleotide at the position corresponding to position 20 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘T’ at the position corresponding to position 27 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘T’ at the position corresponding to position 28 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 21 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 29 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘T’ at the position corresponding to position 21 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 31 of SEQ ID NO:12, and comprises ‘A’ at the position corresponding to position 34 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the positions corresponding to positions 9 to 20 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the positions corresponding to positions 33 to 41 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘CAC’ at the positions corresponding to positions 34 to 36 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘G’ after the position corresponding to position 39 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘GC’ at the positions corresponding to positions 47 to 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘CACCATG’ after the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘C’ at the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘AA’ after the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘CAT’ at the positions corresponding to positions 49 to 51 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘GCC’ at the positions corresponding to positions 46 to 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘CCA’ at the positions corresponding to positions 49 to 51 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘T’ after the position corresponding to position 51 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘GC’ at the positions corresponding to positions 39 to 40 of SEQ P37829 ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘CA’ after the position corresponding to position 40 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘ATG’ at the positions corresponding to positions 43 to 45 of SEQ ID NO:12, and deletion of the position corresponding to position 46 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘GCCACCATG’ after the position corresponding to position 9 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘AGCACCATG’ after the position corresponding to position 15 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 16 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘CACCATG’ after the position corresponding to position 15 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘ATG’ after the position corresponding to position 15 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘ATG’ after the position corresponding to position 21 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘GG’ after the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘AT’ after the position corresponding to position 30 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 31 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘G’ after the position corresponding to position 33 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘A’ after the position corresponding to position 43 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘GG’ at the positions corresponding to positions 45 and 46 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘AT’ after the position corresponding to position 52 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the positions corresponding to positions 49 and 50 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 51 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 40 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 35 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘A’ after the position corresponding to position 39 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 41 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘C’ at the position corresponding to position 43 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘ATG’ after the position corresponding to position 42 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘TG’ after the position corresponding to position 45 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘ATGGA’ after the position corresponding to position 44 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 44 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 49 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the position corresponding to position 50 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding P37829 to position 50 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 44 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the position corresponding to position 49 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the positions corresponding to positions 42 to 44 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘G’ after the position corresponding to position 39 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises deletion of the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises insertion of ‘GA’ after the position corresponding to position 48 of SEQ ID NO:12. In some embodiments, the third nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprises insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12. An example of such a third nucleotide sequence is SEQ ID NO:13. In some embodiments, the third nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprises insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; and (iv) comprises insertion of ‘GCCACC’ after the position corresponding to position 6 of SEQ ID NO:12; and (v) comprises ‘TG’ at the positions corresponding to positions 8 and 9 of SEQ ID NO:12. An example of such a third nucleotide sequence is SEQ ID NO:14. In some embodiments, the third nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:26; and (ii) comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprises deletion of the position corresponding to position 20 of SEQ ID NO:12; and (iv) comprises insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12. An example of such a third nucleotide sequence is SEQ ID NO:27. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:13, 14, 27, 223, 224 and 256 to 329. In some embodiments, a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:13. In some embodiments, a polynucleotide according to the present P37829 disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:14, 27, 223, 224 and 256 to 329. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:13. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:14. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:27. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:223. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:224. Fourth nucleotide sequence The fourth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 intron 7. Accordingly, in some embodiments, the fourth nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having <100% sequence identity to SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:380 at its 5’ end. It will be appreciated that ‘a nucleotide sequence according to SEQ ID NO:380’ refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:380. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:380 include SEQ ID NO:15 and SEQ ID NO:379. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end. In some embodiments, the fourth nucleotide sequence P37829 comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:379 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end, and comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:379 at its 5’ end, and comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end. In some embodiments the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 or SEQ ID NO:379 in the region 5’ to its midpoint. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 in the region 3’ to its midpoint. In some embodiments, the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 or SEQ ID NO:379 accounts for at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region 5’ to the midpoint of the fourth nucleotide sequence. In some embodiments, the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 accounts for at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region 3’ to the midpoint of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 or SEQ ID NO:379 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 or SEQ ID NO:379 comprises the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 comprises the 3’ nucleotide of the fourth nucleotide sequence. An exemplary sequence comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 at its 5’ end is SEQ ID NO:17. Positions 1 to 102 of SEQ ID NO:17 correspond to positions 1 to 102 of SEQ ID NO:15. P37829 An exemplary sequence comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end is SEQ ID NO:18. Positions 295 to 444 of SEQ ID NO:18 correspond to positions 1 to 150 of SEQ ID NO:16. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:17. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence consists of fewer than 444 nucleotides, e.g. one of <425 nucleotides, <400 nucleotides, <350 nucleotides, <300 nucleotides, <250 nucleotides or <200 nucleotides. In some embodiments, the fourth nucleotide sequence consists of fewer than 255 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 252 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 177 nucleotides. In some embodiments, the fourth nucleotide sequence consists of greater than 444 nucleotides, e.g. one of >450 nucleotides, >475 nucleotides or >500 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 508 nucleotides. In some embodiments, the fourth nucleotide sequence comprises ‘C’ at the position corresponding to position 441 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises ‘T’ at the position corresponding to position 441 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises ‘TT’ at the positions corresponding to positions 436 and 437 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises ‘TCCTC’ at the positions corresponding to positions 11 to 15 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises insertion of ‘TTT’ after the position corresponding to position 10 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises insertion of ‘CCC’ after the position corresponding to position 10 of SEQ ID NO:18. In some embodiments, the fourth nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consists of fewer than 500 nucleotides. Examples of such fourth nucleotide sequences include SEQ ID NOs:17 and 18. In some embodiments, the fourth nucleotide sequence comprises: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or P37829 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consists of fewer than 255 nucleotides. An example of such a fourth nucleotide sequence is SEQ ID NO:17. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fourth nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:18, 227, 330 to 370 and 381. In some embodiments, a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:337. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a third nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:18, 227, 330 to 336, 338 to 370 and 381. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fourth nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:18. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fourth nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:227. Fifth nucleotide sequence The fifth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence which is a variant of human SMN2 exon 8. Accordingly, in some embodiments, the fifth nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having <100% sequence identity to SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘GA’, ‘TG’ or ‘TT’. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘GA’. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘TG’. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘TT’. In some embodiments, the fifth nucleotide sequence consists of the trinucleotide ‘GAG’. In some embodiments, the fifth nucleotide sequence consists of the nucleotide ‘A’. P37829 In some embodiments, the fifth nucleotide sequence encodes a polypeptide of interest (e.g. as described hereinbelow). In some embodiments, the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘GA’, 'TG' or ‘TT’ at positions 1 and 2. In some embodiments, the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘GA’ at positions 1 and 2. In some embodiments, the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘TG’ at positions 1 and 2. In some embodiments, the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘TT’ at positions 1 and 2. In some embodiments, the fifth nucleotide sequence encodes a polypeptide of interest, and comprises ‘GAG’ at positions 1 to 3. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:19. It will be appreciated that ‘a nucleotide sequence according to SEQ ID NO:19’ refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:19. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:19 include SEQ ID NOs:20 and 21. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:20. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:21. In some embodiments, the fifth nucleotide sequence comprises ‘GA’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:32 (cf. e.g. positions 1 and 2 of SEQ ID NO:20). In some embodiments, the fifth nucleotide sequence comprises ‘TG’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:32 (cf. e.g. positions 1 and 2 of SEQ ID NO:21). In some embodiments, the fifth nucleotide sequence comprises fewer than 577 nucleotides. In some embodiments the fifth nucleotide sequence consists of <500 nucleotides, e.g. one of <400 nucleotides, <300 nucleotides, <200 nucleotides, <100 nucleotides or <50 nucleotides. In some embodiments, the fifth nucleotide sequence comprises fewer than 25 nucleotides, e.g. one of <20 nucleotides, <15 nucleotides, <10 nucleotides or <5 nucleotides. In some embodiments, the fifth nucleotide sequence comprises 23 nucleotides. In some embodiments, the fifth nucleotide sequence comprises 2 nucleotides. P37829 In some embodiments, the fifth nucleotide sequence comprises 11 nucleotides. In some embodiments, the fifth nucleotide sequence comprises 8 nucleotides. In some embodiments, the fifth nucleotide sequence comprises 5 nucleotides. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥95% or 100%) sequence identity to positions 1 to 23 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises insertion of ‘G’ after the position corresponding to position 2 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. ≥90% or 100%) sequence identity to positions 1 to 11 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.100%) sequence identity to positions 1 to 8 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g.100%) sequence identity to positions 1 to 5 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises, or consists of, positions 1 and 2 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises ‘A’ at the position corresponding to position 1 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises insertion of ‘T’ after the position corresponding to position 1 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises ‘G’ at the position corresponding to position 3 of SEQ ID NO:32. In some embodiments, the fifth nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:19; and (ii) comprises ‘GA’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:32. An example of such a fifth nucleotide sequence is SEQ ID NO:20. In some embodiments, the fifth nucleotide sequence comprises, or consists of: (i) a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:19; and (ii) comprises ‘TG’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:32. An example of such a fifth nucleotide sequence is SEQ ID NO:21. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of: the dinucleotide ‘TG’, the dinucleotide ‘GA’, the dinucleotide ‘TT’, the trinucleotide ‘GAG’, the nucleotide ‘A’, or a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:20, 21 and 371 to 374. P37829 In some embodiments, a polynucleotide according to the present disclosure does not comprise the nucleotide sequence of SEQ ID NO:20. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of: the dinucleotide ‘TG’, the dinucleotide ‘GA’, the dinucleotide ‘TT’, the trinucleotide ‘GAG’, the nucleotide ‘A’, or a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from SEQ ID NOs:21 and 371 to 374. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of the dinucleotide ‘TG’. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of the dinucleotide ‘GA’. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of the dinucleotide ‘TT’. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of the trinucleotide ‘GAG’. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:20. In some embodiments, a polynucleotide according to the present disclosure comprises a nucleotide sequence (e.g. a fifth nucleotide sequence) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:21. Nucleotide sequences encoding a polypeptide of interest In some embodiments, the fifth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a polypeptide of interest. Where present, the sixth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a polypeptide of interest. A polypeptide of interest may be any polypeptide. In some embodiments, a polypeptide of interest according to the present disclosure may be an antigen- binding polypeptide, an aptamer, an antigen-binding polypeptide complex, an antibody or an antigen- binding fragment or derivative thereof, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, a decoy receptor for a ligand, a decoy ligand for a receptor, an enzyme, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, a transcription factor, an epigenetic modifier, a constituent protein of a site-specific nuclease nucleic acid editing system (e.g. a CRISPR/Cas9 system, a CRISPR/Cpf1 system, a CRISPR/C2c1 system, a CRISPR/C2c2 system, a CRISPR/C2c3 system, a ZFN system or a TALEN system), a constituent protein of a ribonucleoprotein, or a viral protein (e.g. a capsid protein or a viral enzyme). P37829 In some embodiments, a polypeptide of interest according to the present disclosure may be an antigen- binding polypeptide or an antigen-binding polypeptide complex. In some embodiments, a polypeptide of interest may be a chimeric antigen-receptor (CAR). In some embodiments, a polypeptide of interest is a polypeptide suitable for use in therapy or prophylaxis of a disease/condition. In some embodiments, a polypeptide of interest is a detectable polypeptide or a polypeptide having detectable activity. A polypeptide suitable for use in therapy or prophylaxis of a disease/condition may be any polypeptide whose administration is useful for the treatment or prevention of a disease/condition. In some embodiments, a polypeptide suitable for use in therapy or prophylaxis of a disease/condition may be a polypeptide for which deficiency thereof is positively associated with, or implicated in the pathology of, a disease or condition. By way of illustration, in some embodiments, the polypeptide of interest may be MeCP2. Deficiency of MeCP2 is associated with Rett syndrome. In some embodiments, a polypeptide suitable for use in therapy or prophylaxis of a disease/condition may be a polypeptide which inhibits the expression and/or activity of a factor whose expression or activity is positively associated with, or implicated in the pathology of, a disease or condition. A detectable polypeptide may be or comprise a fluorescent polypeptide. Fluorescent polypeptides include green fluorescent protein and variants thereof (e.g. enhanced green fluorescent protein), yellow fluorescent protein (e.g. citrine), red fluorescent protein and variants thereof (e.g. mOrange, mCherry), blue fluorescent protein and variants thereof (e.g. TagBFP), cyan fluorescent protein and variants thereof (e.g. mTurquoise, cerulean), allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin. A detectable polypeptide may be or comprise an epitope tag. Epitope tags include e.g. His, (e.g.6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol). A polypeptide having detectable activity may be or comprise an enzymatic moiety. Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. beta-galactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases. A polypeptide of interest expressed from a polynucleotide of the present disclosure may additionally comprises one or more extraneous amino acids added at the N-terminus of the polypeptide, i.e. immediately upstream of the amino acid sequence of the polypeptide of interest. Such extraneous amino acids may be characterised as forming an N-terminal tag on the polypeptide of interest. It may be desirable to minimise the size of, or completely remove, such extraneous amino acids/N-terminal tags on the polypeptide of interest. In some embodiments, the N-terminal tag consists of fewer than 50 amino acids, e.g. one of ≤40 amino acids, ≤30 amino acids, ≤25 amino acids, ≤20 amino acids, ≤15 amino acids, ≤10 amino acids, ≤9 amino acids, ≤8 amino acids, ≤7 amino acids, ≤6 amino acids, ≤5 amino acids, ≤4 amino acids, ≤3 amino acids, ≤2 amino acids or 1 amino acid. In some embodiments, the polypeptide of interest lacks an N-terminal tag. In some embodiments, the polypeptide of the present disclosure comprises one or more cleavage sites. A cleavage site refers to a sequence of amino acids that acts as a substrate for an enzyme capable of cleaving peptide bonds. Many such cleavage sites are known to, and can be employed by, the person skilled in the art of molecular biology. In some embodiments, the cleavage sequence comprises an autocleavage site. Autocleavage sites include the 2A cleavage sequence from Picornavirus ‘NPGP’, which is cleaved at ’G/P’. Further autocleavage sites are described e.g. in Kim et al., PLoS ONE (2011) 6: e18556 (hereby incorporated by reference in its entirety), and include e.g. T2A, E2A, P2A and F2A cleavage sites. The amino acid sequences of T2A, E2A, P2A and F2A cleavage sites are shown in SEQ ID NOs: 107, 108, 109 and 110, respectively. A cleavage site may be included in a polypeptide according to the present disclosure to provide for removal of extraneous amino acids added at the N-terminus of the polypeptide, i.e. immediately upstream of the amino acid sequence of the polypeptide of interest. That is, a cleavage site may be included for the removal of an N-terminal tag as described hereinabove. Accordingly, in some embodiments, a polypeptide according to the present disclosure comprises a cleavage site adjacent to (i.e. in the amino acid sequence of the polypeptide, e.g. immediately downstream of) extraneous amino acid(s), e.g. extraneous amino acid(s) forming an N-terminal tag. In some embodiments, a cleavage site according to the present disclosure is a 2A cleavage site, e.g. selected from a T2A, E2A, P2A and F2A cleavage site. In some embodiments, the cleavage site is a T2A cleavage site. In some embodiments, a polypeptide according to the present disclosure comprises an amino acid sequence having at least 60%, preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:107, 108, 109 or 110. In some embodiments, a nucleotide sequence encoding a polypeptide of interest according to the present disclosure comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:213 or 217. Further nucleotide sequences of the polynucleotide Polynucleotides according to the present disclosure may comprise additional nucleotide sequences and/or sequence features in addition to the first, second, third, fourth, fifth and/or sixth nucleotide sequences as described hereinabove. The polynucleotides of the present disclosure comprise a start codon 5’ to (i.e. upstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a polypeptide of interest. The start codon is preferably the trinucleotide ‘ATG’. In some embodiments, a start codon is provided in the polynucleotide such that following splicing (e.g. where the polynucleotide is a polyribonucleotide, or splicing of a polyribonucleotide transcribed from the polynucleotide where the polynucleotide is a polydeoxyribonucleotide), the start codon is provided in the mature mRNA molecule in such a way as to serve as the initiator codon for translation of the polypeptide of interest (encoded by the fifth or sixth nucleotide sequence). In some embodiments, the splicing referred to in the preceding paragraph is splicing performed in the presence of a splicing modifier (e.g. as described herein). By way of illustration, splicing of the polyribonucleotide having the nucleotide sequence of SEQ ID NO:22 in the presence of RG7800/RG76196 results in the production of a mature mRNA molecule comprising an initiator codon for translation of a polypeptide of interest. In some embodiments, the splicing referred to in the preceding paragraph is splicing performed in the absence of a splicing modifier (e.g. as described herein). By way of illustration, splicing of the polyribonucleotide having the nucleotide sequence of SEQ ID NO:28 in the absence of a splicing modifier results in the production of a mature mRNA molecule comprising an initiator codon for translation of a polypeptide of interest. In some embodiments, the polynucleotide further comprises a Kozak sequence. In preferred embodiments, the Kozak sequence is provided immediately upstream of the start codon for initiating translation of the polypeptide of interest. In some embodiments, the Kozak sequence consists of a nucleotide sequence conforming to the consensus of SEQ ID NO:33. In some embodiments, the Kozak sequence consists of SEQ ID NO:34. In some embodiments, the polynucleotide comprises a nucleotide sequence consisting of SEQ ID NO:35 5’ to the nucleotide sequence encoding a polypeptide of interest. In some embodiments, SEQ ID NO:35 is provided in the polynucleotide such that following splicing (e.g. where the polynucleotide is a polyribonucleotide, or splicing of a polyribonucleotide transcribed from the polynucleotide where the polynucleotide is a polydeoxyribonucleotide), the start codon of SEQ ID NO:35 is provided in the mature mRNA molecule in such a way as to serve as the initiator codon for translation of the polypeptide of interest (encoded by the fifth or sixth nucleotide sequence). In some embodiments, the splicing referred to in the preceding paragraph is splicing performed in the presence of a splicing modifier (e.g. as described herein). In some embodiments, the splicing referred to in the preceding paragraph is splicing performed in the absence of a splicing modifier (e.g. as described herein). In some embodiments, the polynucleotide further comprises a promoter sequence. The promoter sequence is preferably 5’ to the first nucleotide sequence. In some embodiments, the polynucleotide P37829 further comprises one or more enhancer sequences. The one or more enhancer sequences are preferably 5’ to the first nucleotide sequence. In some embodiments, the polynucleotide further comprises a stop codon. The stop codon is preferably provided immediately 3’ to (i.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the trinucleotide encoding the terminal amino acid of the polypeptide encoded by the fifth or sixth nucleotide sequence. In some embodiments, the polynucleotide further comprises a polyadenylation signal sequence. In preferred embodiments, the polyadenylation signal sequence is provided 3’ to (i.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a polypeptide of interest. In some embodiments, the polynucleotide further comprises a terminator sequence. The terminator sequence is preferably 3’ to the nucleotide sequence encoding a polypeptide of interest (and 3’ to the polyadenylation signal sequence, when present). In preferred embodiments, the constituent nucleotide sequences of the polynucleotides (i.e. the first, second, third, fourth, fifth and/or sixth nucleotide sequences as described hereinabove) are provided immediately adjacent to one another. However, in some embodiments, the polynucleotide further comprises one or more linker nucleotide sequences between one or more of the constituent nucleotide sequences of the polynucleotide. Linker nucleotide sequences may comprise, or consist of, 1-10, e.g. one of 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1- 3, or 1-2 nucleotides. Where a polynucleotide according to the present disclosure comprises one more linker nucleotide sequences (i.e. provided between the first, second, third, fourth, fifth and/or sixth nucleotide sequences), the linker sequences are preferably selected such that they do not substantially affect post-transcriptional processing of the polynucleotide when the polynucleotide is a polyribonucleotide. In preferred embodiments, where a polynucleotide according to the present disclosure comprises one more linker sequences, splicing of the polynucleotide when the polynucleotide is a polyribonucleotide is substantially the same as splicing of an equivalent polyribonucleotide lacking the linker nucleotide sequence(s). Similarly, where a polynucleotide according to the present disclosure comprises one more linker nucleotide sequences (i.e. provided between the first, second, third, fourth, fifth and/or sixth nucleotide sequences), the linker sequences are preferably selected such that they do not alter the amino acid sequence of a polypeptide encoded by the polynucleotide. In preferred embodiments, where a polynucleotide according to the present disclosure comprises one more linker sequences, the polynucleotide encodes the same polypeptide as the equivalent polyribonucleotide lacking the linker nucleotide sequence(s). In some embodiments, the polynucleotide further comprises inverted terminal repeat (ITR) sequences. In some embodiments, the polynucleotide comprises an ITR 5’ to the first nucleotide sequence (and 5’ to the P37829 promoter and/or enhancer sequences, when present). In some embodiments, the polynucleotide comprises an ITR 3’ to the nucleotide sequence encoding a polypeptide of interest (and 3’ to the stop codon, polyadenylation signal sequence and/or terminator sequence, when present). In some embodiments, the polynucleotide comprises an ITR sequence at its 5’ end, and an ITR sequences at its 3’ end. In some embodiments, the first nucleotide of the ITR sequence provided at the 5’ end of the polynucleotide is provided within 1 to 25 nucleotides, e.g. within one of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides of the first nucleotide of the polynucleotide (i.e. position 1 of the nucleotide sequence of the polynucleotide). In some embodiments, the final nucleotide of the ITR sequence provided at the 3’ end of the polynucleotide is provided within 1 to 25 nucleotides, e.g. within one of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides of the final nucleotide of the polynucleotide (i.e. the terminal position of the nucleotide sequence of the polynucleotide). In some embodiments, the polynucleotide of the present disclosure (i.e. comprising the first, second, third, fourth, fifth and/or sixth nucleotide sequences described herein) has a size permitting its delivery as a gene therapy, i.e. in a suitable vector. In some embodiments, the polynucleotide consists of a nucleotide sequence having a size within the packaging limit of a vector for delivering the polynucleotide. In some embodiments, the polynucleotide has a size within the packaging limit of an AAV vector. In some embodiments, the polynucleotide has a size within the packaging limit of an AAV vector of one of the following serotypes: AAV9, AAV9.45, AAV-PHP.B, AAV1, AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV10 or AAVrh74. In some embodiments, the polynucleotide has a size within the packaging limit of an AAV vector of one of the following serotypes: AAV9, AAV9.45, AAV-PHP.B, AAV1, AAV2, AAV2.7m8, AAV2i8, AAV5, AAV6, AAV8, AAV10 or AAVrh74. In some embodiments, the polynucleotide of the present disclosure (i.e. comprising the first, second, third, fourth, fifth and/or sixth nucleotide sequences described herein) consists of fewer than 6,000 nucleotides, e.g. one of ≤5,000, ≤4,750, ≤4,500, ≤4,250, ≤4,000, ≤3,750, ≤3,500, ≤3,250, ≤3,000, ≤2,750, ≤2,500, ≤2,250, ≤2,000, ≤1,750, ≤1,500, ≤1,250 or ≤1,000 nucleotides. In preferred embodiments, the polynucleotide of the present disclosure consists of fewer nucleotides than the number of nucleotides of a known SMN2 exon 6 to exon 8-derived transgene expression system (e.g. an SMN2 exon 6 to exon 8-derived transgene expression system described in Zhang, et al., Gene Ther. (2001) 8: 1532-1538, WO 2022/204471 A1, Monteys et al. Nature (2021) 596: 291-295 or WO 2021/163556 A1). In some embodiments, the polynucleotide of the present disclosure consists of fewer nucleotides than a polynucleotide comprising: (i) SEQ ID NO:36 and (ii) a nucleotide sequence encoding a polypeptide of interest (i.e. adjacent and 3’ to SEQ ID NO:36, the context of the complete sequence of the polynucleotide). Such polynucleotides are described e.g. in WO 2021/163556 A1. P37829 Herein, for conciseness, ‘a polynucleotide comprising: (i) SEQ ID NO:36 and (ii) a nucleotide sequence encoding a polypeptide of interest (i.e. adjacent and 3’ to SEQ ID NO:36, in the context of the complete sequence of the polynucleotide)’ is also referred to herein simply as ‘a SMN2ind minigene polynucleotide’. In some embodiments, the polynucleotide of the present disclosure consists of a number of nucleotides that is less than 1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times or ≤0.25 times the number of nucleotides of a SMN2ind minigene polynucleotide. In some embodiments, the region of a polynucleotide of the present disclosure formed by the first, second, third, fourth and fifth nucleotide sequences consists of fewer nucleotides than the number of nucleotides in SEQ ID NO:36. In some embodiments, the region of a polynucleotide of the present disclosure formed by the first, second, third, fourth and fifth nucleotide sequences consists of a number of nucleotides that is less than 1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times or ≤0.25 times the number of nucleotides in SEQ ID NO:36. The polynucleotide of the present disclosure may comprise 5’ cap, 5’ UTR, 3’ UTR and/or PolyA tail nucleotide sequences. In some embodiments, the polynucleotide comprises a 5’ UTR 5’ to (i.e. upstream of, in the context of the nucleotide sequence of the polynucleotide) a start codon. In some embodiments, the polynucleotide comprises a 3’ UTR 3’ to (i.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) a stop codon. In some embodiments, the polynucleotide comprises a 3’ UTR 5’ to a polyadenylation signal sequence. In some embodiments, the polynucleotide comprises a 3’ UTR 3’ to a stop codon and 5’ to a polyadenylation signal sequence. In some embodiments, the polynucleotide of the present disclosure comprises one or more nucleotide sequences encoding a selectable marker, to facilitate identification and/or selection of cells comprising/expressing the polynucleotide. Selectable markers include proteins that confer resistance to antibiotics or other toxins, e.g., blasticidin, ampicillin, neomycin, methotrexate, or tetracycline, and proteins that complement auxotrophic deficiencies. In some embodiments, the polynucleotide of the present disclosure comprises a nucleotide sequence encoding an internal ribosome entry site (IRES). In some embodiments, the polynucleotide comprises a nucleotide sequence permitting two or more polypeptides to be translated separately from a single polyribonucleotide. The polynucleotides of the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material. P37829 Particular exemplary polynucleotides In some embodiments, a polynucleotide according to the present disclosure comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from Column A of Table A; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from Column B of Table A; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from Column C of Table A; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from Column D of Table A; and a fifth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence selected from Column E of Table A. In accordance with the preceding paragraph, in some embodiments the nucleotide sequence selected from Column A of Table A, and the nucleotide sequence selected from Column B of Table A, and the nucleotide sequence selected from Column C of Table A, and the nucleotide sequence selected from Column B of Table A, and the nucleotide sequence selected from Column D of Table A, and the nucleotide sequence selected from Column B of Table A, and the nucleotide sequence selected from Column E of Table A are all selected form the same row of Table A. By way of illustration, in some embodiments the nucleotide sequences selected from Columns A, B, C, D and E of Table A may be selected from row 3 of Table 1, and therefore may be SEQ ID NOs:5, 91418 and 20, respectively. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and comprising ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) consisting of ≤45 nucleotides; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 300 nucleotides; a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) P37829 sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consisting of fewer than 255 nucleotides; and a fifth nucleotide sequence consisting of the dinucleotide ‘GA’. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:106; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:13; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:17; and a fifth nucleotide sequence consisting of the dinucleotide ‘GA’. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:22. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:3; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:13; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:17; and P37829 a fifth nucleotide sequence consisting of the dinucleotide ‘GA’. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:23. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and comprising ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) comprising ‘CTG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 500 nucleotides; a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; and (iv) comprising insertion of ‘GCCACC’ after the position corresponding to position 6 of SEQ ID NO:12; and (v) comprising ‘TG’ at the positions corresponding to positions 8 and 9 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consisting of fewer than 500 nucleotides; and a fifth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:20. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:5; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:9; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:14; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:18; and a fifth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:20. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:24. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and comprising ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) comprising ‘CAG’ at the positions corresponding to positions 109 to 111 of SEQ ID NO:2; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 500 nucleotides; a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consisting of fewer than 500 nucleotides; and a fifth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:21. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:6; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:9; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:13; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:18; and a fifth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:20. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:25. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and comprising ‘GGC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) consisting of ≤45 nucleotides; In some embodiments, the polynucleotide comprises: a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 300 nucleotides; a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consisting of fewer than 255 nucleotides; and P37829 a fifth nucleotide sequence consisting of the trinucleotide ‘GAG’. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:3; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:13; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:17; and a fifth nucleotide sequence consisting of the trinucleotide ‘GAG’. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:28. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:1; and comprising ‘GCC’ at the positions corresponding to positions 82 to 84 of SEQ ID NO:2; and (iii) consisting of ≤45 nucleotides; In some embodiments, the polynucleotide comprises: a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 300 nucleotides; a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising deletion of the position corresponding to position 20 of SEQ ID NO:12; and (iv) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% P37829 (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consisting of fewer than 255 nucleotides; and a fifth nucleotide sequence consisting of the trinucleotide ‘GAG’. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:3; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:27; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:17; and a fifth nucleotide sequence consisting of the trinucleotide ‘GAG’. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:29. In some embodiments, the polynucleotide does not consist of, or does not comprise, SEQ ID NO:36. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to one of SEQ ID NOs:37 to 105. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221; and (ii) consisting of ≤45 nucleotides; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 300 nucleotides; a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) P37829 sequence identity to a nucleotide sequence according to SEQ ID NO:11; and (ii) comprising ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; and (iii) comprising insertion of ‘A’ after the position corresponding to position 48 of SEQ ID NO:12; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consisting of fewer than 255 nucleotides; and a fifth nucleotide sequence consisting of the dinucleotide ‘TG’. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:219; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:13; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:17; and a fifth nucleotide sequence consisting of the dinucleotide ‘TG’. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:111. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221; and (ii) consisting of ≤45 nucleotides; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 300 nucleotides; P37829 a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:225; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:15 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:16 at its 3’ end; and (iii) consisting of fewer than 255 nucleotides; and a fifth nucleotide sequence consisting of the dinucleotide ‘TG’. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:219; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:223; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:17; and a fifth nucleotide sequence consisting of the dinucleotide ‘TG’. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:116. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221; and (ii) consisting of ≤45 nucleotides; a second nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:7 at its 5’ end; and (ii) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:8 at its 3’ end; and (iii) comprising ‘C’ at the position corresponding to position 5766 of SEQ ID NO:30; and (iv) consisting of fewer than 300 nucleotides; P37829 a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:225; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:227; and (iii) consisting of fewer than 280 nucleotides; and a fifth nucleotide sequence consisting of the dinucleotide ‘TG’. In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:220; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:10; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:224; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:227; and a fifth nucleotide sequence consisting of the dinucleotide ‘TG’. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:164. In some embodiments, the polynucleotide comprises: a first nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:221; and (ii) consisting of ≤45 nucleotides; a second nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:229; a third nucleotide sequence (i) comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to a nucleotide sequence according to SEQ ID NO:225; a fourth nucleotide sequence (i) comprising a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:227; and (iii) consisting of fewer than 280 nucleotides; and a fifth nucleotide sequence consisting of the dinucleotide ‘TG’. P37829 In some embodiments, the polynucleotide comprises: a first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:220; a second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:228; a third nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:224; a fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:227; and a fifth nucleotide sequence consisting of the dinucleotide ‘TG’. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:171. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to one of SEQ ID NOs:111 to 200. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to one of SEQ ID NOs:37 to 105, or 111 to 200. Splicing modifiers Aspects and embodiments of the present disclosure pertain to splicing modifiers. Splicing modifiers are molecules that influence splicing of polyribonucleotides. Such molecules are reviewed e.g. in Tang et al., Molecules (2021) 26(8):2263 and Schneider-Poetsch et al., The Journal of Antibiotics (2021) 74:603-616, both of which are hereby incorporated by reference in their entirety. Splicing modifiers generally reduce/prevent association of factors required for normal post-transcriptional processing (e.g. components of the spliceosome) with RNA. Splicing modifiers typically bind to a nucleotide sequence of a polyribonucleotide and either inhibit or promote the association of RNA-binding proteins and/or non-coding RNAs which function as splicing activators or repressors. Through competitive inhibition of the recruitment of splicing activators and splicing repressors to the polyribonucleotide, splicing modifiers can alter the equilibrium of the splicing-regulatory RNA structures, and thus promote an increase or decrease in skipping or inclusion of an exon and/or retention or excision of an intron in the mature RNA molecule produced by splicing. P37829 In some embodiments, a splicing modifier is a small molecule or a splice-switching nucleic acid (e.g. a splice-switching oligonucleotide). A ‘small molecule’ refers to a low molecular weight (<1000 daltons, typically about 300 to about 700 daltons) organic compound. Splice-switching nucleic acids are reviewed e.g. in Haves and Hastings, Nucleic Acids Res. (2016) 44(14): 6549–6563, which is hereby incorporated by reference in its entirety. Splice-switching nucleic acids include e.g. splice-switching oligonucleotides (SSOs). They disrupt the normal splicing of target RNA transcripts by blocking the RNA:RNA base-pairing and/or protein:RNA binding interactions that occur between components of the splicing machinery and pre-mRNA. Splice-switching nucleic acids may be designed to target a specific region of the target transcript, e.g. to promote skipping of exon(s) of interest and/or to promote inclusion of exon(s) of interest and/or to promote retention of introns of interest and/or to promote excision of introns of interest. SSOs often comprise alterations to oligonucleotide sugar-phosphate backbones in order to reduce/prevent RNAseh degradation, such as e.g. phosphorothioate linkages, phosphorodiamidate linkages such as phosphorodiamidate morpholino (PMOs), and may comprise e.g. peptide nucleic acids (PNAs), locked nucleic acids (LNAs), methoxyethyl nucleotide modifications, e.g.2′O-methyl (2′OMe) and 2′-O-methoxyethyl (MOE) ribose modifications and/or 5’-methylcytosine modifications. Small molecule splicing modifiers contemplated in accordance with the present disclosure include RG- 7800 and RG-7916 (also known as risdiplam), and analogs thereof. RG-7800 and RG-7916 bind to the 5’ splice site of intron 7 and exonic splicing enhancer 2 of exon 7 of human SMN2, thereby stabilising the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 snRNP complex, and promoting inclusion of exon 7 in the mature RNA molecule obtained following splicing of pre-mRNA transcribed from SMN2. RG-7800 and RG-7916 are useful to restore functional SMN2 protein expression from SMN2 alleles comprising the spinal muscular atrophy (SMA)-associated polymorphism c.840C>T (which potentiates skipping of exon 7). RG-7916 (DrugBank Acc. No. DB15305) is a close structural analog of RG-7800 having improved potency, pharmacokinetics and safety profile than RG-7800, and is approved by the FDA for the treatment of SMA. Further analogs of RG-7800 and RG-7916 having similar splicing modifier activity include SMN-C2, SMN-C3, SMN-C5 and TEC-1. Branaplam (DrugBank Acc. No. DB14918; also known as LMI-070) is another small molecule splicing modifier that promotes inclusion of exon 7 in the mature RNA expressed from SMN2. Like RG-7800 and RG-7916, branaplam binds to the 5’ splice site of intron 7 of human SMN2. PK4C9 (also known as homocarbonyltopsentin) is another small molecule splicing modifier that promotes inclusion of exon 7 in the mature RNA expressed from SMN2. It is thought to bind to the 5’ splice site of exon 7 and TSL2, and improve accessibility of the 5′ splice site via stabilising a triloop structure of TSL2. Small molecule splicing modifiers that promote inclusion of exon 7 in the mature RNA expressed from SMN2 are described e.g. in WO 2015/173181 A1 and WO 2009/151546 A2, which are hereby P37829 incorporated by reference in their entirety. Further small molecule splicing modifiers that promote inclusion of exon 7 in the mature RNA expressed from SMN2 are described e.g. in WO 2022/204471 A1, which is hereby incorporated by reference in its entirety. Nusinersen (DrugBank Acc. No. DB13161) is a splice-switching oligonucleotide that promotes exon 7 retention in mature RNA expressed from human SMN2. Nusinersen is an 18-mer 2’-MOe phosphorothioate antisense oligonucleotide that hybridises to intronic splicing silencer site 1 of intron 7, occupying the site and thereby inhibiting the association of the splicing suppressor ribonucleoproteins hnRNPs A1/A2, thus promoting inclusion of exon 7 in the mature RNA molecule. The splicing modifier according to the present disclosure preferably promotes inclusion of SMN2 exon 7 in RNA obtained following splicing of pre-mRNA transcribed from human SMN2. Such molecules increase the proportion of RNA molecules comprising SMN2 exon 7 among RNA molecules obtained following splicing of pre-mRNA transcribed from human SMN2 (i.e. relative to the proportion obtained in the absence of the splicing modifier). For conciseness, ‘a splicing modifier that promotes inclusion of SMN2 exon 7 in RNA obtained following splicing of pre-mRNA transcribed from human SMN2’ may be referred to herein simply as ‘a splicing modifier that promotes SMN2 exon 7 inclusion’ In some embodiments, the splicing modifier promotes inclusion of exon 7 in RNA obtained following splicing of pre-mRNA transcribed from an allele of human SMN2 comprising c.840C>T. That is, in some embodiments, the splicing modifier increases the proportion of RNA molecules comprising SMN2 exon 7 among RNA molecules obtained following splicing of pre-mRNA transcribed from an allele of human SMN2 comprising c.840C>T (i.e. relative to the proportion obtained in the absence of the splicing modifier). In some embodiments, the splicing modifier increases the level of human SMN2 protein comprising amino acids encoded by exon 7 of human SMN2 (i.e. relative to the level detected in the absence of the splicing modifier). In some embodiments, the splicing modifier increases the proportion of polypeptides comprising amino acids encoded by exon 7 of human SMN2 among polypeptides expressed from human SMN2 (i.e. relative to the proportion obtained in the absence of the splicing modifier). In some embodiments, the splicing modifier increases the proportion of polypeptides comprising amino acids encoded by exon 7 of human SMN2 among polypeptides expressed from an allele of human SMN2 comprising c.840C>T (i.e. relative to the proportion obtained in the absence of the splicing modifier). In some embodiments, the splicing modifier promotes inclusion of the third nucleotide sequence in the product of splicing of a polynucleotide according to the present disclosure (i.e. where the polynucleotide is a polyribonucleotide). In some embodiments, the splicing modifier increases the proportion of molecules comprising the third nucleotide sequence among molecules obtained following splicing of a polynucleotide according to the present disclosure (i.e. where the polynucleotide is a polyribonucleotide). In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:13 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence P37829 of SEQ ID NO:22. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:13 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:22. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:13 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:23. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:13 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:23. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:14 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:24. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:14 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:24. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:13 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:25. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:13 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:25. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:13 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:28. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:13 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:28. In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:27 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:29. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:27 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:29. Splicing modifiers having such functional properties can be identified by analysis e.g. in suitable in vitro assays. Such assays may comprise culturing cells in vitro in the presence or absence of a candidate splicing modifier, and analysing the RNA and/or protein produced after an appropriate period of time for an effect of the candidate splicing modifier to be observed. By way of illustration, cells can be transfected with a vector comprising DNA having the sequence of SEQ ID NO:22 and cultured in the presence or absence of a candidate splicing modifier for a suitable period of time (e.g.24, 48, 72 hours). RNA can subsequently be isolated from the cells, and analysed (e.g. by qRT-PCR) to determine the P37829 level/proportion of mature RNA molecules comprising the nucleotide sequence consisting of SEQ ID NO:13. In some embodiments, a splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound of formula (I) of WO 2015/173181 A1. In some embodiments, a splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound selected from those listed in claim 39 of WO 2015/173181 A1. In some embodiments, a splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound selected from those listed in claim 40 of WO 2015/173181 A1. In some embodiments, the splicing modifier according to the present disclosure is selected from: RG- 7916, RG-7800, SMN-C2, SMN-C3, SMN-C5, TEC-1, branaplam, PK4C9 and nusinersen. In some embodiments, the splicing modifier is selected from: RG-7916, RG-7800, SMN-C2, SMN-C3, SMN-C5 and TEC-1. In some embodiments, the splicing modifier is selected from RG-7916 and RG-7800. In preferred embodiments, the splicing modifier is risdiplam (RG-7916). Functional properties of the polynucleotides In aspects and embodiments of the present disclosure, the polynucleotides of the present disclosure may be characterised by reference to one or more functional properties. In some embodiments, where the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. In some embodiments, where the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides comprising the third nucleotide sequence. In some embodiments, in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, inclusion of the third nucleotide sequence is promoted in splicing of the polyribonucleotide, favouring the production of mature RNA molecules encoding the polypeptide of interest, and thus expression of the polypeptide of interest at the protein level. Such polynucleotides may be referred to herein as ‘ON-switch’ polynucleotides. In some embodiments, cells comprising an ON-switch polynucleotide of the present disclosure substantially do not express the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, cells comprising the ON-switch polynucleotide express the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion. Exemplary ON-switch polynucleotides according to the present disclosure include polynucleotides comprising SEQ ID NO:22, 23, 24 or 25. Further exemplary ON-switch polynucleotides according to the present disclosure include polynucleotides comprising one of SEQ ID NOs:37 to 105. P37829 Inclusion of the third nucleotide sequence as described herein in the products of splicing of a polyribonucleotide according to the present disclosure can be evaluated using methods that are well known to the person skilled in the art. Such methods include qRT-PCR based methods for the detection and/or quantification of RNA molecules comprising nucleotide sequences of interest. For example, cells may be transduced in vitro with a vector comprising a polynucleotide according to the present disclosure and subsequently cultured in vitro in the presence or absence of a splicing modifier that promotes SMN2 exon 7 inclusion for a period of time appropriate for an effect on splicing of a polyribonucleotide according to the present disclosure to be observed. After such period of time, total RNA may be isolated from the cells, cDNA may be prepared from the total RNA, and the number/proportion of mature RNA molecules comprising the third nucleotide sequence may be evaluated by qPCR using oligonucleotides providing for the specific amplification and/or detection of the products of splicing of the polyribonucleotide (e.g. oligonucleotides hybridising to nucleotide sequences spanning exon:exon boundaries). The qPCR analysis may employ oligonucleotides enabling distinction between RNA molecules comprising the third nucleotide sequence, and RNA molecules lacking the third nucleotide sequence. Such qRT-PCR-based methods for analysing the products of splicing of a polyribonucleotide are described in the experimental examples of the present disclosure. Expression of a polypeptide of interest can be evaluated using any suitable technique for the detection and/or quantification of the relevant polypeptide. Such techniques include e.g. antibody-based methods, (for example flow cytometry, immunocytochemistry, western blot, ELISA), fluorescence microscopy and flow cytometry. In some embodiments, expression of a polypeptide of interest can be evaluated as described in the experimental examples of the present disclosure. In preferred embodiments, expression of a polypeptide of interest may be evaluated by flow cytometry. Herein, within a plurality/population of polyribonucleotides obtained after splicing that ‘substantially lack’ the third nucleotide sequence, the third nucleotide sequence may be present in fewer than 15%, e.g. one of ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of the polyribonucleotides. Conversely, within a plurality/population of polyribonucleotides obtained after splicing that ‘comprise’ the third nucleotide sequence, the third nucleotide sequence may be present in more than 80%, e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% of the polyribonucleotides. Herein, within a plurality/population of cells that ‘substantially do not express’ a polypeptide of interest, the polypeptide may be expressed by fewer than 15%, e.g. one of ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of the cells. Conversely, within a plurality/population of cells that ‘express’ a polypeptide of interest, the polypeptide may be expressed by more than 80%, e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% of the cells. In some embodiments, cells that ‘substantially do not express’ a polypeptide of interest may display a level of expression of the polypeptide of interest which is less than 0.2 times, e.g. one of ≤0.1 times, ≤0.09 times, ≤0.08 times, ≤0.07 times, ≤0.06 times, ≤0.05 times, ≤0.04 times, ≤0.03 times, ≤0.02 times, or ≤0.01 times the level of expression by cells that express the polypeptide of interest. In some P37829 embodiments, cells that ‘express’ a polypeptide of interest may display a level of expression of the polypeptide of interest which is greater than 5 times, e.g. one of ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times or ≥10000 times level of expression by cells that ‘substantially do not express’ the polypeptide of interest. In some embodiments, the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 in cells comprising a polynucleotide according to the present disclosure following culture in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times or ≥10000 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 in cells comprising a polynucleotide according to the present disclosure following culture in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, e.g. one of ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times or ≤1 times the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 in equivalent cells not comprising the polynucleotide. In some embodiments, the level of a polypeptide of interest in cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times or ≥10000 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the level of expression of a polypeptide of interest by cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, e.g. one of ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times or ≤1 times the level of the polypeptide of interest in equivalent cells not comprising the polynucleotide. In some embodiments, the proportion of cells expressing a polypeptide of interest within a population of cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times or ≥10000 times the proportion of such cells expressing the polypeptide of interest cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the proportion of cells expressing a polypeptide of interest within a population of cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 P37829 times, e.g. one of ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times or ≤1 times the proportion of such cells expressing the polypeptide of interest cultured in the presence of the splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, inclusion of the third nucleotide sequence is promoted in splicing of the polyribonucleotide, favouring the production of mature RNA molecules encoding a premature stop codon, and thus preventing expression of the polypeptide of interest at the protein level. Such polynucleotides may be referred to herein as ‘OFF- switch’ polynucleotides. In some embodiments, cells comprising an OFF-switch polynucleotide of the present disclosure express the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, cells comprising the OFF-switch polynucleotide do not express the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion. Exemplary OFF-switch polynucleotides according to the present disclosure include polynucleotides comprising SEQ ID NO:28 or 29. In some embodiments, the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26 in cells comprising a polynucleotide according to the present disclosure following culture in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times or ≥10000 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26 in cells comprising a polynucleotide according to the present disclosure following culture in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, e.g. one of ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times or ≤1 times the level of the polypeptide of interest in equivalent cells not comprising the polynucleotide. In some embodiments, the level of a polypeptide of interest in cells comprising a polynucleotide according to the present disclosure (e.g. an OFF-switch polynucleotide) in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times or ≥10000 times the level in equivalent cells cultured in the presence of the splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the level of expression of a polypeptide of interest by cells comprising a polynucleotide according to the present disclosure (e.g. an OFF-switch polynucleotide) in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, e.g. one of ≤50 times, P37829 ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times or ≤1 times the level of the polypeptide of interest in equivalent cells not comprising the polynucleotide. In some embodiments, the proportion of cells expressing a polypeptide of interest within a population of cells comprising a polynucleotide according to the present disclosure (e.g. an OFF-switch polynucleotide) cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times or ≥10000 times the proportion of such cells expressing the polypeptide of interest cultured in the presence of the splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the proportion of cells expressing a polypeptide of interest within a population of cells comprising a polynucleotide according to the present disclosure (e.g. an OFF-switch polynucleotide) cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, e.g. one of ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times or ≤1 times the proportion of such cells expressing the polypeptide of interest cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion. The polynucleotides of the present disclosure possess novel and/or improved properties relative to known transgene expression systems, e.g. known transgene expression systems comprising a SMSM-mediated switch derived from SMN2 exons 6 to 8 (e.g. SMN2 exon 6 to exon 8-derived transgene expression systems described in Zhang, et al., Gene Ther. (2001) 8: 1532-1538, WO 2022/204471 A1, Monteys et al. Nature (2021) 596: 291-295 or WO 2021/163556 A1). In some embodiments, the polynucleotides of the present disclosure possess novel and/or improved properties relative to a SMN2ind minigene polynucleotide (which is defined hereinabove). In some embodiments, cells comprising a polynucleotide of the present disclosure cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion produce a population of RNA molecules having an increased proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26, compared to the population of RNA molecules produced by cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. That is, in some embodiments the polynucleotide of the present disclosure is more effective at promoting inclusion of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to a SMN2ind minigene polynucleotide. That is, the polynucleotides of the present disclosure are more responsive to induction of SMN2 exon 7 variant inclusion in the products of splicing in response to a splicing modifier that promotes SMN2 exon 7 inclusion than SMN2ind minigene polynucleotides. In some embodiments, the proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 among RNA molecules obtained from cells comprising a polynucleotide of the present disclosure cultured in the P37829 presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times or ≥10 times the proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 among RNA molecules obtained from cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. It will be appreciated that the functional properties described herein are evaluated employing the same experimental conditions for the evaluation of cells comprising the different polynucleotides (i.e. the polynucleotide of the present disclosure, and the SMN2ind minigene polynucleotide). For example, the same cell type, the same splicing modifier that promotes SMN2 exon 7 inclusion, the same concentration of the splicing modifier that promotes SMN2 exon 7 inclusion, etc. are used, the same culture period is provided, and the cells are analysed in order to determine the proportion of RNA molecules comprising the relevant nucleotide sequence and/or the level of the polypeptide of interest in the same way. In some embodiments, cells comprising a polynucleotide of the present disclosure cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion produce a population of RNA molecules having a decreased proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26, compared to the population of RNA molecules produced by cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. That is, in some embodiments the polynucleotide of the present disclosure is more effective at excluding a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to a SMN2ind minigene polynucleotide. That is, the polynucleotides of the present disclosure are less ‘leaky’ with respect to SMN2 exon 7 variant inclusion in the products of splicing in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to SMN2ind minigene polynucleotides. Leaky production of mature RNA molecules comprising an SMN2 exon 7 variant (i.e. in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion) may also be referred to herein as ‘background’ production of such RNA molecules. In some embodiments, the proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or 26 among RNA molecules obtained from cells comprising a polynucleotide of the present disclosure cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the proportion of RNA molecules comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11 or P37829 26 among RNA molecules obtained from cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. In some embodiments, cells comprising a polynucleotide of the present disclosure (e.g. an ON-switch polynucleotide) cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion display an increased level of protein expression of the polypeptide of interest, compared to the level of protein expression of the polypeptide of interest by cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. That is, in some embodiments the polynucleotide of the present disclosure is more effective at promoting protein expression of the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to a SMN2ind minigene polynucleotide. That is, the polynucleotides of the present disclosure are more responsive to induction of expression of the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to SMN2ind minigene polynucleotides. It will be appreciated that for the purposes of such comparison, the different polynucleotides (i.e. the polynucleotide of the present disclosure, and the SMN2ind minigene polynucleotide) preferably encode the same polypeptide of interest. In some embodiments, the level of protein expression of the polypeptide of interest from cells comprising a polynucleotide of the present disclosure (e.g. an ON-switch polynucleotide) cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, e.g. one of ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times or ≥10 times the level of protein expression of the polypeptide of interest from cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. In some embodiments, cells comprising a polynucleotide of the present disclosure (e.g. an ON-switch polynucleotide) cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion display a decreased level of protein expression of the polypeptide of interest, compared to the level of protein expression of the polypeptide of interest by cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. That is, in some embodiments the polynucleotide of the present disclosure is more effective at preventing protein expression of the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to a SMN2ind minigene polynucleotide. That is, the polynucleotides of the present disclosure are less ‘leaky’ with respect to expression of the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion, compared to SMN2ind minigene polynucleotides. Leaky expression of the polypeptide of interest (i.e. in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion) may also be referred to herein as ‘background’ expression of the polypeptide. In some embodiments, the level of protein expression of the polypeptide of interest from cells comprising a polynucleotide of the present disclosure (e.g. an ON-switch polynucleotide) cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 1 times, e.g. ≤0.99 times, ≤0.95 times, P37829 ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level of protein expression of the polypeptide of interest from cells comprising a SMN2ind minigene polynucleotide and cultured under the same conditions. In some embodiments, a polypeptide of interest expressed from a polynucleotide of the present disclosure comprises fewer extraneous additional amino acids (i.e. amino acids additional to the amino acid sequence of the polypeptide of interest) compared to a polypeptide of interest expressed from SMN2ind minigene polynucleotide. In some embodiments, a polypeptide of interest expressed from a polynucleotide of the present disclosure comprises a smaller N-terminal tag (i.e. formed of extraneous, additional amino acids in addition to the amino acid sequence of the polypeptide of interest) than a polypeptide of interest expressed from SMN2ind minigene polynucleotide. That is, in some embodiments, the polynucleotides of the present disclosure provide for the inducible expression of polypeptides of interest comprising fewer extraneous additional amino acids, relative to polypeptides of interest expressed from SMN2ind minigene polynucleotide. Vectors The present disclosure provides vectors comprising the polynucleotides according to the present disclosure. It will be appreciated that a vector is also a polynucleotide, and so in some embodiments a polynucleotide according to the present disclosure may be a vector. A ‘vector’ as used herein refers to a polynucleotide used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell (i.e. the vector may be an expression vector). Such vectors may include a promoter sequence operably linked to the nucleotide sequence to be expressed. Vectors may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used in a vector according to the present disclosure. The term ‘operably linked’ may include the situation where nucleic acid encoding a polypeptide of interest according to the present disclosure and regulatory nucleic acid sequence(s) (e.g. a promoter and/or enhancers) are covalently linked in such a way as to place the expression of the nucleic acid encoding a polypeptide under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into the desired polypeptide(s). Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno- associated virus vectors, vaccinia virus vectors, baculoviral vectors and herpesvirus vectors), transposon- based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus P37829 et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety. In some embodiments, the vector may be a eukaryotic vector, i.e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression. In some embodiments, the vector comprises a CMV (e.g. mCMV), SV40, RSV or PGK promoter. In some embodiments, the polynucleotide according to the present disclosure (e.g. the vector according to the present disclosure) comprises a CMV promoter, a CAG promoter, a hEF1a promoter, a hUbiC promoter, an RSV promoter, a TK promoter, a PGK promoter, or a CAG minimal promoter. In some embodiments, the polynucleotide according to the present disclosure (e.g. the vector according to the present disclosure) comprises a promoter having a nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% (e.g. one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) sequence identity to SEQ ID NO:201, 202, 203, 204, 205, 206, 207 or 208. In some embodiments, a vector is selected based on tropism for a cell type/tissue/organ to which it is desired to deliver the polynucleotide according to the present disclosure. In some embodiments, a vector is selected based on tropism for a cell type/tissue/organ in which it is desired to express the polypeptide of interest. For example, it may be desired to deliver the polynucleotide to, and/or express the polypeptide of interest in, a cell type/tissue/organ affected by a disease/condition to be treated/prevented in accordance with the present disclosure (e.g. a cell type/tissue/organ in which the symptoms of the disease/condition manifest). For example, it might be desirable to deliver a polynucleotide of the present disclosure encoding a polypeptide of interest to neuronal cells/tissue, and vectors having a tropism for such cells/tissue may be employed in such instances (i.e. neurotropic vectors). In preferred embodiments, the vector is an adeno-associated virus (AAV) vector. Adeno-associated virus vectors and their use to vector gene therapy is reviewed e.g. in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378 and Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272, both of which are hereby incorporated by reference in their entirety. In some embodiments, a vector may be an adeno-associated virus vector described in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378. In some embodiments, a vector may be an adeno-associated virus vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272. In some embodiments, the vector is a self-complementary adeno-associated virus (scAAV) vector. Self- complementary adeno-associated virus vectors are described e.g. in McCarty, Mol Ther. (2008) 16(10):1648-56, which is hereby incorporated by reference in its entirety. Conventional AAV have a P37829 single-stranded DNA genome, and depend on the DNA replication machinery of a transduced cell to synthesise the complementary strand, delaying transgene expression. By contrast, scAAV contain complementary sequences that spontaneously anneal upon infection, eliminating the requirement for DNA synthesis in the transduced host cell. Compared to classical, single-stranded AAV vectors, scAAV vectors have been shown to provide for accelerated onset of transgene expression, and an increased level of transgene expression. In some embodiments, a vector may be an adeno-associated viral vector of one of the following serotypes: AAV9 (including AAV9 variants AAV-PHP.B and AAV9.45), AAV1, AAV2 (including AAV2 variant AAV2i8), AAV5, AAV6, AAV8, AAV10 or AAVrh74. In some embodiments, a vector may be an adeno-associated viral vector of one of the following serotypes: AAV9 (including AAV9 variants AAV- PHP.B and AAV9.45), AAV1, AAV2 (including AAV2 variants AAV2.7m8 and AAV2i8), AAV5, AAV6, AAV8, AAV10 or AAVrh74. In some embodiments, the vector is an AAV9 vector. In some embodiments a vector comprises modification to increase binding to and/or transduction of a cell-type of interest (i.e. as compared to the level of binding/transduction by the unmodified vector). In some embodiments modification is to a capsid protein. In some embodiments a vector comprises a capsid protein comprising a cell-targeting peptide. In some embodiments the cell-targeting peptide is a cell-targeting peptide described in Büning and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12: 248-265, which is hereby incorporated by reference in its entirety, e.g. a cell-targeting peptide shown in Table 1, 2, 3 or 4 thereof. In some embodiments a vector comprises a capsid protein comprising substitution to one or more tyrosine residues, e.g. one or more surface-exposed tyrosine residues. In some embodiments, one or more tyrosine residues of the capsid protein are substituted with phenylalanine. In some embodiments a vector comprises a capsid protein in which one or more tyrosine residues are substituted with another amino acid as described in Iida et al., Biomed Res Int. (2013) 2013: 974819, which is hereby incorporated by reference in its entirety. In some embodiments, a vector may be an adeno-associated virus vector described in Büning and Srivastava, supra. In some embodiments, a vector may be an adeno-associated virus vector described in Iida et al., supra. In some embodiments the vector comprises a control element for inducible expression of the polynucleotide of the disclosure. A sequence for controlling expression of the polynucleotide may provide for expression of the polynucleotide by cells of a particular type or tissue. For example, expression may be under the control of a cell type- or tissue-specific promoter. P37829 Promoters for cell type- or tissue-specific expression of a polynucleotide in accordance with the present disclosure can be selected in accordance with a disease/condition to be treated/prevented. For example, the promoter may drive expression in a cell type/tissue/an organ affected by the disease/condition (e.g. a cell type/tissue/an organ in which the symptoms of the disease/condition manifest). In some embodiments, a promoter may provide for expression of the polynucleotide in neuronal cells/tissue. In some embodiments, a promoter may be a neuron-specific promoter (e.g. a CaMKII, NSE or SynI-miniCMV promoter) In some embodiments, a promoter may provide for expression of the polynucleotide in muscle cells/tissue (e.g. cardiac and/or skeletal muscle cells/tissue). In some embodiments, a promoter may be a cardiac or cardiomyocte-specific promoter (e.g. a cTNT, α-MHC or MLC2v promoter). In some embodiments, a promoter may be a skeletal muscle/striated muscle cell- specific promoter (e.g. a MCK, MHCK7 or desmin promoter). In some embodiments, a promoter may be a vascular endothelial cell-specific promoter (e.g. a Tie2 promoter). In some embodiments, a promoter may be a vascular smooth muscle cell-specific promoter (e.g. a SM22a promoter). In some embodiments, a promoter may be a monocyte/macrophage-specific promoter (e.g. a LysM promoter). A sequence for controlling expression of the polynucleotide may provide for expression of the polynucleotide in response to e.g. a given agent/signal. For example, expression may be under the control of inducible promoter. The agent may provide for inducible expression of the polynucleotide in vivo by administration of the agent to a subject having been administered with a modified cell according to the disclosure, or ex vivo/in vitro by administration of the agent to cells in culture ex vivo or in vitro. In some embodiments a polynucleotide or vector according to the present disclosure may employ a conditional expression system for controlling expression of the polynucleotide by cells comprising the polynucleotide/vector. ‘Conditional expression’ may also be referred to herein as ‘inducible expression’, and refers to expression contingent on certain conditions, e.g. the presence of a particular agent. Conditional expression systems are well known in the art and are reviewed e.g. in Ryding et al. Journal of Endocrinology (2001) 171, 1-14, which is hereby incorporated by reference in its entirety. Cells The present disclosure also provides a cell comprising or expressing a polynucleotide according to the present disclosure. Also provided is a cell comprising or expressing vector according to the present disclosure. A polynucleotide according to the present disclosure (e.g. a polyribonucleotide) may be produced within a cell by transcription from a polynucleotide (e.g. a polydeoxyribonucleotide) encoding the polynucleotide. The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate). In preferred embodiments, the cell may be a human cell. P37829 The cell may be an immune cell. The cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be e.g. a T cell, B cell, NK cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof. The cell may express e.g. CD3 polypeptides (e.g. CD3γ CD3ε CD3ζ or CD3δ), TCR polypeptides (TCRα or TCRβ), CD27, CD28, CD4 or CD8. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)). The present disclosure also provides a method for producing a cell comprising or expressing a polynucleotide/vector according to the present disclosure, the method comprising introducing a polynucleotide/vector of the present disclosure into a cell. In some embodiments, introducing a polynucleotide/vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. adeno-associated viral transduction). In some embodiments, the polynucleotide/vector is introduced to the cell in vivo, e.g. by administration of a vector according to the present disclosure (e.g. a viral vector, e.g. an adeno-associated viral vector) to a subject. In some embodiments, the polynucleotide/vector is introduced into cells in culture ex vivo or in vitro. Any suitable method may be employed to produce a cell according to the present disclosure. Such methods may comprise nucleic acid transfer for permanent (i.e. stable) or transient expression of the polynucleotide of the present disclosure. In some embodiments, following introduction into a cell, the polynucleotide may be integrated into or form part of the genomic DNA of the cell. In some embodiments, following introduction into a cell, the polynucleotide may be maintained extrachromosomally. Any suitable genetic engineering platform may be used, and include gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol. (2014) 32:189-225, hereby incorporated by reference in its entirety. Methods also include those described e.g. in Wang and Rivière Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety. Suitable methods for introducing nucleic acid(s)/vector(s) into cells include transduction, transfection and electroporation. In some embodiments, the methods additionally comprise maintaining the cell under conditions suitable for expression of the polynucleotide/vector by the cell. The present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure. Compositions The present disclosure also provides compositions comprising the polynucleotides, vectors and cells described herein. In particular, the present disclosure provides pharmaceutical compositions and medicaments comprising the polynucleotides, vectors and cells of the present disclosure. P37829 Such compositions may comprise the relevant article (i.e. the polynucleotide/vector/cell) in a formulation suitable for clinical use. The present disclosure is concerned in particular with pharmaceutical compositions/medicaments comprising polynucleotides and vectors according to the present disclosure. The compositions of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents/excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide). The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed.(A)Adejare), 23rd Edition (2020), Academic Press. The pharmaceutical compositions/medicaments according to the present disclosure may be formulated for administration to a subject, e.g. administration via a route of administration as appropriate for the nature of the therapeutic agent and the disease to be treated/prevented. In some embodiments, a pharmaceutical composition/medicament may be formulated for parenteral, systemic, topical, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal,, oral or transdermal administration. In some embodiments, a pharmaceutical composition/medicament may be formulated for administration by injection or infusion, or administration by ingestion. Medicaments and pharmaceutical compositions may be formulated for administration to a blood vessel, or to a tissue/organ of interest (e.g. a tissue/organ affected by a disease/condition, e.g. a tissue/organ in which symptoms of the disease/condition manifest). The pharmaceutical compositions/medicaments may comprise the polynucleotide/vector/cell in a sterile or isotonic medium. The pharmaceutical compositions/medicaments may be provided in fluid, including gel, P37829 form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via cannula) to a blood vessel, or a selected region of the human or animal body. The pharmaceutical compositions/medicaments may be provided in solid form, e.g. in lyophilised form. The present disclosure also provides methods for producing pharmaceutical compositions/medicaments according to the present disclosure. Such methods may comprise mixing a polynucleotide/vector/cell described herein with a pharmaceutically-acceptable carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent. Such methods generally include the step of bringing into association the polynucleotide/vector/cell with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary. Polynucleotides, vectors, cells and compositions according to the present disclosure may be modified and/or formulated to facilitate delivery to, and/or uptake by, a cell type/tissue/organ of interest (e.g. a cell type/tissue/organ in which symptoms of a disease/condition manifest). Strategies for targeted delivery of polynucleotides are reviewed e.g. in Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated by reference in their entirety. In some embodiments, articles of the present disclosure may be encapsulated in a nanoparticle or a liposome. In some embodiments, articles of the present disclosure may be (covalently or non-covalently) associated with a cell-penetrating peptide (e.g. a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide), a cationic polymer, a cationic lipid or a viral carrier. Nanoparticles may be organic, e.g. micelles, liposomes, proteins, solid-lipid particles, solid polymer particles, dendrimers, and polymer therapeutics. Nanoparticles may be inorganic, e.g. such as nanotubes or metal particles, optionally with organic molecules added. In some embodiments, a nanoparticle is a nanoparticle described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, which is hereby incorporated by reference in its entirety. In some embodiments, a nanoparticle is a PLGA, polypeptide, poly(β-amino ester), DOPE, β-cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan or polyphosophoester nanoparticle. In some embodiments, polynucleotides and vectors according to the present disclosure comprise modification to incorporate one or more moieties facilitating delivery to, and/or uptake by, a cell type, organ or tissue of interest (e.g. a cell type/tissue/organ in which symptoms of a disease/condition manifest). In some embodiments, polynucleotides or vectors according to the present disclosure are linked (e.g. chemically conjugated to) one or more moieties facilitating delivery to, and/or uptake by, a cell type, tissue or organ of interest. P37829 Moieties facilitating delivery to, and/or uptake by, cell types, tissues or organs of interest are described e.g. in Benizri et al., Bioconjug Chem. (2019) 30(2): 366–383, which is hereby incorporated by reference in its entirety. Such moieties include e.g. N-acetylgalactosamine (GalNAc), α-tocopherol, cell-penetrating peptides, nucleic acid aptamers, antibodies and antigen-binding fragments/derivatives thereof, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g. palmitic acid) and nucleolipid moieties. Articles of the present disclosure may be formulated in a sustained release delivery system, in order to release the polynucleotide, vector, cell or composition at a predetermined rate. Sustained release delivery systems may maintain a constant drug/therapeutic/prophylactic concentration for a specified period of time. In some embodiments, articles of the present disclosure are formulated in a liposome, gel, implant, device, or drug-polymer conjugate e.g. hydrogel. In some embodiments, a composition according to the present disclosure may further comprise a splicing modifier that promotes SMN2 exon 7 inclusion (e.g. as described herein). Therapeutic/prophylactic applications The polynucleotides, vectors, cells and compositions of the present disclosure find use in therapy and prophylaxis. Accordingly, the present disclosure provides a polynucleotide, vector, cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is a polynucleotide, vector, cell or composition described herein for use in a method of treating or preventing a disease/condition described herein. Also provided is the use of a polynucleotide, vector, cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically- or prophylactically- effective amount of a polynucleotide, vector, cell or composition described herein. The intervention described in the preceding paragraph may be effective to reduce the development or progression of a disease/condition, alleviate the symptoms of a disease/condition or reduce the pathology of a disease/condition. The intervention may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition. In some embodiments, the intervention may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition. In some embodiments, the intervention may prevent progression/development of the disease/condition a later stage (e.g. a chronic stage). It will be appreciated that the polynucleotides, vectors, cells and compositions described herein may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from an increase in the level of the polypeptide of interest (i.e. the polypeptide of interest encoded by the polynucleotide). P37829 For example, the disease/condition may be a disease/condition associated with and/or characterised by deficiency/insufficiency of the polypeptide of interest. Deficiency/insufficiency of the polypeptide of interest may be positively associated with the onset, development or progression of the disease/condition, and/or positively associated with the severity of one or more symptoms of the disease/condition. Deficiency/insufficiency of the polypeptide of interest may be a risk factor for the onset, development or progression of the disease/condition. The disease/condition may be characterised by a decreased level of expression or activity of the polypeptide of interest, e.g. as compared to the level of expression/activity in the absence of the disease/condition. In some embodiments, the disease/condition may be characterised by a decrease in the number/proportion/activity of cells expressing the polypeptide of interest, e.g. as compared to the level/number/proportion/activity in the absence of the disease/condition (e.g. in a healthy subject, or in equivalent non-diseased tissue). By way of illustration, in some embodiments, the polypeptide of interest may be MeCP2, and the disease/condition to be treated/prevented in accordance with the present disclosure may be a disease/condition caused by deficiency/insufficiency of MeCP2, e.g. Rett syndrome. By way of further example, in embodiments wherein the polypeptide of interest is a polypeptide capable of inhibiting the expression and/or activity of a target antigen of interest, the disease/condition may be a disease/condition in which the target antigen, or cells comprising/expressing the target antigen are pathologically-implicated, e.g. a disease/condition in which an increased level/activity of the target antigen, or an increase in the number/proportion/activity of cells comprising/expressing the target antigen is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition. In some embodiments, an increased level/activity of the target antigen, or an increase in the number/proportion/activity of cells comprising/expressing the target antigen may be a risk factor for the onset, development or progression of the disease/condition. The disease/condition may be characterised by an increase in the level of expression or activity of the target antigen, e.g. as compared to the level of expression/activity in the absence of the disease/condition. In some embodiments, the disease/condition may be characterised by an increase in the number/proportion/activity of cells expressing the target antigen, e.g. as compared to the level/number/proportion/activity in the absence of the disease/condition (e.g. in a healthy subject, or in equivalent non-diseased tissue). Therapeutic/prophylactic intervention in accordance with the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of the target antigen; a reduction in the activity of the target antigen; and/or a reduction in the number/proportion/activity of cells comprising/expressing the target antigen. By way of further example, the disease/condition may be a disease/condition to be treated by nucleic acid editing, and the polypeptide of interest may be a constituent protein of an appropriate site-specific nuclease nucleic acid editing system. The present disclosure provides the articles of the present disclosure for use, uses of articles of the present disclosure, and methods comprising administering polynucleotides, vectors, cells and P37829 compositions according to the present disclosure to a subject (e.g. a subject in need of treatment). In some embodiments, the methods comprise administering to the subject a splicing modifier that promotes SMN2 exon 7 inclusion (e.g. a splicing modifier that promotes SMN2 exon 7 inclusion as described herein). Administration of the articles of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease/condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed.(A)Adejare), 23rd Edition (2020), Academic Press. Administration of the articles of the present disclosure may be parenteral, systemic, intravenous, intra- arterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral. In some cases, the articles of the present disclosure may be formulated for targeted delivery to specific cells, a tissue, an organ and/or a tumor. Multiple doses of an article of the present disclosure may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. In some embodiments, the present disclosure provides the articles of the present disclosure for use, uses of articles of the present disclosure, and methods comprising administering: (i) a polynucleotide, vector, cell or composition according to the present disclosure, and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, to a subject (e.g. a subject in need of treatment). In embodiments in accordance with aspects of the preceding paragraph, provision of (i) and (ii) may be as a combination therapy. In some embodiments, (i) and (ii) may be provided simultaneously or sequentially. Simultaneous administration refers to administration of the two or more agents (e.g. a polynucleotide, vector, cell or composition according to the present disclosure, and a splicing modifier that promotes SMN2 exon 7 inclusion) together, for example as a pharmaceutical composition containing both agents (i.e. as a combined preparation), or immediately after each other (e.g. within 1, 4, 6, 8 or 12 hours), and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. Sequential administration refers to administration of one of the agents followed after a given time interval P37829 by separate administration of another agent. It is not required that the agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval. In some embodiments, a splicing modifier that promotes SMN2 exon 7 inclusion is administered to a subject after administration of a polynucleotide, vector, cell or composition according to the present disclosure. In some embodiments, a polynucleotide, vector, cell or composition according to the present disclosure is administered to a subject, and a splicing modifier that promotes SMN2 exon 7 inclusion is administered continuously to the subject thereafter. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is administered in a quantity, and/or with a periodicity, selected to achieve a desired level of expression of the polypeptide of interest. That is, in some embodiments the quantity of, and/or periodicity with which, a splicing modifier that promotes SMN2 exon 7 inclusion administered to a subject in accordance with the present disclosure is selected to achieve a desired level of expression of the polypeptide of interest. In some embodiments, the quantity of, and/or periodicity with which, a splicing modifier that promotes SMN2 exon 7 inclusion is administered to a subject is adjusted through the course of its administration to change (i.e. decrease or increase) the level of expression of the polypeptide of interest in the subject. In embodiments wherein the polynucleotide of the disclosure is an ON-switch polynucleotide, the quantity and/or periodicity of administration of a splicing modifier that promotes SMN2 exon 7 inclusion may be increased to increase the level of expression of the polypeptide of interest. Conversely, the quantity and/or periodicity of administration of a splicing modifier that promotes SMN2 exon 7 inclusion may be decreased to decrease the level of expression of the polypeptide of interest. In embodiments wherein the polynucleotide of the disclosure is an OFF-switch polynucleotide, the quantity and/or periodicity of administration of a splicing modifier that promotes SMN2 exon 7 inclusion may be decreased to increase the level of expression of the polypeptide of interest. Conversely, the quantity and/or periodicity of administration of a splicing modifier that promotes SMN2 exon 7 inclusion may be increased to decrease the level of expression of the polypeptide of interest. Further methods The present disclosure also provides a method for modifying a cell to comprise or express a polynucleotide according to the present disclosure, comprising introducing into a cell a polynucleotide or vector according to the present disclosure. In some embodiments, introducing a polynucleotide or vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction). Transfection relates to the process of introducing nucleic acid into cells using means other than viral infection and is hence a non-viral method. Transfection may be performed by physical/mechanical methods (including electroporation, sonoporation, magnetofection, gene microinjection and laser irradiation) or chemical methods (liposomal-based or non-liposomal based). Liposomal-based transfection P37829 reagents are chemicals which enable the formation of positively charged lipid aggregates, which can then merge with the phospholipid bilayer of the cell to facilitate the entry of foreign genetic material. Examples of liposomal-based transfection reagents include, but are not limited to Oligofectamine®, Lipofectamine® and DharmaFECT®. Non-liposomal transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendrimers and non-liposomal lipids. One example of a non- liposomal transfection reagent is polyethylenimine (PEI). Electroporation may be performed e.g. as described in Koh et al., Molecular Therapy – Nucleic Acids (2013) 2, e114, which is hereby incorporated by reference in its entirety. Transduction is a process by which nucleic acids may be introduced into a cell by a virus or a viral vector. Accordingly, in some embodiments the polynucleotide is, or is comprised in, a viral vector, or the vector is a viral vector. Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola- Ila, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety. Agents may be employed in the methods of the present disclosure to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs). In some embodiments the methods comprise centrifuging the cells into which it is desired to introduce polynucleotide or vector according to the present disclosure in the presence of cell culture medium comprising viral vector(s) comprising the polynucleotide (referred to in the art as ‘spinfection’). In some embodiments, the methods comprise culturing the cell under conditions suitable for expression of the polynucleotide or vector by the cell. In some embodiments, the methods comprise culturing the cell under conditions suitable for transcription of a polydeoxyribonucleotide. In some embodiments, the methods comprise culturing the cell under conditions suitable for post-transcriptional processing (e.g. splicing) of a polyribonucleotide. In some embodiments, the methods comprise culturing the cell under conditions suitable for translation of a polypeptide from a polyribonucleotide. Methods for culturing (including generating and/or expanding) populations of cells in vitro/ex vivo – including suitable culture conditions (i.e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), cell numbers, culture periods etc. – are well known to the skilled person. Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. The present disclosure also provides a method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell a polynucleotide or vector according to the present disclosure. Where the polynucleotide is, or wherein the vector comprises/encodes, an OFF-switch, the cell may express the polypeptide of interest following introduction of the polynucleotide/vector into the cell. P37829 Where the polynucleotide is, or wherein the vector comprises/encodes, an ON-switch, the method may further comprise contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion as described herein. Thus, in some embodiments, the method comprises (i) introducing into a cell a polynucleotide or vector according to the present disclosure; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion. ‘Contacting’ a cell with a splicing modifier may comprise bringing a cell into contact with a splicing modifier in a cell culture, and may be achieved by applying the splicing modifier to the cells in culture. The present disclosure also provides a method for inhibiting expression of a polypeptide of interest in a cell. The method comprises contacting a cell comprising a polynucleotide or vector according to the present disclosure comprising/encoding an OFF-switch with a splicing modifier that promotes SMN2 exon 7 inclusion as described herein. Any suitable quantity/concentration of a splicing modifier may be employed in the methods of the present disclosure. It will be appreciated that the quantity/concentration of the splicing modifier is preferably selected such as to achieve the desired effect, i.e. increased inclusion of the SMN2 exon 7 variant nucleotide sequence in the products of splicing of the relevant polyribonucleotide. Subjects A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use). The subject to be administered with an article of the present disclosure (e.g. in accordance with therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. A subject may have (e.g. may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for one or more markers of such a disease/condition. Kits The present disclosure also provides kits of parts. In some aspects and embodiments, a kit of parts according to the present disclosure comprises (i) a polynucleotide, vector, or a pharmaceutical composition according to the present disclosure, and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion. P37829 Kits of parts according to the present disclosure may comprise a predetermined quantity of articles according to (i) and/or (ii), as described in the preceding paragraph. In some embodiments, articles according to (i) and/or (ii) are provided in containers (e.g. in vials or bottles). The kit may provide articles according to (i) and/or (ii) together with instructions (e.g. a protocol) as to how to employ them in accordance with a therapeutic or prophylactic intervention as described herein. In some embodiments, the kit of parts may comprise a polynucleotide or vector according to the present disclosure, and optionally materials for introducing the polynucleotide/vector into a cell. In some embodiments, the kit of parts may comprise a system for producing a cell according to the present disclosure. In some embodiments, the kit of parts may comprise a (closed) bag cell incubation system in which a polynucleotide or vector can be introduced into a cell. In some embodiments, the kit of parts may comprise materials for formulating a polynucleotide or vector according to the present disclosure to a pharmaceutical composition, e.g. a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. In some embodiments, the kit of parts further comprises reagents, buffers and/or standards required for execution of a method according to the present disclosure. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein. The manufacture of kits of parts according to the present disclosure preferably follows standard procedures which are known to the person skilled in the art. Sequence identity The ‘sequence identity’ between a given nucleotide sequence (e.g. of a polynucleotide) and a reference nucleotide sequence is calculated by determining the percentage of the nucleotides in the given nucleotide sequence that are identical to those of the reference nucleotide sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percentage sequence identity between the two sequences. Similarly, ‘sequence identity’ between a given amino acid sequence (e.g. of a polypeptide) and a reference amino acid sequence is calculated by determining the percentage of the amino acids in the given amino acid sequence that are identical to those of the reference amino acid sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percentage sequence identity between the two sequences. Pairwise and multiple sequence alignment for the purposes of evaluating sequence identity between two or more nucleotide or amino acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al.2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772–780) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. P37829 Sequences SEQ ID DESCRIPTION SEQUENCENO: GCX1-2AATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATX3-4G 1 SMN2 exon 6 variant consensus 1 wherein X1-2 = absent or TT; X3-4 = AT, CT or CA 2 SMN2 exon 6 (wildtype) ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATG 45 nucleotide SMN2 exon 6 comprising 3 ATG>GGC at positions GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATG 82 to 84 (SMN2 exon 6 variant 1) GCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCX1G 4 SMN2 exon 6 variant consensus 2 wherein X1 = T or A SMN2 exon 6 comprising ATG>GGC at positions 64 to 66, ATG>GGC at 5 positions 82 to 84 and ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA ATG>CTG at positions AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTG 109 to 111 (SMN2 exon 6 variant 2) SMN2 exon 6 comprising ATG>GGC at positions 64 to 66, 6 ATG>GGC at positions ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA 82 to 84 and ATG>CAG AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAG at positions 109 to 111 (SMN2 exon 6 variant 3) 7 Positions 1 to 102 of GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT SMN2 intron 6 AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACT 3’ 162 positions of SMN2 intron 6, comprising A>C TTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTT 8 4 positions from the 3’ GTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTAT end (position 5766 of ATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAG intron 6) 414 nucleotide SMN2 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT intron 6, comprising A>C AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATAC 4 po AACTGTCTGAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAA 9 sitions from the 3’ end (position 5766 of AAGCTTTCATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGT intron 6) (intron 6 variant ACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGT 1) CTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATC TATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAG 264 nucleotide SMN2 intron 6, comprising A>C GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT 4 position AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT 410 s from the 3’ end (position 5766 of CTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACA intron 6) (intron 6 variant TCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTT 2) TATTTTCCTTCCAG GATTTTX1-6AX7-8CAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA 11 SMN2 exon 7 variant consensus 1 wherein X1-6 = absent or GCCACC; X7-8 = GA or TG 12 SMN2 exon 7 (wildtype) GGTTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAAGGA SMN2 exon 7 comprising G>A at position 2, and GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA 13 insertion of A after position 48 (exon 7 variant 1) SMN2 exon 7 comprising 14 G>A at position 2, GATTTTGCCACCATGCAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGG insertion of GCCACC A P37829 after position 6, GA>TG at positions 8 and 9, and insertion of A after position 48 (exon 7 variant 2) Positions 1 to 102 of GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA SMN2 intron 7 AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAA 3’ 150 positions of SMN2 CTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGA intron 7 AGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCA AGCCTCTGGTTCTAATTTCTCATTTGCAG GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA 252 nucleotide SMN2 AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG intron 7 GATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT CATTTGCAG GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAA ACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCC 444 nucleotide SMN2 TACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAG intron 7 (wildtype) AGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTAT TGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAG TCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGT TCTAATTTCTCATTTGCAG X1-2AATGCTGGCATAGAGCAGCAC Positions 1 to 23 of SMN2 exon 8 consensus wherein X1-2 = GA or TG; Positions 1 to 23 of SMN2 exon 8 (exon 8 GAAATGCTGGCATAGAGCAGCAC variant 1) Positions 1 to 23 of SMN2 exon 8, comprising GA>TG at TGAATGCTGGCATAGAGCAGCAC positions 1 to 2 (exon 8 variant 2) GCCACCATGGCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGT AATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTT ATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGAT CATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT pMM112 ON-switch TCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAA GGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTG GAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATA TGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTT TAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATT TCTCATTTGCAGGA GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM130 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGA ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC pMM59 ON-switch ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC AGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAA TCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTT CAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCA P37829 AAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGG TTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATC ACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTG CACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGAC ATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTG GCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM198 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACT TTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGT TAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTAT TAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTG GAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTA GGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTGAATGCTGGCATAGA GCAGCAC GATTTTAGACAAAATCAAAX1AGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant consensus 2 wherein X1 = absent or A SMN2 exon 7 comprising G>A at position 2, comprising deletion of GATTTTAGACAAAATCAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA position 20, and comprising insertion of A after position 48 (exon 7 variant 3) GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM193 OFF-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM194 OFF-switch ATTTTAGACAAAATCAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCT GCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATG TTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCT AGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTC CACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCA GGAG GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATAC AACTGTCTGAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAA AAGCTTTCATGCTATTGTTAGATTATTTTGATTATACACTTTTGAATTGAAATTATACTTTTT CTAAATAATGTTTTAATCTCTGATTTGAAATTGATTGTAGGGAATGGAAAAGATGGGATAA SMN2 intron 6 (wildtype) TTTTTCATAAATGAAAAATGAAATTCTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTTGC TCTGTTGCCCAGGCTGGAGTGCAATGGCGTGATCTTGGCTCACAGCAAGCTCTGCCTCC TGGATTCACGCCATTCTCCTGCCTCAGCCTCAGAGGTAGCTGGGACTACAGGTGCCTGC AATTTTTTTAATGCACAAAGATCTGGGGTAATGTGTACCACATTGAACCTTGGGGAGTAT GGCTTCAAACTTGTCACTTTATACGTTAGTCTCCTACGGACATGTTCTATTGTATTTTAGT P37829 CAGAACATTTAAAATTATTTTATTTTATTTTATTTTTTTTTTTTTTTTGAGACGGAGTCTCGC TCTGTCACCCAGGCTGGAGTACAGTGGCGCAGTCTCGGCTCACTGCAAGCTCCGCCTC CCGGGTTCACGCCATTCTCCTGCCTCAGCCTCTCCGAGTAGCTGGGACTACAGGCGCC CGCCACCACGCCCGGCTAATTTTTTTTTATTTTTAGTAGAGACGGGGTTTCACCGTGGTC TCGATCTCCTGACCTCGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAAG CGTGAGCCACCGCGCCCGGCCTAAAATTATTTTTAAAAGTAAGCTCTTGTGCCCTGCTAA AATTATGATGTGATATTGTAGGCACTTGTATTTTTAGTAAATTAATATAGAAGAAACAACT GACTTAAAGGTGTATGTTTTTAAATGTATCATCTGTGTGTGCCCCCATTAATATTCTTATTT AAAAGTTAAGGCCAGACATGGTGGCTTACAACTGTAATCCCAACAGTTTGTGAGGCCGA GGCAGGCAGATCACTTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGATGAAAC CTTGTCTCTACTAAAAATACCAAAAAAAATTTAGCCAGGCATGGTGGCACATGCCTGTAA TCCGAGCTACTTGGGAGGCTGTGGCAGGAAAATTGCTTTAATCTGGGAGGCAGAGGTTG CAGTGAGTTGAGATTGTGCCACTGCACTCCACCCTTGGTGACAGAGTGAGATTCCATCT CAAAAAAAGAAAAAGGCCTGGCACGGTGGCTCACACCTATAATCCCAGTACTTTGGGAG GTAGAGGCAGGTGGATCACTTGAGGTTAGGAGTTCAGGACCAGCCTGGCCAACATGGT GACTACTCCATTTCTACTAAATACACAAAACTTAGCCCAGTGGCGGGCAGTTGTAATCCC AGCTACTTGAGAGGTTGAGGCAGGAGAATCACTTGAACCTGGGAGGCAGAGGTTGCAG TGAGCCGAGATCACACCGCTGCACTCTAGCCTGGCCAACAGAGTGAGAATTTGCGGAG GGAAAAAAAAGTCACGCTTCAGTTGTTGTAGTATAACCTTGGTATATTGTATGTATCATGA ATTCCTCATTTTAATGACCAAAAAGTAATAAATCAACAGCTTGTAATTTGTTTTGAGATCA GTTATCTGACTGTAACACTGTAGGCTTTTGTGTTTTTTAAATTATGAAATATTTGAAAAAAA TACATAATGTATATATAAAGTATTGGTATAATTTATGTTCTAAATAACTTTCTTGAGAAATA ATTCACATGGTGTGCAGTTTACCTTTGAAAGTATACAAGTTGGCTGGGCACAATGGCTCA CGCCTGTAATCCCAGCACTTTGGGAGGCCAGGGCAGGTGGATCACGAGGTCAGGAGAT CGAGACCATCCTGGCTAACATGGTGAAACCCCGTCTCTACTAAAAGTACAAAAACAAATT AGCCGGGCATGTTGGCGGGCACCTTTTGTCCCAGCTGCTCGGGAGGCTGAGGCAGGA GAGTGGCGTGAACCCAGGAGGTGGAGCTTGCAGTGAGCCGAGATTGTGCCAGTGCACT CCAGCCTGGGCGACAGAGCGAGACTCTGTCTCAAAAAATAAAATAAAAAAGAAAGTATA CAAGTCAGTGGTTTTGGTTTTCAGTTATGCAACCATCACTACAATTTAAGAACATTTTCAT CACCCCAAAAAGAAACCCTGTTACCTTCATTTTCCCCAGCCCTAGGCAGTCAGTACACTT TCTGTCTCTATGAATTTGTCTATTTTAGATATTATATATAAACGGAATTATACGATATGTGG TCTTTTGTGTCTGGCTTCTTTCACTTAGCATGCTATTTTCAAGATTCATCCATGCTGTAGA ATGCACCAGTACTGCATTCCTTCTTATTGCTGAATATTCTGTTGTTTGGTTATATCACATTT TATCCATTCATCAGTTCATGGACATTTAGGTTGTTTTTATTTTTGGGCTATAATGAATAATG TTGCTATGAACATTCGTTTGTGTTCTTTTTGTTTTTTTGGTTTTTTGGGTTTTTTTTGTTTTG TTTTTGTTTTTGAGACAGTCTTGCTCTGTCTCCTAAGCTGGAGTGCAGTGGCATGATCTT GGCTTACTGCAAGCTCTGCCTCCCGGGTTCACACCATTCTCCTGCCTCAGCCCGACAAG TAGCTGGGACTACAGGCGTGTGCCACCATGCACGGCTAATTTTTTGTATTTTTAGTAGAG ATGGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCTGCCTG CCTAGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGCACCTGGCCTTAAGT GTTTTTAATACGTCATTGCCTTAAGCTAACAATTCTTAACCTTTGTTCTACTGAAGCCACG TGGTTGAGATAGGCTCTGAGTCTAGCTTTTAACCTCTATCTTTTTGTCTTAGAAATCTAAG CAGAATGCAAATGACTAAGAATAATGTTGTTGAAATAACATAAAATAGGTTATAACTTTGA TACTCATTAGTAACAAATCTTTCAATACATCTTACGGTCTGTTAGGTGTAGATTAGTAATG AAGTGGGAAGCCACTGCAAGCTAGTATACATGTAGGGAAAGATAGAAAGCATTGAAGCC AGAAGAGAGACAGAGGACATTTGGGCTAGATCTGACAAGAAAAACAAATGTTTTAGTATT AATTTTTGACTTTAAATTTTTTTTTTATTTAGTGAATACTGGTGTTTAATGGTCTCATTTTAA TAAGTATGACACAGGTAGTTTAAGGTCATATATTTTATTTGATGAAAATAAGGTATAGGCC GGGCACGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGATC ACCTGAGGTCGGGAGTTAGAGACTAGCCTCAACATGGAGAAACCCCGTCTCTACTAAAA AAAATACAAAATTAGGCGGGCGTGGTGGTGCATGCCTGTAATCCCAGCTACTCAGGAGG CTGAGGCAGGAGAATTGCTTGAACCTGGGAGGTGGAGGTTGCGGTGAGCCGAGATCAC CTCATTGCACTCCAGCCTGGGCAACAAGAGCAAAACTCCATCTCAAAAAAAAAAAAATAA GGTATAAGCGGGCTCAGGAACATCATTGGACATACTGAAAGAAGAAAAATCAGCTGGGC GCAGTGGCTCACGCCGGTAATCCCAACACTTTGGGAGGCCAAGGCAGGCGAATCACCT GAAGTCGGGAGTTCCAGATCAGCCTGACCAACATGGAGAAACCCTGTCTCTACTAAAAA TACAAAACTAGCCGGGCATGGTGGCGCATGCCTGTAATCCCAGCTACTTGGGAGGCTG AGGCAGGAGAATTGCTTGAACCGAGAAGGCGGAGGTTGCGGTGAGCCAAGATTGCACC ATTGCACTCCAGCCTGGGCAACAAGAGCGAAACTCCGTCTCAAAAAAAAAAGGAAGAAA AATATTTTTTTAAATTAATTAGTTTATTTATTTTTTAAGATGGAGTTTTGCCCTGTCACCCA GGCTGGGGTGCAATGGTGCAATCTCGGCTCACTGCAACCTCCGCCTCCTGGGTTCAAG TGATTCTCCTGCCTCAGCTTCCCGAGTAGCTGTGATTACAGCCATATGCCACCACGCCC AGCCAGTTTTGTGTTTTGTTTTGTTTTTTGTTTTTTTTTTTTGAGAGGGTGTCTTGCTCTGT CCCCCAAGCTGGAGTGCAGCGGCGCGATCTTGGCTCACTGCAAGCTCTGCCTCCCAGG TTCACACCATTCTCTTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGCCCGCCACC ACACCCGGCTAATTTTTTTGTGTTTTTAGTAGAGATGGGGTTTCACTGTGTTAGCCAGGA TGGTCTCGATCTCCTGACCTTTTGATCCACCCGCCTCAGCCTCCCCAAGTGCTGGGATT ATAGGCGTGAGCCACTGTGCCCGGCCTAGTCTTGTATTTTTAGTAGAGTCGGGATTTCT CCATGTTGGTCAGGCTGTTCTCCAAATCCGACCTCAGGTGATCCGCCCGCCTTGGCCTC CAAAAGTGCAAGGCAAGGCATTACAGGCATGAGCCACTGTGACCGGCAATGTTTTTAAA P37829 TTTTTTACATTTAAATTTTATTTTTTAGAGACCAGGTCTCACTCTATTGCTCAGGCTGGAGT GCAAGGGCACATTCACAGCTCACTGCAGCCTTGACCTCCAGGGCTCAAGCAGTCCTCTC ACCTCAGTTTCCCGAGTAGCTGGGACTACAGTGATAATGCCACTGCACCTGGCTAATTTT TATTTTTATTTATTTATTTTTTTTTGAGACAGAGTCTTGCTCTGTCACCCAGGCTGGAGTG CAGTGGTGTAAATCTCAGCTCACTGCAGCCTCCGCCTCCTGGGTTCAAGTGATTCTCCT GCCTCAACCTCCCAAGTAGCTGGGATTAGAGGTCCCCACCACCATGCCTGGCTAATTTT TTGTACTTTCAGTAGAAACGGGGTTTTGCCATGTTGGCCAGGCTGTTCTCGAACTCCTGA GCTCAGGTGATCCAACTGTCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCA CTGTGCCTAGCCTGAGCCACCACGCCGGCCTAATTTTTAAATTTTTTGTAGAGACAGGGT CTCATTATGTTGCCCAGGGTGGTGTCAAGCTCCAGGTCTCAAGTGATCCCCCTACCTCC GCCTCCCAAAGTTGTGGGATTGTAGGCATGAGCCACTGCAAGAAAACCTTAACTGCAGC CTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTC TGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACAT CCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTT ATTTTCCTTACAG GAAATGCTGGCATAGAGCAGCACTAAATGACACCACTAAAGAAACGATCAGACAGATCT GGAATGTGAAGCGTTATAGAAGATAACTGGCCTCATTTCTTCAAAATATCAAGTGTTGGG AAAGAAAAAAGGAAGTGGAATGGGTAACTCTTCTTGATTAAAAGTTATGTAATAACCAAAT GCAATGTGAAATATTTTACTGGACTCTATTTTGAAAAACCATCTGTAAAAGACTGAGGTG SMN2 exon 8 (wildtype) GGGGTGGGAGGCCAGCACGGTGGTGAGGCAGTTGAGAAAATTTGAATGTGGATTAGAT TTTGAATGATATTGGATAATTATTGGTAATTTTATGAGCTGTGAGAAGGGTGTTGTAGTTT ATAAAAGACTGTCTTAATTTGCATACTTAAGCATTTAGGAATGAAGTGTTAGAGTGTCTTA AAATGTTTCAAATGGTTTAACAAAATGTATGTGAGGCGTATGTGGCAAAATGTTACAGAAT CTAACTGGTGGACATGGCTGTTCATTGTACTGTTTTTTTCTATCTTCTATATGTTTAAAAGT ATATAATAAAAATATTTAATTTTTTTTTAAATTA GCCX1CC Kozak consensus wherein X1 = A or G Kozak GCCACC Kozak plus start codon GCCACCATG ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGTAAGTAATC ACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAA AATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCTG AGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTCA TGCTATTGTTAGATTATTTTGATTATACACTTTTGAATTGAAATTATACTTTTTCTAAATAAT GTTTTAATCTCTGATTTGAAATTGATTGTAGGGAATGGAAAAGATGGGATAATTTTTCATA AATGAAAAATGAAATTCTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTTGCTCTGTTGC CCAGGCTGGAGTGCAATGGCGTGATCTTGGCTCACAGCAAGCTCTGCCTCCTGGATTCA CGCCATTCTCCTGCCTCAGCCTCAGAGGTAGCTGGGACTACAGGTGCCTGCCACCACG CCTGGCTAGCTGGGATTAGAGGTCCCCACCACCATGCCTGGCTAATTTTTTGTACTTTCA GTAGAAACGGGGTTTTGCCATGTTGGCCAGGCTGTTCTCGAACTCCTGAGCTCAGGTGA SMN2ind minigene of TCCAACTGTCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGTGCCTAGC WO 2021/163556 A1 CTGAGCCACCACGCCGGCCTAATTTTTAAATTTTTTGTAGAGACAGGGTCTCATTATGTT (SEQ ID NO:2 of WO GCCCAGGGTGGTGTCAAGCTCCAGGTCTCAAGTGATCCCCCTACCTCCGCCTCCCAAA 2021/163556 A1) GTTGTGGGATTGTAGGCATGAGCCACTGCAAGAAAACCTTAACTGCAGCCTAATAATTGT TTTCTTTGGGATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTCTGATCATATTT TGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAG CTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTC CAGGATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGT AAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAA CAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAAC AATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTA CTAGAATTCTCATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAGAG CTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTG TGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTC AAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGATCTGACATATGAAGTGCTCT AGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGTAAGTAATC ACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAA AATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCTG pLS41 ON-switch AGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTCA CAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCT ATTTTTTTTAACTTCCTTTATTTTCCTTACAGGGTTTTAGACAAAATCAAAAAGAAGGAAGG P37829 TGCTCACATTCCTTAAATATAAGGAGAAATGCTGGCATAGAGCAGCACGTAAGTCTGCCA GCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTT GAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTA AAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTC ATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATAT GATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGAT AACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTG GTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCAT TTGCAG ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGTAAGTAATC ACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAA AATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCTG AGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTCA TGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAAA GACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAA CAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCT pLS76 ON-switch ATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAGG TGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTT TTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTT AGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATT AGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGG AAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAG GCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTAC ACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTA ACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGAG CAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGTAAGTAATC ACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAA AATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATAT TTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAA pLS159 ON-switch GCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTT CCAGGATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAG TAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAA ACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGG ATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAAC TGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTC ATTTGCAGGAAATGCTGGCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATGGTAAGTAATC ACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAA AATACGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATA pLS160 ON-switch TAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAG ACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAG CATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTG AACTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAA CTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGAGC AGCAC GATTCTCTTGATGATGCTGATGCTTTGGGAAGTATGTTAATTTCATGGTACATGAGTGGC TATCATACTGGCTATTATATGGTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGT AGTTATGTGACTTTGTTTTGTAAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAA AAAATAAAAATAAAAAAATACAACTGTCTGAGGCTTAAATTACTCTTGCATTGTCCCTAAG TATAATTTTAGTTAATTTTAAAAAGCTTTCATGCTATCTTAACTGCAGCCTAATAATTGTTT TCTTTGGGATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTG TTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTA TCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAG pLS167 ON-switch GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAG TCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAA ATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAAT CAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTA GAATTCTCATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTT TAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGA TATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAG TTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAA pLS168 ON-switch ACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAA AATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCTG AGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTCA P37829 TGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAAA GACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAA CAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCT ATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAGG TGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTT TTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTT AGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATT AGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGG AAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAG GCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTAC ACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTA ACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGAG CAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pLS179 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM70 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGCATAGAG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM71 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM72 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCT GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM73 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CAGGAAAT pMM143 ON-switch GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA P37829 GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCTCCTCTTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM144 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCAAGGCTTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM145 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCTAGTCTTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM146 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCTTTCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAAC AAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGAT AACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTG GTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCAT TTGCAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM147 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCCCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAAC AAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGAT AACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTG GTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCAT TTGCAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM136 ON-switch ATTTTAGACAAAATCAAAAAGAAGGTAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTC TGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAAT GTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACC TAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGT CCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGC AGGAAATGCTGGCATAGAGCAGCAC pMM137 ON-switch TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT P37829 CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGATGGTGCTCACATTCCTTAAATATAAGGAGTAAGTC TGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAAT GTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACC TAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGT CCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGC AGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM138 ON-switch ATTTTAGACAAAATCAAAAGGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM139 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAAGTGCTCACATTCCTTAAATATAAGGAGTAAGTC TGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAAT GTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACC TAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGT CCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGC AGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM140 ON-switch ATTTTAGACAAAATCAAAATGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTC TGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAAT GTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACC TAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGT CCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGC AGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM141 ON-switch ATTTTAGACAAAATCAAAAAGAGGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM142 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGAGCACACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGAAATGCTGGCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT pMM151 ON-switch ATTTTAGAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTA TGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACAT TTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTG CACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGAC ATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTG GCATAGAGCAGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM152 ON-switch ATTTTAGAGAAGGAAGGTGCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGA ATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGT TCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACA CTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAA CTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGAGC AGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pLS174 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACT TTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGT TAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTAT TAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTG GAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTA GGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGA GCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM56 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCCACCATGTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM60 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAGCCACCATGATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGA ATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGT TCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCC AAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTG GTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATAT CATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCT GGCATAGAGCAGCAC pMM61 ON-switch ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAACAACATGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM62 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAGCCACCATGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM63 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACAGCCACCATGAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTT ACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGA TGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAAC TATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGT GTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTA GTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACT GTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGG TTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pLS176 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTGCCACCATGCAAAATCAAAAAGA AGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAA TCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTT CAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCA AAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGG TTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATC ACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTG CACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGAC ATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTG P37829 GCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM108 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTGCCACCATGCAAAATCAAAAAGA AGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAA TCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTT CAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCA AAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGG TTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATC ACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTG CACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGAC ATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTG GCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATGTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM110 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTGCCACCATGCAAAATCAAAAAGA AGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAA TCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTT CAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCA AAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGG TTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATC ACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTG CACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGAC ATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTG GCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATTTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM111 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTGCCACCATGCAAAATCAAAAAGA AGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAA TCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTT CAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCA AAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGG TTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATC ACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTG CACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGAC ATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTG GCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA pMM242 ON-switch ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGAGCCACCATGCAAAATCAAAA AGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGT CCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGT TGGTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTAT ATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATA P37829 CTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAG GACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATG CTGGCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM243 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATAGCACCATGCAAAA AGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGT GAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAA GTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATG CCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGT TGGTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTAT ATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATA CTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAG GACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATG CTGGCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM244 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATAGCACCATGGAAAA AGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGT GAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAA GTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATG CCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGT TGGTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTAT ATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATA CTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAG GACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATG CTGGCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM245 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCACCATGGAAAAAG AAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGA ATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGT TCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCC AAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTG GTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATAT CACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACT GCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGA CATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCT GGCATAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA pMM246 ON-switch AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC TACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAG ATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAA CTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTG TGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACT AGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCAC TGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATG GTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCA TAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM247 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAATGGAAGG AAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCT TACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAG ATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAA CTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTG TGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACT AGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCAC TGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATG GTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCA TAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pLS175 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACT TTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGT TAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTAT TAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTG GAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTA GGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGA GCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM123 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAATGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTAC TTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATG TTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTA TTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGT GGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGT AGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGT ACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTT TAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAG AGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC pMM124 ON-switch ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATGGTAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM125 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GATGGCGTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCT TACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAG ATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAA CTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTG TGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACT AGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCAC TGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATG GTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCA TAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM126 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATGTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM127 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTATGGATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT pMM128 ON-switch AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG P37829 GTGCTCACATTCCTTAAATATAAGATGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM129 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATGAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTT TTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTT AGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATT AGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGG AAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAG GCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTAC ACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTA ACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGAG CAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM201 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATGGCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM202 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTAACATGGCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTA CTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGAT GTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACT ATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTG TGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAG TAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT pMM203 ON-switch GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA P37829 ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATATGCCTAAATTAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTAC TTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATG TTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTA TTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGT GGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGT AGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGT ACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTT TAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAG AGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM204 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCATGTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCT TACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAG ATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAA CTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTG TGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACT AGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCAC TGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATG GTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCA TAGAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM205 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTATGAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTT ACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGA TGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAAC TATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGT GTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTA GTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACT GTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGG TTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATA GAGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM206 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTATGGAAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAAT CTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTC AGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAA AACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGT TGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCA CTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGC ACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACA TGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGG CATAGAGCAGCAC pMM248 ON-switch CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC P37829 ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATTAAGATGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTAC TTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATG TTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTA TTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGT GGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGT AGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGT ACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTT TAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAG AGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM249 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTAAAATGAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTT TTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTT AGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATT AGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGG AAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAG GCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTAC ACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTA ACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGAG CAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM250 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTGAAATGAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTT TTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTT AGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATT AGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGG AAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAG GCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTAC ACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTA ACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGGAAATGCTGGCATAGAG CAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM251 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCAAAATGAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTT GTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTA GAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATTA GATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGGA AGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAGG CAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACA CTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAA AGCAC GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA pMM252 ON-switch GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT P37829 TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGGA GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM254 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATGTCCTTAAATATAAGGAGTAAG TCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAA ATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAA CCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGT GTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAGGA GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM255 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGATGAGTAAG TCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAA ATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAA CCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGT GTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAGGA GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM256 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATGAGGAGTAAGTCT GCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATG TTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCT AGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTC CACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCA GGA GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM257 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATGATAAGGAGTAAG TCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAA ATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAA CCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGT GTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAGGA ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM199 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACT TTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGT TAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTAT TAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTG GAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTA GGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTCCAGTGAATGCTGGCATAGA GCAGCAC P37829 ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM200 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACT TTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGT TAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTAT TAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTG GAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTA GGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTTTTTTTCAGTGAATGCTGGCATAGAG CAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM236 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACT TTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGT TAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTAT TAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTG GAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTA GGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGATGAATGCTGGCATAG AGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATTAGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC pMM237 ON-switch TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACT TTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGT TAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTAT TAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTG GAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTA GGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGATGAATGCTGGCATAG AGCAGCAC ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATTTGGTAAGTAAT CACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATA AAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATACAACTGTCT GAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAAAAGCTTTC ATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAA AGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAA pMM238 ON-switch ACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGC TATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAG GTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACT TTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGT TAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTAT TAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTG GAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTA GGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT P37829 AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGATGAATGCTGGCATAG AGCAGCAC 45 nucleotide SMN2 exon 6 comprising ATG>GGC at positions 82 to 84, additionally GCCACCATGGCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATG comprising 5’ ‘GCCACCATG’ T2A cleavage site GSGEGRGSLLTCGDVEENPGP E2A linker GSGQCTNYALLKLAGDVESNPGP P2A cleavage site GSGATNFSLLKQAGDVEENPGP F2A cleavage site GSGVKQTLNFDLLKLAGDVESNPGP GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM273 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM362 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAAGAGTAAGTG GCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATG TTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCT AGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTC CACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCA GTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM363 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATGATGAGTAAGAT TGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAAT GTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACC TAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGT CCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGC AGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM364 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT GTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM358 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAG CATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTG AACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCA TACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA pMM359 ON-switch GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT P37829 TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTAT GAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATT TAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGC ACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACA TGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM360 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAA GTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAA AAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTG TACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGT TTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM361 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATC TTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCA GATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTC TGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTG GAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM365 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATG AAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTT AAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCA CTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACAT GGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM366 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGA AAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTA AAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCAC TGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATG GTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM367 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAA AGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAA AAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACT GTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGG TTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM368 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATT ATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAAC ATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATAC pMM369 ON-switch GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC P37829 TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCA TTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAA CATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATA CTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAG GACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM370 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGC ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCAT ACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGA GGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM371 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCC AGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTT TGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGG CATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCAC AGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM372 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAG TGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAA AGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGT ACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTT TAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM373 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTG AATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAG TTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTAC ACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTA ACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM374 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAA TCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTT CAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACAC TCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAAC TGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM375 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCGGTGCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGA ATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGT CTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG pMM376 ON-switch GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA P37829 GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGA AAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTA AAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCAC TGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATG GTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM377 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAG TGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAA AGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGT ACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTT TAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM378 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGT GAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAA GTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM379 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTA TGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACAT TTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTG CACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGAC ATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM380 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTTAAATATAAGGAGTAAGTCTGCCAGCA TTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAA CATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATA CTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAG GACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM381 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGAGAAGGAAGGTGCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGA ATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGT TCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACA CTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAA CTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM382 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGT AAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAGTG P37829 GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM386 ON-switch ATTTTAGACAAAATCAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTA GGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCC ACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG TG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM387 ON-switch ATTTTAGACAAAATCAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCT GCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATG TTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCT AGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTC CACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCA GTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM436 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAAGGCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATT ATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAAC ATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGCATAC TGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGG ACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM437 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAACTCACATTCCTTAAATATAAGGAGTAAGTCTGCC AGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTT TGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGG CATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCAC AGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM438 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAACACATTCCTTAAATATAAGGAGTAAGTCTGCCA GCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTT GAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAACCTAGGC ATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACA GAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM477 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGG AATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT pMM478 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA P37829 TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCAC AGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM479 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTCTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCAC AGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM480 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACAACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCAC AGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM481 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGTGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCAC AGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM482 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGCTCTAGTCAAGTTTAACTGGTGTCCAC AGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT pMM483 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCAC AGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM484 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGGCATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA pMM485 ON-switch TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG P37829 ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTTGAATTAGGAGGGGAGGATTCATTGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTAAAAAGTACATTAAAAGACTATCAACTTAATTTCTGA TCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCA TATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATT pMM464 ON-switch TTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATA AGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGT GGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTAGCAGACTTTTT TTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGC TCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCT CTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTAAAAAATAAATTAAAGTACATTAAAAGACTATCAACT TAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTT TTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAAC pMM465 ON-switch TTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTC CTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTT TATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGA AAGAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTA CACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAACTTTTAAAGTACATTAAAAG ACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAAC AAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTA pMM466 ON-switch TTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAGGT GCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTT TGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTA GAAAGTTGAAAGGTTAATAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAAC CTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTG TCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTG CAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAAGGGATAACTTTTAAAGT ACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGT CTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATC pMM467 ON-switch TATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAA GAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGTCTGCCAGCATTATGAAAGTG AATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAG TTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAACTAGTAGGCAGACCAGCAGACTTTTT TTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGC TCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCT CTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM468 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTAGCAGACTTTTTTTTATTGTGAT ATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGT TTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAAT TTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC pMM469 ON-switch CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT P37829 CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGAGACCAGCAGACTTTTT TTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGC TCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCT CTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM470 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATAGGCAGACCAG CAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACAT ATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTC GTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM471 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAGCAGACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT CATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM472 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGA CTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGA AGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCA AGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM473 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTAGACCAGCAGACTTTTTTTTATT GTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGT CAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTT CTAATTTCTCATTTGCAGTG GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM474 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGAGTAAGT CTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAA TGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTTAGGCAGACCAGCAGACTTTTT TTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGC TCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCT CTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAACTTTTAAAGTACATTAAAAG ACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAAC pMM567 ON-switch AAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTA AAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGA AAGTTGAAAGGTTAATAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCT P37829 AGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTC CACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCA GTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM568 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATAGGCAGACCAGCA GACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATAT GAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGT CAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM569 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGC CTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM570 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTTAGGCAGACCAGCAGACTTTTTTTT ATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCT AGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM591 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG GCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGC CTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM592 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM593 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAAAATATATAGGAGTAA GTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACA GAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGT CAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG P37829 GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM594 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGTCCTTATAAGGAGTAAGTCTGCCAGCATT ATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAAC ATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTATT GTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGT CAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTT CTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM595 ON-switch AATAATTAAAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGC CTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCAGGATTT pMM596 ON-switch TAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAG CATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTG AACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTT ATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCT AGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM597 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGAATAGGAGTAAGTCTGCCAGCATTATGAAAG TGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAA AGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATG GGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTA ACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTC TCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCATT pMM598 ON-switch TTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCC AGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTT TGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTT TTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCT CTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTC TGGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM599 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CAAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGC pMM600 ON-switch GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT P37829 TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAACAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGC CTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM601 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAGCCAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGC CTCTGGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCGCATC TTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATACTACTTG CTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATA AAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATAT ATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTA pMM642 ON-switch GACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGCA TTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAA CATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTAT TGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAG TCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGT TCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATACTACTT GCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAAT AAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATA TATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTT pMM643 ON-switch AGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGC ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTA TTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATACTACTTG CTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATA AAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATAT ATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTA pMM709 ON-switch GACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGCA TTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAA CATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTAT TGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAG TCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGT TCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGATGTGACTTTGTTTTGTAAATTTATAAAATACTACTT GCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAAT AAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATA TATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTT pMM644 ON-switch AGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGC ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTA TTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTAAATTTATAAAATACTACTT pMM645 ON-switch TATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTT AGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGC P37829 ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTA TTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATTTTGTTGAAT AAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATA TATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTT pMM646 ON-switch AGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGC ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTA TTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTGAAT AAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATA TATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTT pMM647 ON-switch AGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGC ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTA TTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT AAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATA TATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTT pMM648 ON-switch AGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGC ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTA TTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTATCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATA TATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTT pMM649 ON-switch AGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGC ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTA TTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTAAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTT pMM650 ON-switch AGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGC ATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGA ACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTA TTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATAAAATAAGT AAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTAT pMM651 ON-switch CTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAAT AAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATAT GGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTT P37829 AACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTT CTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM634 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAATGTTTTTG AACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTT ATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCT AGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM711 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT AACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTT ATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCT AGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM635 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTT ATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCT AGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM636 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGTACTAGTAGGCAGACCAGCAGACTTTTTTTT ATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCT AGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM637 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTA GTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM638 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT GTTCTAATTTCTCATTTGCAGTG pMM639 ON-switch GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA P37829 GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCGCACTGTACACTCTGACATATGAAGTGCTCT AGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM640 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCT GGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM641 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAAATTCGTCAAGCCTCTG GTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM712 ON-switch ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCT GGTTCTAATTTCTCATTTGCAGTG GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG pMM579 ON-switch ATTTTAGACAAAATCGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGCATTATGA AAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTA AAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGAT ATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGT TTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAAT TTCTCATTTGCAGTG GGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCAGCATCTTTTCCTGAC AATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATACTACTTGCTTCTCTCTT TATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAA AATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCT ATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCA pMM618 ON-switch AAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGCATTATGAAAGT GAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAA GTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATG GGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTA ACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTC TCATTTGCAGTG pMM619 ON-switch ACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAAC AAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTA P37829 TTTTTTTTAACTTCCTTTATTTTCCTTCCAGGATTTTAGACAAAATCAAAAAGAAGGAAGGT GCTCACATTCCTTAAATAGGAGTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGT AAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGA AAGTTACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCA TACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAG AGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAGTG CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT TGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAG CMV promoter TACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGG GGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAA CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCG TGTACGGTGGGAGGTCTATATAAGCAGAGCT ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTT TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAA GTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATT AGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCT CCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGAT GGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGG GCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCC GAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGC GCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCG CCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGC GGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCT CAG promoter TTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGA GCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCC GCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCG CAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGC TGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGG TGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGA GCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCG TGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCG GGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGT CGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAG GGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCC CCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGG GAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCT GTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTC TGGCGTGTGACCGGCGGC GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG GGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATA AGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT AAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTG CCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGG TTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGC TTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTT CGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCT GCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGG hEF1a promoter TATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGT TCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAA GCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGG GCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCC GGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTC CACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCAAGCTTTTGGAGTACGT CGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGT GGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTT TGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCA TTTCAGGTGTCGTGA hUbiC promoter GACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGAC ATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAAC AGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGT P37829 GAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGC CGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTAGC GGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAA GCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAA CTGGGGGTTGGGGGGAGCGCAGCAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGA CGCTTGTGAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGC AAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATG GGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGG TTTGTCGTCTGTTGCGGGGGCGGCAGTTATGGCGGTGCCGTTGGGCAGTGCACCCGTA CCTTTGGGAGCGCGCGCCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAAT GCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACG CAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAG GGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGT AGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTT TTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAGACTA GTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGAC AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACAT GCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACG RSV promoter ATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAA TTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGA CCATTCACCACATTGGTGTGCAC AAATGAGTCTTCGGACCTCGCGGGGGCCGCTTAAGCGGTGGTTAGGGTTTGTCTGACG CGGGGGGAGGGGGAAGGAACGAAACACTCTCATTCGGAGGCGGCTCGGGGTTTGGTC TTGGTGGCCACGGGCACGCAGAAGAGCGCCGCGATCCTCTTAAGCACCCCCCCGCCCT CCGTGGAGGCGGGGGTTTGGTCGGCGGGTGGTAACTGGCGGGCCGCTGACTCGGGC GGGTCGCGCGCCCCAGAGTGTGACCTTTTCGGTCTGCTCGCAGACCCCCGGGCGGCG CCGCCGCGGCGGCGACGGGCTCGCTGGGTCCTAGGCTCCATGGGGACCGTATACGTG TK promoter GACAGGCTCTGGAGCATCCGCACGACTGCGGTGATATTACCGGAGACCTTCTGCGGGA CGAGCCGGGTCACGCGGCTGACGCGGAGCGTCCGTTGGGCGACAAACACCAGGACGG GGCACAGGTACACTATCTTGTCACCCGGAGGCGCGAGGGACTGCAGGAGCTTCAGGGA GTGGCGCAGCTGCTTCATCCCCGTGGCCCGTTGCTCGCGTTTGCTGGCGGTGTCCCCG GAAGAAATATATTTGCATGTCTTTAGTTCTATGATGACACAAACCCCGCCCAGCGTCTTG TCATTGGCGAATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCCAGGTCCACTT CGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCT TAA AAGCTTGGGAGTTCCGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGC GGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACA TTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCC GGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGC PGK promoter CGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCC AGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAG CAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGG GGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCG GAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC CAG minimal promoter AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTA TTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCC CCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGCAGCGATGGGGGCGGGGGG GGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTAT GGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG GTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGA CCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAA CTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTC ATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAA Nluc coding sequence GGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGG TAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATC GCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAAT TATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACG GAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCG AATTCTCACGGCTTTCCGCCTGAGGTTGAAGAGCAAGCCGCCGGTACATTGCCTATGTC PEST coding sequence CTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTGCTTCAGCTCGCATC AACGTC Nluc-PEST coding sequence CCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAA CTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTC P37829 ATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAA GGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGG TAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATC GCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAAT TATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACG GAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGAATTCTCACGGCTTTCCGCC TGAGGTTGAAGAGCAAGCCGCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGT ATGGACCGGCACCCAGCCGCTTGTGCTTCAGCTCGCATCAACGTC Bipartite NLS AAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTC TGATAAAAAGTACTCAATAGGTCTGGACATAGGGACAAATTCAGTTGGTTGGGCAGTGAT CACAGATGAATACAAGGTTCCTTCTAAGAAGTTTAAGGTGCTGGGTAATACTGATCGACA CTCTATCAAGAAAAACTTGATTGGGGCACTTCTCTTCGACTCCGGGGAGACTGCAGAAG CTACACGGCTGAAGAGAACAGCTCGGCGACGATACACCAGACGAAAAAACCGCATTTGT TATCTTCAAGAAATCTTTTCCAATGAAATGGCAAAAGTGGATGATAGTTTCTTCCATCGGC TTGAGGAGAGTTTCTTGGTGGAGGAAGATAAGAAGCACGAAAGGCACCCAATATTTGGA AATATAGTTGATGAGGTAGCCTACCATGAGAAATACCCAACTATCTACCATCTTCGGAAA AAACTTGTGGACTCTACAGATAAGGCTGACTTGAGGCTCATTTATCTGGCACTGGCTCAC ATGATAAAATTCAGAGGTCATTTTTTGATCGAGGGTGACCTGAATCCAGATAACTCCGAT GTCGATAAACTCTTTATCCAACTCGTGCAGACATATAATCAGCTTTTTGAGGAAAATCCCA TAAACGCTTCTGGGGTTGACGCCAAAGCCATCCTCTCAGCTCGCCTTTCCAAGTCTCGA AGACTGGAAAATCTCATAGCTCAACTCCCAGGGGAAAAGAAAAACGGACTGTTCGGTAA TCTCATAGCTCTTTCACTCGGTCTGACTCCTAATTTTAAGAGCAATTTTGACCTTGCCGAA GACGCTAAACTTCAGCTCTCAAAGGACACTTATGACGATGATTTGGACAATTTGCTTGCC CAAATTGGTGACCAGTACGCTGACCTGTTTCTTGCCGCAAAAAATCTTTCAGATGCTATT CTCTTGAGTGACATCCTGAGAGTAAATACCGAAATAACCAAGGCCCCACTCAGTGCTTC CATGATTAAGCGCTATGACGAGCATCATCAAGACCTTACTCTGTTGAAGGCATTGGTAAG GCAGCAATTGCCAGAAAAATACAAAGAAATATTTTTCGACCAGTCAAAAAATGGTTATGC AGGTTACATCGACGGTGGCGCCTCACAGGAGGAGTTTTATAAGTTCATCAAACCTATCTT GGAGAAAATGGATGGCACCGAGGAACTCCTTGTCAAATTGAACCGGGAAGATTTGCTTA GGAAACAGCGGACATTTGATAACGGCAGCATACCACACCAAATCCATCTGGGTGAGCTG CATGCAATTCTCCGGCGACAGGAGGATTTTTACCCTTTCCTCAAAGATAATAGAGAAAAG ATCGAAAAGATTCTCACTTTCCGGATTCCCTACTATGTTGGGCCCCTCGCACGAGGCAAT TCTCGGTTTGCTTGGATGACAAGAAAGTCTGAAGAGACCATCACTCCCTGGAATTTTGAA GAAGTCGTAGACAAAGGGGCATCAGCACAGTCCTTCATAGAGCGGATGACAAATTTTGA TAAGAACCTTCCAAACGAGAAAGTCCTTCCAAAACACTCACTCTTGTACGAATACTTTACT GTATATAACGAACTGACAAAGGTTAAATATGTGACCGAGGGCATGCGAAAACCAGCCTT CCTTTCCGGTGAGCAAAAAAAGGCTATTGTCGATCTGTTGTTTAAGACAAATAGAAAAGT TACTGTGAAACAATTGAAGGAGGATTACTTTAAGAAAATCGAATGCTTTGATAGCGTGGA AATCTCTGGCGTGGAGGACCGCTTCAATGCATCATTGGGAACTTACCATGACCTGCTCA Cas9 coding sequence AGATTATCAAAGATAAAGATTTTCTGGATAATGAAGAAAATGAGGACATTCTTGAAGATAT TGTACTCACTCTGACCCTTTTTGAGGACAGGGAGATGATTGAAGAAAGACTGAAGACCTA CGCCCATCTGTTCGACGACAAAGTAATGAAGCAATTGAAGCGAAGGAGGTACACAGGGT GGGGACGGCTTTCTAGGAAGCTGATAAATGGCATCCGGGATAAACAGTCCGGCAAGAC TATACTGGATTTTCTGAAGTCTGACGGTTTTGCCAATCGCAATTTCATGCAATTGATACAC GACGATTCACTGACCTTTAAGGAGGATATTCAGAAGGCACAGGTTTCAGGGCAAGGTGA CAGTTTGCACGAGCATATTGCAAATCTCGCAGGGTCTCCCGCCATAAAGAAGGGGATAC TGCAGACAGTTAAGGTCGTAGATGAGCTGGTGAAGGTGATGGGGCGGCACAAGCCCGA GAATATCGTTATTGAAATGGCACGAGAAAATCAAACAACTCAAAAAGGTCAGAAGAACTC ACGAGAACGGATGAAGCGCATTGAAGAAGGTATCAAGGAACTCGGTAGCCAAATATTGA AGGAACACCCTGTTGAAAACACCCAGCTGCAAAACGAGAAGTTGTACCTGTATTATCTGC AGAATGGTAGAGATATGTATGTAGATCAAGAACTCGACATTAACCGACTTTCTGATTACG ATGTTGACCATATCGTTCCACAATCCTTTCTTAAGGATGATTCCATTGACAATAAGGTCTT GACACGCTCCGATAAGAATCGAGGTAAAAGTGATAATGTACCTTCAGAGGAGGTGGTGA AGAAGATGAAAAACTACTGGCGCCAGCTCCTTAATGCCAAGCTCATTACTCAACGAAAAT TTGACAATTTGACCAAGGCTGAACGGGGAGGACTTTCAGAGCTGGATAAAGCCGGATTT ATTAAGCGCCAACTTGTAGAAACTAGGCAAATCACAAAGCACGTTGCCCAGATCCTCGAT AGTCGAATGAATACCAAGTATGATGAGAATGACAAACTCATTAGAGAGGTTAAGGTTATT ACTCTTAAGAGTAAGCTCGTTTCTGATTTCAGGAAAGATTTTCAATTTTATAAAGTCCGAG AAATCAACAATTACCACCACGCACACGACGCTTACCTCAATGCAGTAGTCGGGACAGCT CTCATCAAGAAATATCCCAAATTGGAATCCGAATTTGTTTATGGGGATTACAAAGTGTAC GATGTAAGGAAAATGATAGCTAAATCAGAGCAGGAGATTGGCAAGGCTACTGCAAAGTA TTTCTTCTACTCAAACATAATGAATTTCTTTAAAACAGAGATTACTCTCGCCAACGGAGAA ATCCGAAAACGGCCTCTCATCGAAACAAACGGCGAGACTGGAGAAATCGTTTGGGACAA AGGCCGAGATTTTGCTACTGTTAGAAAGGTACTGTCAATGCCTCAAGTTAATATAGTAAA GAAAACCGAGGTACAAACCGGGGGCTTTTCTAAAGAATCAATACTCCCTAAGCGAAATTC TGATAAGCTCATTGCCAGAAAGAAAGATTGGGACCCAAAGAAGTATGGCGGGTTCGACT CTCCTACAGTCGCATACAGCGTGTTGGTAGTCGCTAAAGTAGAGAAAGGGAAATCAAAA AAACTTAAGTCTGTAAAAGAATTGTTGGGTATCACTATAATGGAACGGTCAAGTTTCGAG AAGAACCCCATAGACTTCCTGGAGGCCAAAGGATACAAAGAAGTCAAGAAAGACCTCAT CATCAAGCTCCCAAAATATTCTCTTTTCGAGTTGGAAAACGGCCGGAAGAGGATGCTCG P37829 CTAGTGCTGGGGAGCTCCAAAAAGGAAACGAGTTGGCACTCCCCAGTAAGTATGTAAAC TTTCTTTACCTGGCAAGCCACTACGAGAAGTTGAAGGGAAGTCCTGAGGATAACGAGCA GAAACAACTGTTCGTGGAGCAACATAAACATTACCTTGACGAGATAATTGAGCAAATTAG CGAGTTTTCAAAGCGCGTAATCCTGGCCGACGCAAACCTCGACAAAGTACTTAGCGCAT ACAACAAACACAGGGACAAACCAATCCGAGAACAAGCTGAAAACATCATACATTTGTTTA CCCTCACAAACTTGGGTGCACCCGCAGCATTTAAGTACTTTGACACAACCATTGACAGG AAGAGATATACCAGTACCAAAGAAGTACTGGATGCCACCTTGATTCACCAATCTATTACC GGTCTGTATGAGACCAGAATAGATCTGAGTCAGCTGGGAGGAGAT Linker coding sequence GGGAGTGG 3xSV40_NLS GCCCCCAAGAAAAAAAGGAAGGTTGGTAGCGCAGGCAGTGCCGCAGGGTCTGGTCCAA AAAAAAAGCGGAAGGTCGAGGACCCTAAGAAGAAACGGAAAGTGGAC AAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCGGGA GTGGTGATAAAAAGTACTCAATAGGTCTGGACATAGGGACAAATTCAGTTGGTTGGGCA GTGATCACAGATGAATACAAGGTTCCTTCTAAGAAGTTTAAGGTGCTGGGTAATACTGAT CGACACTCTATCAAGAAAAACTTGATTGGGGCACTTCTCTTCGACTCCGGGGAGACTGC AGAAGCTACACGGCTGAAGAGAACAGCTCGGCGACGATACACCAGACGAAAAAACCGC ATTTGTTATCTTCAAGAAATCTTTTCCAATGAAATGGCAAAAGTGGATGATAGTTTCTTCC ATCGGCTTGAGGAGAGTTTCTTGGTGGAGGAAGATAAGAAGCACGAAAGGCACCCAATA TTTGGAAATATAGTTGATGAGGTAGCCTACCATGAGAAATACCCAACTATCTACCATCTT CGGAAAAAACTTGTGGACTCTACAGATAAGGCTGACTTGAGGCTCATTTATCTGGCACTG GCTCACATGATAAAATTCAGAGGTCATTTTTTGATCGAGGGTGACCTGAATCCAGATAAC TCCGATGTCGATAAACTCTTTATCCAACTCGTGCAGACATATAATCAGCTTTTTGAGGAAA ATCCCATAAACGCTTCTGGGGTTGACGCCAAAGCCATCCTCTCAGCTCGCCTTTCCAAG TCTCGAAGACTGGAAAATCTCATAGCTCAACTCCCAGGGGAAAAGAAAAACGGACTGTT CGGTAATCTCATAGCTCTTTCACTCGGTCTGACTCCTAATTTTAAGAGCAATTTTGACCTT GCCGAAGACGCTAAACTTCAGCTCTCAAAGGACACTTATGACGATGATTTGGACAATTTG CTTGCCCAAATTGGTGACCAGTACGCTGACCTGTTTCTTGCCGCAAAAAATCTTTCAGAT GCTATTCTCTTGAGTGACATCCTGAGAGTAAATACCGAAATAACCAAGGCCCCACTCAGT GCTTCCATGATTAAGCGCTATGACGAGCATCATCAAGACCTTACTCTGTTGAAGGCATTG GTAAGGCAGCAATTGCCAGAAAAATACAAAGAAATATTTTTCGACCAGTCAAAAAATGGT TATGCAGGTTACATCGACGGTGGCGCCTCACAGGAGGAGTTTTATAAGTTCATCAAACC TATCTTGGAGAAAATGGATGGCACCGAGGAACTCCTTGTCAAATTGAACCGGGAAGATT TGCTTAGGAAACAGCGGACATTTGATAACGGCAGCATACCACACCAAATCCATCTGGGT GAGCTGCATGCAATTCTCCGGCGACAGGAGGATTTTTACCCTTTCCTCAAAGATAATAGA GAAAAGATCGAAAAGATTCTCACTTTCCGGATTCCCTACTATGTTGGGCCCCTCGCACGA GGCAATTCTCGGTTTGCTTGGATGACAAGAAAGTCTGAAGAGACCATCACTCCCTGGAA TTTTGAAGAAGTCGTAGACAAAGGGGCATCAGCACAGTCCTTCATAGAGCGGATGACAA ATTTTGATAAGAACCTTCCAAACGAGAAAGTCCTTCCAAAACACTCACTCTTGTACGAATA CTTTACTGTATATAACGAACTGACAAAGGTTAAATATGTGACCGAGGGCATGCGAAAACC Bipartite NLS-Cas9- AGCCTTCCTTTCCGGTGAGCAAAAAAAGGCTATTGTCGATCTGTTGTTTAAGACAAATAG Linker-3xSV40_NLS- AAAAGTTACTGTGAAACAATTGAAGGAGGATTACTTTAAGAAAATCGAATGCTTTGATAG STOP CGTGGAAATCTCTGGCGTGGAGGACCGCTTCAATGCATCATTGGGAACTTACCATGACC TGCTCAAGATTATCAAAGATAAAGATTTTCTGGATAATGAAGAAAATGAGGACATTCTTGA AGATATTGTACTCACTCTGACCCTTTTTGAGGACAGGGAGATGATTGAAGAAAGACTGAA GACCTACGCCCATCTGTTCGACGACAAAGTAATGAAGCAATTGAAGCGAAGGAGGTACA CAGGGTGGGGACGGCTTTCTAGGAAGCTGATAAATGGCATCCGGGATAAACAGTCCGG CAAGACTATACTGGATTTTCTGAAGTCTGACGGTTTTGCCAATCGCAATTTCATGCAATT GATACACGACGATTCACTGACCTTTAAGGAGGATATTCAGAAGGCACAGGTTTCAGGGC AAGGTGACAGTTTGCACGAGCATATTGCAAATCTCGCAGGGTCTCCCGCCATAAAGAAG GGGATACTGCAGACAGTTAAGGTCGTAGATGAGCTGGTGAAGGTGATGGGGCGGCACA AGCCCGAGAATATCGTTATTGAAATGGCACGAGAAAATCAAACAACTCAAAAAGGTCAGA AGAACTCACGAGAACGGATGAAGCGCATTGAAGAAGGTATCAAGGAACTCGGTAGCCAA ATATTGAAGGAACACCCTGTTGAAAACACCCAGCTGCAAAACGAGAAGTTGTACCTGTAT TATCTGCAGAATGGTAGAGATATGTATGTAGATCAAGAACTCGACATTAACCGACTTTCT GATTACGATGTTGACCATATCGTTCCACAATCCTTTCTTAAGGATGATTCCATTGACAATA AGGTCTTGACACGCTCCGATAAGAATCGAGGTAAAAGTGATAATGTACCTTCAGAGGAG GTGGTGAAGAAGATGAAAAACTACTGGCGCCAGCTCCTTAATGCCAAGCTCATTACTCA ACGAAAATTTGACAATTTGACCAAGGCTGAACGGGGAGGACTTTCAGAGCTGGATAAAG CCGGATTTATTAAGCGCCAACTTGTAGAAACTAGGCAAATCACAAAGCACGTTGCCCAG ATCCTCGATAGTCGAATGAATACCAAGTATGATGAGAATGACAAACTCATTAGAGAGGTT AAGGTTATTACTCTTAAGAGTAAGCTCGTTTCTGATTTCAGGAAAGATTTTCAATTTTATAA AGTCCGAGAAATCAACAATTACCACCACGCACACGACGCTTACCTCAATGCAGTAGTCG GGACAGCTCTCATCAAGAAATATCCCAAATTGGAATCCGAATTTGTTTATGGGGATTACA AAGTGTACGATGTAAGGAAAATGATAGCTAAATCAGAGCAGGAGATTGGCAAGGCTACT GCAAAGTATTTCTTCTACTCAAACATAATGAATTTCTTTAAAACAGAGATTACTCTCGCCA ACGGAGAAATCCGAAAACGGCCTCTCATCGAAACAAACGGCGAGACTGGAGAAATCGTT TGGGACAAAGGCCGAGATTTTGCTACTGTTAGAAAGGTACTGTCAATGCCTCAAGTTAAT ATAGTAAAGAAAACCGAGGTACAAACCGGGGGCTTTTCTAAAGAATCAATACTCCCTAAG CGAAATTCTGATAAGCTCATTGCCAGAAAGAAAGATTGGGACCCAAAGAAGTATGGCGG GTTCGACTCTCCTACAGTCGCATACAGCGTGTTGGTAGTCGCTAAAGTAGAGAAAGGGA P37829 AATCAAAAAAACTTAAGTCTGTAAAAGAATTGTTGGGTATCACTATAATGGAACGGTCAA GTTTCGAGAAGAACCCCATAGACTTCCTGGAGGCCAAAGGATACAAAGAAGTCAAGAAA GACCTCATCATCAAGCTCCCAAAATATTCTCTTTTCGAGTTGGAAAACGGCCGGAAGAG GATGCTCGCTAGTGCTGGGGAGCTCCAAAAAGGAAACGAGTTGGCACTCCCCAGTAAG TATGTAAACTTTCTTTACCTGGCAAGCCACTACGAGAAGTTGAAGGGAAGTCCTGAGGAT AACGAGCAGAAACAACTGTTCGTGGAGCAACATAAACATTACCTTGACGAGATAATTGAG CAAATTAGCGAGTTTTCAAAGCGCGTAATCCTGGCCGACGCAAACCTCGACAAAGTACT TAGCGCATACAACAAACACAGGGACAAACCAATCCGAGAACAAGCTGAAAACATCATAC ATTTGTTTACCCTCACAAACTTGGGTGCACCCGCAGCATTTAAGTACTTTGACACAACCA TTGACAGGAAGAGATATACCAGTACCAAAGAAGTACTGGATGCCACCTTGATTCACCAAT CTATTACCGGTCTGTATGAGACCAGAATAGATCTGAGTCAGCTGGGAGGAGATGCCCCC AAGAAAAAAAGGAAGGTTGGTAGCGCAGGCAGTGCCGCAGGGTCTGGTCCAAAAAAAA AGCGGAAGGTCGAGGACCCTAAGAAGAAACGGAAAGTGGACTGAA GCCGCCGCCGCCGCCGCCGCGCCGAGCGGAGGAGGAGGAGGAGGCGAGGAGGAGA GACTGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAA GTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAGCCCGTGC AGCCATCAGCCCACCACTCTGCTGAGCCCGCAGAGGCAGGCAAAGCAGAGACATCAGA AGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCG CTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGG ACACGGAAGCTTAAGCAAAGGAAATCTGGCCGCTCTGCTGGGAAGTATGATGTGTATTT GATCAATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAA GGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGA GCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGG AACTGGCAGAGGCCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCGGC CACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTT MeCP2 coding sequence GTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCA CATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCCGA CCCTCAGGCCATTCCCAAGAAACGGGGCCGAAAGCCGGGGAGTGTGGTGGCAGCCGC TGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAG ACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTCAAGG AAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAA GACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAG CAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCC CCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGA GCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAA AGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCCCCAAGGAGCC AGCTAAGACTCAGCCCGCGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTACAAACAC CGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAG AGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCTGA GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAGGTAAGTAATCACTCA GCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATTTATAAAATAC TACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGATCATATTTTGT TGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAGG ATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGAGTAAGTCTG CCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGT TTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAGACTTT TTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGT GCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGC CTCTGGTTCTAATTTCTCATTTGCAGTGGCCGCCGCCGCCGCCGCCGCGCCGAGCGGA GGAGGAGGAGGAGGCGAGGAGGAGAGACTGGAAGAAAAGTCAGAAGACCAGGACCTC CAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGA GAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCGCA pMM569 ON-switch with GAGGCAGGCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAA MeCP2 coding sequence GCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATG ATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGCTTAAGCAAAGGAAATCTGGCCG CTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAA AGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATT TTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAA GAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCCCCAAAGG GAGCGGCACCACGAGACCCAAGGCGGCCACGTCAGAGGGTGTGCAGGTGAAAAGGGT CCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGG GCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATCAAACGCC CCGGCAGGAAGCGAAAAGCTGAGGCCGACCCTCAGGCCATTCCCAAGAAACGGGGCC GAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGA AGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACC CGGGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCC TCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAA GGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCA CCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCACCC P37829 CTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGC CCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACT GGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCGC CGCCACGGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTT TCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGA CCGAGAGAGTTAGCTGA 45 nucleotide SMN2 GCAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATCAG exon 6 comprising ATG>GGC at positions 82 to 84, and AT>CA 109 to 110 (SMN2 exon 6 variant 4) 45 nucleotide SMN2 GCAATTTCCTGGTACGGCAGTGGCTATCATACTGGCTATTATCAG exon 6 comprising A>C at position 75, ATG>GGC at positions 82 to 84, and AT>CA 109 to 110 (SMN2 exon 6 variant 5) SMN2 exon 6 variant GCAATTTCX1TGGTACGGCAGTGGCTATCATACTGGCTATTATCAG consensus 3 wherein X1 = A or C SMN2 exon 6 variant GCX1-2AATTTCX3TGGTACGGCAGTGGCTATCATACTGGCTATTX4-5X6-7G consensus 4 wherein X1-2 = absent or TT; X3 = A or C; X4-5 = AT or CA; X6-7 = AT, CT or CA SMN2 exon 7 comprising GATTTTAGACAAAATCGGTGCTCACATTCCTTAAATATAAGGA G>A at position 2, deletion of AAAAAGAAGGAA at positions 17-28, and insertion of A after position 48 (exon 7 variant 4) SMN2 exon 7 comprising GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGA G>A at position 2, and insertion of TA after position 48 (exon 7 variant 5) SMN2 exon 7 variant GATTTTAGACAAAATCX1-12GGTGCTCACATTCCTTAAATAX13-15GGA consensus 3 wherein X1-12 = absent or AAAAAGAAGGAA; X13-15 = TAA or absent SMN2 exon 7 variant GATTTTX1-6AX7-8CAAAATCX9-20GGTGCTCACATTCCTTAAATAX21-23GGA consensus 4 wherein X1-6 = absent or GCCACC; X7-8 = GA or TG; X9-20 = absent, AAAAAGAAGGAA or AAAAGAAGGAA; X21-23 = TAA or absent 277 nucleotide SMN2 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA intron 7 (intron 7 variant AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA 2) GCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGAC ATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAAT TCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG 260 nucleotide SMN2 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT intron 6, comprising AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT deletion of CCTT at CTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACA positions 5762-5765 of TCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTT intron 6, and A>C 4 TATTTTCCAG positions from the 3’ end (position 5766 of intron 6) (intron 6 variant 3) GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT SMN2 intron 6 variant CTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACA consensus TCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTT TATTTTCCX1-4AG wherein X1-6 = absent or TTCC SMN2 exon 6 variant 6 GATTCTCTTGATGATGCTGATGCTTTGGGAAGTATGTTAATTTCATGGTACATGAGTGGC P37829 TATCATACTGGCTATTATATG SMN2 exon 6 variant 7 AGTATGTTAATTTCATGGTACATGAGTGGCTATCATACTGGCTATTATATG SMN2 exon 6 variant 8 ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATGTG SMN2 exon 6 variant 9 ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATTTG SMN2 exon 6 variant 10 ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATATG SMN2 exon 6 variant 11 ATAATTCCCCCACCACCTCCCATATGTCCAGATTCTCTTGATGATGCTGATGCTTTGGGA AGTGGCTTAATTTCATGGTACGGCAGTGGCTATCATACTGGCTATTATTAG SMN2 exon 6 variant 12 ACTGGCTATTATCAG SMN2 exon 6 variant 13 GGCAGTGGCTATCATACTGGCTATTATCAG SMN2 intron 6 variant 4 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATAC AACTGTCTGAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAA AAGCTTTCATGCTATCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGT ACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGT CTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATC TATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTACAG SMN2 intron 6 variant 5 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGC TATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCC AG SMN2 intron 6 variant 6 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAATAC AACTGTCTGAGGCTTAAATTACTCTTGCATTGTCCCTAAGTATAATTTTAGTTAATTTTAAA AAGCTTTCATGCTATTGTTAGATTATTTTGATTATACACTTTTGAATTGAAATTATACTTTTT CTAAATAATGTTTTAATCTCTGATTTGAAATTGATTGTAGGGAATGGAAAAGATGGGATAA TTTTTCATAAATGAAAAATGAAATTCTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTTGC TCTGTTGCCCAGGCTGGAGTGCAATGGCGTGATCTTGGCTCACAGCAAGCTCTGCCTCC TGGATTCACGCCATTCTCCTGCCTCAGCCTCAGAGGTAGCTGGGACTACAGGTGCCTGC CACCACGCCTGGCTAGCTGGGATTAGAGGTCCCCACCACCATGCCTGGCTAATTTTTTG TACTTTCAGTAGAAACGGGGTTTTGCCATGTTGGCCAGGCTGTTCTCGAACTCCTGAGC TCAGGTGATCCAACTGTCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTG TGCCTAGCCTGAGCCACCACGCCGGCCTAATTTTTAAATTTTTTGTAGAGACAGGGTCTC ATTATGTTGCCCAGGGTGGTGTCAAGCTCCAGGTCTCAAGTGATCCCCCTACCTCCGCC TCCCAAAGTTGTGGGATTGTAGGCATGAGCCACTGCAAGAAAACCTTAACTGCAGCCTA ATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTCTGA TCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCA TATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATT TTCCTTCCAG SMN2 intron 6 variant 7 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAAAAAGTACATTAAAAGACTAT CAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAAT GCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTT TTAACTTCCTTTATTTTCCTTCCAG SMN2 intron 6 variant 8 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAATTAAAGTACATTA AAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTG AAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATA GCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAG SMN2 intron 6 variant 9 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAACTTTTA AAGTACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAA ATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTA TATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAG SMN2 intron 6 variant 10 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTAAAAAATAAAAATAAAAAAAGG GATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATA AAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATAT ATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAG SMN2 intron 6 variant 11 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT CTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACA TCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTT TATTTTCCTTCCA SMN2 intron 6 variant 12 GTAAGTAATCGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATT TATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGAT CATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCAT P37829 ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 13 GTAAGTAATCACTCAGCATCTTTTCAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATT TATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGAT CATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 14 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGATGTGACTTTGTTTTGTAAATT TATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGAT CATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 15 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTAAATT TATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGAT CATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 16 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT CTGATCATATTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 17 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT CTGATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 18 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT CTGATCATATTTTGTAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 19 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT CTGATCATATTTTGTTGAATAAAATAAGTATCTTGTGAAACAAAATGCTTTTTAACATCCAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 20 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT CTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACA TCCATATAAAGCTATCTAAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG SMN2 intron 6 variant 21 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT CTGATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACATCCATATAAAGCTAT CTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTTTCCTTCCAG SMN2 exon 7 variant 6 GGTTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 7 GATTTTAGACAAAATCAAAAAGAAGGTAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 8 GATTTTAGACAAAATCAAAAAGAAGGATGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 9 GATTTTAGACAAAATCAAAAGGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 10 GATTTTAGACAAAATCAAAAAGAAGGAAAGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 11 GATTTTAGACAAAATCAAAATGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 12 GATTTTAGACAAAATCAAAAAGAGGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 13 GATTTTAGACAAAATCAAAAAGAAGGAAGGAGCACACATTCCTTAAATATAAGGA SMN2 exon 7 variant 14 GATTTTAGAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 15 GATTTTAGAGAAGGAAGGTGCTTAAATATAAGGA SMN2 exon 7 variant 16 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAAGA SMN2 exon 7 variant 17 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATGATGA SMN2 exon 7 variant 18 GATTTTAGACAAAATCAAAAAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 19 GATTTTAGACAAAATCCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 20 GATTTTAGACTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 21 GATTTTAGACAAAATCGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 22 GATTTTAGACAAAATCTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 23 GATTTTAGACAAAATCGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 24 SMN2 exon 7 variant 25 SMN2 exon 7 variant 26 GATTTTAGACAAAATCGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 27 GATTTTAGACAAAATCGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA P37829 SMN2 exon 7 variant 28 GATTTTAGACAAAATCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 29 GATTTTAGACAAAATCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 30 GATTTTAGACAAAATCATTCCTTAAATATAAGGA SMN2 exon 7 variant 31 GATTTTAGACAAAATCGGTGCTTAAATATAAGGA SMN2 exon 7 variant 32 GATTTTAGACAAAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 33 GATTTTAGACGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 34 GATTTTAGAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 35 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTTAAATATAAGGA SMN2 exon 7 variant 36 GATTTTAGCTTAAATATAAGGA SMN2 exon 7 variant 37 GATTTTAGACAAAATCAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 38 GATTTTAGACAAAATCAAAAAAGGCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 39 GATTTTAGACAAAATCAAAAAGAAGGAACTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 40 GATTTTAGACAAAATCAAAAAGAAGGAACACATTCCTTAAATATAAGGA SMN2 exon 7 variant 41 GATTTTAGACAAAATCGGTGCTCACATTCCTTAAATAGGA SMN2 exon 7 variant 42 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAAAATATATAGGA SMN2 exon 7 variant 43 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGTCCTTATAAGGA SMN2 exon 7 variant 44 GAATAATTAAAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGA SMN2 exon 7 variant 45 TTTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATAGGA SMN2 exon 7 variant 46 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAACAGGA SMN2 exon 7 variant 47 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAGCCAGGA SMN2 exon 7 variant 48 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCCACCATGTCCTTAAATATAAGGA SMN2 exon 7 variant 49 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAGCCACCATGATAAG GA SMN2 exon 7 variant 50 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAACAACATGGA SMN2 exon 7 variant 51 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACAGCCACCATGAATATAAGGA SMN2 exon 7 variant 52 GATTTTAGAGCCACCATGCAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATA AGGA SMN2 exon 7 variant 53 GATTTTAGACAAAATAGCACCATGCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATA AGGA SMN2 exon 7 variant 54 GATTTTAGACAAAATAGCACCATGGAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATA AGGA SMN2 exon 7 variant 55 GATTTTAGACAAAATCAAAAAATGGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 56 GATTTTAGACAAAATCACCATGGAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAG GA SMN2 exon 7 variant 57 GATTTTAGACAAAATATGGAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 58 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAATGGA SMN2 exon 7 variant 59 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATGGTAAGGA SMN2 exon 7 variant 60 GATTTTAGACAAAATCAAAAAGAAGGAAGGATGGCGTCACATTCCTTAAATATAAGGA SMN2 exon 7 variant 61 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTATGGATATAAGGA SMN2 exon 7 variant 62 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATATAAGATGA SMN2 exon 7 variant 63 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATGTCCTTAAATATAAGGA SMN2 exon 7 variant 64 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATGAGGA SMN2 exon 7 variant 65 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATGATAAGGA SMN2 exon 7 variant 66 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATGGCCTTAAATATAAGGA SMN2 exon 7 variant 67 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTAACATGGCCTTAAATATAAGGA SMN2 exon 7 variant 68 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATATGCCTAAATTAAGGA SMN2 exon 7 variant 69 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCATGTTAAATATAAGGA SMN2 exon 7 variant 70 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTATGAATATAAGGA SMN2 exon 7 variant 71 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTATGGAAAATATAAGGA SMN2 exon 7 variant 72 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAAATTAAGATGA SMN2 exon 7 variant 73 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTAAAATGAGGA SMN2 exon 7 variant 74 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTGAAATGAGGA SMN2 exon 7 variant 75 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCAAAATGAGGA SMN2 exon 7 variant 76 GATTTTAGACAAAATCAAAAAGAAGGAAGGAATAGGA SMN2 exon 7 variant 77 GATTTTAGACAAAATCAAAAAGAAGGAAGGTGCTCACATTCCTTAGCCACCATGGA SMN2 intron 7 variant 3 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAG AGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 4 GTAAGTCTGCTCCTCTTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT CATTTGCAG SMN2 intron 7 variant 5 AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT P37829 CATTTGCAG SMN2 intron 7 variant 6 GTAAGTCTGCTAGTCTTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT CATTTGCAG SMN2 intron 7 variant 7 GTAAGTCTGCTTTCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTG GAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATA TGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTT TAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATT TCTCATTTGCAG SMN2 intron 7 variant 8 GTAAGTCTGCCCCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTG GAAAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATA TGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTT TAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATT TCTCATTTGCAG SMN2 intron 7 variant 9 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAA ACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCC TACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAG AGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTAT TGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAG TCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGT TCTAATTTCTCATTTCCAG SMN2 intron 7 variant 10 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAA ACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCC TACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAG AGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTAT TGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAG TCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGATCTGACATATGAAGTGCT CTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTC TGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 11 GTAAGTGGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAA ACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGGG ATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAAC TGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTC ATTTGCAG SMN2 intron 7 variant 12 GTAAGATTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT CATTTGCAG SMN2 intron 7 variant 13 GTAAGTGTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT CATTTGCAG SMN2 intron 7 variant 14 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTT AACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 15 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGT GTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAG SMN2 intron 7 variant 16 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTCTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGT GTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAG SMN2 intron 7 variant 17 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACAACATATGAAGTGCTCTAGTCAAGTTTAACTGGT GCAG SMN2 intron 7 variant 18 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG P37829 GATAACCTAGGCATACTGCACTGTACACTCTGTGAAGTGCTCTAGTCAAGTTTAACTGGT GTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAG SMN2 intron 7 variant 19 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTCTGACATATGCTCTAGTCAAGTTTAACTGGT GTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAG SMN2 intron 7 variant 20 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTATATGAAGTGCTCTAGTCAAGTTTAACTGGT GTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTT GCAG SMN2 intron 7 variant 21 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTGCACTGTACACTCTGGCATATGAAGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT CATTTGCAG SMN2 intron 7 variant 22 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAACTTTTTTTTATTGTGATATGG GATAACCTAGGCATACTTGAATTAGGAGGGGAGGATTCATTGTGCTCTAGTCAAGTTTAA CTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCT CATTTGCAG SMN2 intron 7 variant 23 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTAGCAGACTTTTTTTTAT TGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAG TCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGT TCTAATTTCTCATTTGCAG SMN2 intron 7 variant 24 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGAGACCAGCAGA CTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGA AGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCA AGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 25 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATAGGCA GACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACT CTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACT GGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 26 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAA CTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGC ACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACA TGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 27 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAGCAGACTTTTTTTTATTGTGA TATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAG TTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAA TTTCTCATTTGCAG SMN2 intron 7 variant 29 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTAGACCAGCAGACTTTT TTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTG CTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCC TCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 30 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTTAGGCAGACCAGCAGA CTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGA AGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCA AGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 31 GTAAGTCTGGCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA GCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGAC ATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAAT TCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 32 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA ATATGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 33 GTAAGTCTGCAAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA P37829 AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA GCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGAC ATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAAT TCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 34 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA ATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAG ACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATG AAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 35 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCAGCAG ACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATG AAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 36 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTGTTAGAAAGTTACTAGTAGGCAGACCAGCAGA CTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGA AGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCA AGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 37 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGTACTAGTAGGCAGACCAGCAG ACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATG AAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 38 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA GCAGACTTTTTTTTATTGTGATATAACCTAGGCATACTGCACTGTACACTCTGACATATGA AGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCA AGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 39 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA GCAGACTTTTTTTTATTGTGATATGGGATAGGCATACTGCACTGTACACTCTGACATATGA AGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCA AGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 40 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA GCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCGCACTGTACACTCTGACATATG AAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 41 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA GCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGAC ATATGAAGTGCTCTAGTCTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 42 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA GCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGAC ATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 intron 7 variant 43 GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTACTAGTAGGCAGACCA GCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGAC ATATGAAGTGCTCTAGTCAAGTTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTC AAGCCTCTGGTTCTAATTTCTCATTTGCAG SMN2 exon 8 variant 3 GAAATGCTGGCATAGAG SMN2 exon 8 variant 4 GAAATGCTGGC SMN2 exon 8 variant 5 GAAATGCT SMN2 exon 8 variant 6 GAAAT SMN2 intron 6 variant 22 GTAAGTAATCACTCAGCATCTTTTCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGT AAATTTATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTT CTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAAACAAAATGCTTTTTAACA TCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTT TATTTTCCTTCCAT SMN2 intron 6 variant 23 GTAAGTAATCACTCAGCATCCTGACAATTTTTTTGTAGTTATGTGACTTTGTTTTGTAAATT TATAAAATACTACTTGCTTCTCTCTTTATATTACTTTAAAAGACTATCAACTTAATTTCTGAT ATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTATTT TCCTTCCAG 3’ 162 positions of SMN2 TTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTT P37829 intron 6, comprising GTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTAT deletion of TTCC from 6 ATAGCTATTTTTTTTAACTTCCTTTATTTTCCAG positions upstream of 3’ end TTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTT 3’ 162 positions of SMN2 GTGAAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTAT intron 6, consensus ATAGCTATTTTTTTTAACTTCCTTTATTTTCCX1-X4AG wherein X1-4 = absent or TTCC Positions 1 to 102 of SMN2 intron 7, GTAAGTGTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA comprising C>G at AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAA position 7 Positions 1 to 102 of GTAAGTX1TGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGA SMN2 intron 7, AAACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAA consensus wherein X1 = C or G GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAA AACAAATGTTTTTGAACATTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAA ACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCC SMN2 intron 7 variant 44 TACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAG AGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTAT TGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAG TCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGT TCTAATTTCTTTTTTTCAG Numbered statements The following numbered paragraphs (paras) describe particular aspects and embodiments of the present disclosure: 1. A polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:222, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:378 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:226; (iv) a fourth nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:380 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end; (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘GA’, ‘TG’ or ‘TT’, or (b) comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:19, wherein the nucleotide sequence comprises ‘GA’, ‘TG’ or ‘TT’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:19, or (c) encoding a polypeptide of interest, and comprising ‘GA’, ‘TG’ or ‘TT’ at positions 1 and 2; and (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a) or (v)(b), a sixth nucleotide sequence encoding a polypeptide wherein the polynucleotide comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. P37829 2. The polynucleotide according to para 1, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. 3. The polynucleotide according to para 1 or para 2, wherein the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:222. 4. The polynucleotide according to any one of paras 1 to 3, wherein the first nucleotide sequence comprises, or consists of, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:106, SEQ ID NO:219 or SEQ ID NO:220. 5. The polynucleotide according to any one of paras 1 to 4, wherein the second nucleotide sequence comprises SEQ ID NO:7 at its 5’ end, and comprises SEQ ID NO:8 or SEQ ID NO:377 at its 3’ end. 6. The polynucleotide according to any one of paras 1 to 5, wherein the second nucleotide sequence consists of fewer than 500 nucleotides. 7. The polynucleotide according to any one of paras 1 to 6, wherein the second nucleotide sequence comprises, or consists of, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:228. 8. The polynucleotide according to any one of paras 1 to 7, wherein the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:226. 9. The polynucleotide according to any one of paras 1 to 8, wherein the third nucleotide sequence consists of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:223 or SEQ ID NO:224. 10. The polynucleotide according to any one of paras 1 to 9, wherein the fourth nucleotide sequence comprises SEQ ID NO:15 or SEQ ID NO:379 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end. 11. The polynucleotide according to any one of paras 1 to 10, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides. 12. The polynucleotide according to any one of paras 1 to 11, wherein the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:227 or SEQ ID NO:340. 13. The polynucleotide according to any one of paras 1 to 12, wherein the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’ or ‘TG’, or (b) comprises, or consists of, a nucleotide sequence according to SEQ ID NO:19. P37829 14. The polynucleotide according to any one of paras 1 to 13, wherein the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’ or ‘TG’, or (b) comprises, or consists of, SEQ ID NO:20 or SEQ ID NO:21. 15. The polynucleotide according to any one of paras 1 to 14, wherein the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:164 and SEQ ID NO:171. 16. The polynucleotide according to any one of paras 1 to 15, wherein the polynucleotide further comprises a promoter sequence 5’ to the start codon. 17. The polynucleotide according to any one of paras 1 to 16, wherein the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a polypeptide of interest. 18. The polynucleotide according to any one of paras 1 to 17, wherein the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. 19. A vector comprising a polynucleotide according to any one of paras 1 to 18. 20. The vector according to para 19, wherein the vector is an adeno-associated virus (AAV) vector. 21. A pharmaceutical composition comprising a polynucleotide according to any one of paras 1 to 18, or a vector according to para 19 or para 20, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. 22. A cell comprising a polynucleotide according to any one of paras 1 to 18, or a vector according to para 19 or para 20. 23. The cell according to para 22, wherein the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 24. A method for modifying a cell to express a polypeptide of interest, comprising: (i) introducing into a cell a polynucleotide according to any one of paras 1 to 18, or a vector according to para 19 or para 20; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 25. A method for expressing a polypeptide of interest in a cell, comprising contacting a cell according to para 22 with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. P37829 26. A polynucleotide according to any one of paras 1 to 18, a vector according to para 19 or para 20, or a pharmaceutical composition according to para 21, for use in a method of medical treatment or prophylaxis. 27. A polynucleotide according to any one of paras 1 to 18, a vector according to para 19 or para 20, or a pharmaceutical composition according to para 21, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 28. Use of a polynucleotide according to any one of paras 1 to 18, a vector according to para 19 or para 20, or a pharmaceutical composition according to para 21, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 29. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject a polynucleotide according to any one of paras 1 to 18, a vector according to para 19 or para 20, or a pharmaceutical composition according to para 21. 30. The polynucleotide, vector, or pharmaceutical composition for use according to para 27, the use according to para 28, or the method according to para 29, wherein treating or preventing the disease or condition further comprises administering to the subject a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 31. The polynucleotide, vector, or pharmaceutical composition for use according to para 27 or para 30, the use according to para 28 or para 30, or the method according to para 29 or para 30, wherein the disease or condition is a disease or condition characterised by deficiency of the polypeptide of interest. 32. A kit, comprising: (i) a polynucleotide according to any one of paras 1 to 18, a vector according to para 19 or para 20, or a pharmaceutical composition according to para 21; and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 33. A polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:1, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 at its 3’ end, and consisting of fewer than 1044 nucleotides; P37829 (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26, wherein the third nucleotide sequence comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; (iv) a fourth nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end; (v) a fifth nucleotide sequence: (a) consisting of the trinucleotide ‘GAG’ or (b) encoding a polypeptide of interest, and comprising ‘GAG’ at positions 1 to 3; and (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a), a sixth nucleotide sequence encoding a polypeptide of interest; wherein the polynucleotide comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. 34. The polynucleotide according to para 33, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. 35. The polynucleotide according to para 33 or para 34, wherein the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:1. 36. The polynucleotide according to any one of paras 33 to 35, wherein the first nucleotide sequence comprises, or consists of, SEQ ID NO:3. 37. The polynucleotide according to any one of paras 33 to 36, wherein the second nucleotide sequence comprises SEQ ID NO:7 at its 5’ end, and comprises SEQ ID NO:8 at its 3’ end. 38. The polynucleotide according to any one of paras 33 to 37, wherein the second nucleotide sequence consists of fewer than 500 nucleotides. 39. The polynucleotide according to any one of paras 33 to 38, wherein the second nucleotide sequence comprises, or consists of, SEQ ID NO:10. 40. The polynucleotide according to any one of paras 33 to 39, wherein the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:26. 41. The polynucleotide according to any one of paras 33 to 40, wherein the third nucleotide sequence consists of SEQ ID NO:13 or SEQ ID NO:27. 42. The polynucleotide according to any one of paras 33 to 41, wherein the fourth nucleotide sequence comprises SEQ ID NO:15 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end. P37829 43. The polynucleotide according to any one of paras 33 to 42, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides. 44. The polynucleotide according to any one of paras 33 to 43, wherein the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17. 45. The polynucleotide according to any one of paras 33 to 44, wherein the polynucleotide comprises at least 80% sequence identity to SEQ ID NO:28 or SEQ ID NO:29. 46. The polynucleotide according to any one of paras 33 to 45, wherein the polynucleotide further comprises a promoter sequence 5’ to the start codon. 47. The polynucleotide according to any one of paras 33 to 46, wherein the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a polypeptide of interest. 48. The polynucleotide according to any one of paras 33 to 47, wherein the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. 49. A vector comprising a polynucleotide according to any one of paras 33 to 48. 50. The vector according to para 49, wherein the vector is an adeno-associated virus (AAV) vector. 51. A pharmaceutical composition comprising a polynucleotide according to any one of paras 33 to 48, or a vector according to para 49 or para 50, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. 52. A cell comprising a polynucleotide according to any one of paras 33 to 48, or a vector according to para 49 or para 50. 53. The cell according to para 52, wherein the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 54. A method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell a polynucleotide according to any one of paras 33 to 48, or a vector according to para 49 or para 50. 55. A method for inhibiting expression of a polypeptide of interest in a cell, comprising contacting a cell according to para 52 with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. P37829 56. A polynucleotide according to any one of paras 33 to 48, a vector according to para 49 or para 50, or a pharmaceutical composition according to para 51, for use in a method of medical treatment or prophylaxis. 57. A polynucleotide according to any one of paras 33 to 48, a vector according to para 49 or para 50, or a pharmaceutical composition according to para 51, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 58. Use of a polynucleotide according to any one of paras 33 to 48, a vector according to para 49 or para 50, or a pharmaceutical composition according to para 51, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 59. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject a polynucleotide according to any one of paras 33 to 48, a vector according to para 49 or para 50, or a pharmaceutical composition according to para 51. 60. The polynucleotide, vector, or pharmaceutical composition for use according to para 57, the use according to para 58, or the method according to para 59, wherein the disease or condition is a disease or condition characterised by deficiency of the polypeptide of interest. 61. A kit, comprising: (i) a polynucleotide according to any one of paras 33 to 48, a vector according to para 49 or para 50, or a pharmaceutical composition according to para 51; and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 1A. A polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:1, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:11, wherein the third nucleotide sequence comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12, and wherein the third nucleotide sequence comprises insertion of ‘A’ immediately after the position corresponding to position 48 of SEQ ID NO:12; P37829 (iv) a fourth nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end; (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘GA’, ‘TG’ or ‘TT’, or (b) comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:19, wherein the nucleotide sequence comprises ‘GA’, ‘TG’ or ‘TT’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:19, or (c) encoding a polypeptide of interest, and comprising ‘GA’, ‘TG’ or ‘TT’ at positions 1 and 2; and (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a) or (v)(b), a sixth nucleotide sequence encoding a polypeptide of interest; wherein the polynucleotide comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. 2A. The polynucleotide according to para 1A, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. 3A. The polynucleotide according to para 1A or para 2A, wherein the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:1. 4A. The polynucleotide according to any one of paras 1A to 3A, wherein the first nucleotide sequence comprises, or consists of, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. 5A. The polynucleotide according to any one of paras 1A to 4A, wherein the second nucleotide sequence comprises SEQ ID NO:7 at its 5’ end, and comprises SEQ ID NO:8 at its 3’ end. 6A. The polynucleotide according to any one of paras 1A to 5A, wherein the second nucleotide sequence consists of fewer than 500 nucleotides. 7A. The polynucleotide according to any one of paras 1A to 6A, wherein the second nucleotide sequence comprises, or consists of, SEQ ID NO:9 or SEQ ID NO:10. 8A. The polynucleotide according to any one of paras 1A to 7A, wherein the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:11. 9A. The polynucleotide according to any one of paras 1A to 8A, wherein the third nucleotide sequence consists of SEQ ID NO:13 or SEQ ID NO:14. 10A. The polynucleotide according to any one of paras 1A to 9A, wherein the fourth nucleotide sequence comprises SEQ ID NO:15 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end. P37829 11A. The polynucleotide according to any one of paras 1A to 10A, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides. 12A. The polynucleotide according to any one of paras 1A to 11A, wherein the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17 or SEQ ID NO:18. 13A. The polynucleotide according to any one of paras 1A to 12A, wherein the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’, or (b) comprises, or consists of, a nucleotide sequence according to SEQ ID NO:19. 14A. The polynucleotide according to any one of paras 1A to 13A, wherein the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’, or (b) comprises, or consists of, SEQ ID NO:20 or SEQ ID NO:21. 15A. The polynucleotide according to any one of paras 1A to 14A, wherein the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25. 16A. The polynucleotide according to any one of paras 1A to 15A, wherein the polynucleotide further comprises a promoter sequence 5’ to the start codon. 17A. The polynucleotide according to any one of paras 1A to 16A, wherein the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a polypeptide of interest. 18A. The polynucleotide according to any one of paras 1A to 17A, wherein the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. 19A. A vector comprising a polynucleotide according to any one of paras 1A to 18A. 20A. The vector according to para 19A, wherein the vector is an adeno-associated virus (AAV) vector. 21A. A pharmaceutical composition comprising a polynucleotide according to any one of paras 1A to 18A, or a vector according to para 19A or para 20A, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. 22A. A cell comprising a polynucleotide according to any one of paras 1A to 18A, or a vector according to para 19A or para 20A. 23A. The cell according to para 22A, wherein the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 24A. A method for modifying a cell to express a polypeptide of interest, comprising: P37829 (i) introducing into a cell a polynucleotide according to any one of paras 1A to 18A, or a vector according to para 19A or para 20A; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 25A. A method for expressing a polypeptide of interest in a cell, comprising contacting a cell according to para 22A with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 26A. A polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A, for use in a method of medical treatment or prophylaxis. 27A. A polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 28A. Use of a polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 29A. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject a polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A. 30A. The polynucleotide, vector, or pharmaceutical composition for use according to para 27A, the use according to para 28A, or the method according to para 29A, wherein treating or preventing the disease or condition further comprises administering to the subject a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 31A. The polynucleotide, vector, or pharmaceutical composition for use according to para 27A or para 30A, the use according to para 28A or para 30A, or the method according to para 29A or para 30A, wherein the disease or condition is a disease or condition characterised by deficiency of the polypeptide of interest. 32A. A kit, comprising: (i) a polynucleotide according to any one of paras 1A to 18A, a vector according to para 19A or para 20A, or a pharmaceutical composition according to para 21A; and P37829 (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 33A. A polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:1, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26, wherein the third nucleotide sequence comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; (iv) a fourth nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end; (v) a fifth nucleotide sequence: (a) consisting of the trinucleotide ‘GAG’ or (b) encoding a polypeptide of interest, and comprising ‘GAG’ at positions 1 to 3; and (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a), a sixth nucleotide sequence encoding a polypeptide of interest; wherein the polynucleotide comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. 34A. The polynucleotide according to para 33A, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. 35A. The polynucleotide according to para 33A or para 34A, wherein the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:1. 36A. The polynucleotide according to any one of paras 33A to 35A, wherein the first nucleotide sequence comprises, or consists of, SEQ ID NO:3. 37A. The polynucleotide according to any one of paras 33A to 36A, wherein the second nucleotide sequence comprises SEQ ID NO:7 at its 5’ end, and comprises SEQ ID NO:8 at its 3’ end. 38A. The polynucleotide according to any one of paras 33A to 37A, wherein the second nucleotide sequence consists of fewer than 500 nucleotides. 39A. The polynucleotide according to any one of paras 33A to 38A, wherein the second nucleotide sequence comprises, or consists of, SEQ ID NO:10. P37829 40A. The polynucleotide according to any one of paras 33A to 39A, wherein the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:26. 41A. The polynucleotide according to any one of paras 33A to 40A, wherein the third nucleotide sequence consists of SEQ ID NO:13 or SEQ ID NO:27. 42A. The polynucleotide according to any one of paras 33A to 41A, wherein the fourth nucleotide sequence comprises SEQ ID NO:15 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end. 43A. The polynucleotide according to any one of paras 33A to 42A, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides. 44A. The polynucleotide according to any one of paras 33A to 43A, wherein the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17. 45A. The polynucleotide according to any one of paras 33A to 44A, wherein the polynucleotide comprises at least 80% sequence identity to SEQ ID NO:28 or SEQ ID NO:29. 46A. The polynucleotide according to any one of paras 33A to 45A, wherein the polynucleotide further comprises a promoter sequence 5’ to the start codon. 47A. The polynucleotide according to any one of paras 33A to 46A, wherein the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a polypeptide of interest. 48A. The polynucleotide according to any one of paras 33A to 47A, wherein the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. 49A. A vector comprising a polynucleotide according to any one of paras 33A to 48A. 50A. The vector according to para 49A, wherein the vector is an adeno-associated virus (AAV) vector. 51A. A pharmaceutical composition comprising a polynucleotide according to any one of paras 33A to 48A, or a vector according to para 49A or para 50A, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. 52A. A cell comprising a polynucleotide according to any one of paras 33A to 48A, or a vector according to para 49A or para 50A. 53A. The cell according to para 52A, wherein the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. P37829 54A. A method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell a polynucleotide according to any one of paras 33A to 48A, or a vector according to para 49A or para 50A. 5 55A. A method for inhibiting expression of a polypeptide of interest in a cell, comprising contacting a cell according to para 52A with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 56A. A polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 10 50A, or a pharmaceutical composition according to para 51A, for use in a method of medical treatment or prophylaxis. 57A. A polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 50A, or a pharmaceutical composition according to para 51A, for use in treating or preventing a disease 15 or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 58A. Use of a polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 50A, or a pharmaceutical composition according to para 51A, in the manufacture of a medicament 20 for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 59A. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising 25 administering to a subject a polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 50A, or a pharmaceutical composition according to para 51A. 60A. The polynucleotide, vector, or pharmaceutical composition for use according to para 57A, the use according to para 58A, or the method according to para 59A, wherein the disease or condition is a 30 disease or condition characterised by deficiency of the polypeptide of interest. 61A. A kit, comprising: (i) a polynucleotide according to any one of paras 33A to 48A, a vector according to para 49A or para 50A, or a pharmaceutical composition according to para 51A; and 35 (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. Table A Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence 1 pMM112 ON-switch SEQ ID NO:106 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 GA P37829 Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence (SEQ ID NO:22) pMM130 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 GA 2 (SEQ ID NO:23) pMM59 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:20 3 (SEQ ID NO:24) pMM198 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:21 4 (SEQ ID NO:25) pMM193 OFF-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 GAG 5 (SEQ ID NO:28) pMM194 OFF-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:27 SEQ ID NO:17 GAG 6 (SEQ ID NO:29) pLS41 ON-switch SEQ ID NO:2 SEQ ID NO:238 SEQ ID NO:256 SEQ ID NO:18 SEQ ID NO:20 7 (SEQ ID NO:37) pLS76 ON-switch SEQ ID NO:2 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:20 8 (SEQ ID NO:38) pLS159 ON-switch SEQ ID NO:2 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:20 9 (SEQ ID NO:39) pLS160 ON-switch SEQ ID NO:2 SEQ ID NO:239 SEQ ID NO:13 SEQ ID NO:330 SEQ ID NO:20 10 (SEQ ID NO:40) pLS167 ON-switch SEQ ID NO:230 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:20 11 (SEQ ID NO:41) pLS168 ON-switch SEQ ID NO:231 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:20 12 (SEQ ID NO:42) pLS179 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:20 13 (SEQ ID NO:43) pMM70 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:20 14 (SEQ ID NO:44) pMM71 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:20 15 (SEQ ID NO:45) pMM72 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:20 16 (SEQ ID NO:46) pMM73 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:20 17 (SEQ ID NO:47) pMM143 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:331 SEQ ID NO:20 18 (SEQ ID NO:48) pMM144 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:332 SEQ ID NO:20 19 (SEQ ID NO:49) pMM145 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:333 SEQ ID NO:20 20 (SEQ ID NO:50) pMM146 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:334 SEQ ID NO:20 21 (SEQ ID NO:51) pMM147 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:335 SEQ ID NO:20 22 (SEQ ID NO:52) 23 pMM136 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:257 SEQ ID NO:17 SEQ ID NO:20 P37829 Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence (SEQ ID NO:53) pMM137 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:258 SEQ ID NO:17 SEQ ID NO:20 24 (SEQ ID NO:54) pMM138 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:259 SEQ ID NO:17 SEQ ID NO:20 25 (SEQ ID NO:55) pMM139 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:260 SEQ ID NO:17 SEQ ID NO:20 26 (SEQ ID NO:56) pMM140 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:261 SEQ ID NO:17 SEQ ID NO:20 27 (SEQ ID NO:57) pMM141 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:262 SEQ ID NO:17 SEQ ID NO:20 28 (SEQ ID NO:58) pMM142 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:263 SEQ ID NO:17 SEQ ID NO:20 29 (SEQ ID NO:59) pMM151 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:264 SEQ ID NO:17 SEQ ID NO:20 30 (SEQ ID NO:60) pMM152 ON-switch SEQ ID NO:3 SEQ ID NO:10 SEQ ID NO:265 SEQ ID NO:17 SEQ ID NO:20 31 (SEQ ID NO:61) pLS174 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:20 32 (SEQ ID NO:62) pMM56 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:299 SEQ ID NO:18 SEQ ID NO:20 33 (SEQ ID NO:63) pMM60 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:301 SEQ ID NO:18 SEQ ID NO:20 34 (SEQ ID NO:64) pMM61 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:302 SEQ ID NO:18 SEQ ID NO:20 35 (SEQ ID NO:65) pMM62 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:329 SEQ ID NO:18 SEQ ID NO:20 36 (SEQ ID NO:66) pMM63 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:303 SEQ ID NO:18 SEQ ID NO:20 37 (SEQ ID NO:67) pLS176 ON-switch SEQ ID NO:234 SEQ ID NO:9 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:20 38 (SEQ ID NO:68) pMM108 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:20 39 (SEQ ID NO:69) pMM110 ON-switch SEQ ID NO:232 SEQ ID NO:9 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:20 40 (SEQ ID NO:70) pMM111 ON-switch SEQ ID NO:233 SEQ ID NO:9 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:20 41 (SEQ ID NO:71) pMM242 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:304 SEQ ID NO:18 SEQ ID NO:20 42 (SEQ ID NO:72) pMM243 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:305 SEQ ID NO:18 SEQ ID NO:20 43 (SEQ ID NO:73) pMM244 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:306 SEQ ID NO:18 SEQ ID NO:20 44 (SEQ ID NO:74) 45 pMM245 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:308 SEQ ID NO:18 SEQ ID NO:20 P37829 Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence (SEQ ID NO:75) pMM246 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:309 SEQ ID NO:18 SEQ ID NO:20 46 (SEQ ID NO:76) pMM247 ON-switch SEQ ID NO:5 SEQ ID NO:9 SEQ ID NO:307 SEQ ID NO:18 SEQ ID NO:20 47 (SEQ ID NO:77) pLS175 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:20 48 (SEQ ID NO:78) pMM123 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:310 SEQ ID NO:18 SEQ ID NO:20 49 (SEQ ID NO:79) pMM124 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:311 SEQ ID NO:18 SEQ ID NO:20 50 (SEQ ID NO:80) pMM125 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:312 SEQ ID NO:18 SEQ ID NO:20 51 (SEQ ID NO:81) pMM126 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:315 SEQ ID NO:18 SEQ ID NO:20 52 (SEQ ID NO:82) pMM127 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:313 SEQ ID NO:18 SEQ ID NO:20 53 (SEQ ID NO:83) pMM128 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:314 SEQ ID NO:18 SEQ ID NO:20 54 (SEQ ID NO:84) pMM129 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:316 SEQ ID NO:18 SEQ ID NO:20 55 (SEQ ID NO:85) pMM201 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:318 SEQ ID NO:18 SEQ ID NO:20 56 (SEQ ID NO:86) pMM202 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:319 SEQ ID NO:18 SEQ ID NO:20 57 (SEQ ID NO:87) pMM203 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:320 SEQ ID NO:18 SEQ ID NO:20 58 (SEQ ID NO:88) pMM204 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:321 SEQ ID NO:18 SEQ ID NO:20 59 (SEQ ID NO:89) pMM205 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:322 SEQ ID NO:18 SEQ ID NO:20 60 (SEQ ID NO:90) pMM206 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:323 SEQ ID NO:18 SEQ ID NO:20 61 (SEQ ID NO:91) pMM248 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:324 SEQ ID NO:18 SEQ ID NO:20 62 (SEQ ID NO:92) pMM249 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:325 SEQ ID NO:18 SEQ ID NO:20 63 (SEQ ID NO:93) pMM250 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:326 SEQ ID NO:18 SEQ ID NO:20 64 (SEQ ID NO:94) pMM251 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:327 SEQ ID NO:18 SEQ ID NO:20 65 (SEQ ID NO:95) pMM252 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 GA 66 (SEQ ID NO:96) 67 pMM254 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:315 SEQ ID NO:17 GA P37829 Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence (SEQ ID NO:97) pMM255 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:314 SEQ ID NO:17 GA 68 (SEQ ID NO:98) pMM256 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:316 SEQ ID NO:17 GA 69 (SEQ ID NO:99) pMM257 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:317 SEQ ID NO:17 GA 70 (SEQ ID NO:100) pMM199 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:336 SEQ ID NO:21 71 (SEQ ID NO:101) pMM200 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:381 SEQ ID NO:21 72 (SEQ ID NO:102) pMM236 ON-switch SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:21 73 (SEQ ID NO:103) pMM237 ON-switch SEQ ID NO:235 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:21 74 (SEQ ID NO:104) pMM238 ON-switch SEQ ID NO:233 SEQ ID NO:9 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:21 75 (SEQ ID NO:105) pMM273 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:17 TG 76 (SEQ ID NO:111) pMM362 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:266 SEQ ID NO:338 TG 77 (SEQ ID NO:112) pMM363 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:267 SEQ ID NO:339 TG 78 (SEQ ID NO:113) pMM364 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:340 TG 79 (SEQ ID NO:114) pMM358 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:268 SEQ ID NO:17 TG 80 (SEQ ID NO:115) pMM359 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:223 SEQ ID NO:17 TG 81 (SEQ ID NO:116) pMM360 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:269 SEQ ID NO:17 TG 82 (SEQ ID NO:117) pMM361 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:270 SEQ ID NO:17 TG 83 (SEQ ID NO:118) pMM365 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:271 SEQ ID NO:17 TG 84 (SEQ ID NO:119) pMM366 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:272 SEQ ID NO:17 TG 85 (SEQ ID NO:120) pMM367 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:273 SEQ ID NO:17 TG 86 (SEQ ID NO:121) pMM368 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:274 SEQ ID NO:17 TG 87 (SEQ ID NO:122) pMM369 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:275 SEQ ID NO:17 TG 88 (SEQ ID NO:123) 89 pMM370 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:276 SEQ ID NO:17 TG P37829 Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence (SEQ ID NO:124) pMM371 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:277 SEQ ID NO:17 TG 90 (SEQ ID NO:125) pMM372 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:278 SEQ ID NO:17 TG 91 (SEQ ID NO:126) pMM373 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:279 SEQ ID NO:17 TG 92 (SEQ ID NO:127) pMM374 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:280 SEQ ID NO:17 TG 93 (SEQ ID NO:128) pMM375 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:281 SEQ ID NO:17 TG 94 (SEQ ID NO:129) pMM376 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:282 SEQ ID NO:17 TG 95 (SEQ ID NO:130) pMM377 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:283 SEQ ID NO:17 TG 96 (SEQ ID NO:131) pMM378 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:284 SEQ ID NO:17 TG 97 (SEQ ID NO:132) pMM379 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:264 SEQ ID NO:17 TG 98 (SEQ ID NO:133) pMM380 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:285 SEQ ID NO:17 TG 99 (SEQ ID NO:134) pMM381 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:265 SEQ ID NO:17 TG 100 (SEQ ID NO:135) pMM382 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:286 SEQ ID NO:17 TG 101 (SEQ ID NO:136) pMM386 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:287 SEQ ID NO:17 TG 102 (SEQ ID NO:137) pMM387 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:27 SEQ ID NO:17 TG 103 (SEQ ID NO:138) pMM436 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:289 SEQ ID NO:17 TG 104 (SEQ ID NO:139) pMM437 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:290 SEQ ID NO:17 TG 105 (SEQ ID NO:140) pMM438 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:291 SEQ ID NO:17 TG 106 (SEQ ID NO:141) pMM477 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:341 TG 107 (SEQ ID NO:142) pMM478 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:342 TG 108 (SEQ ID NO:143) pMM479 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:343 TG 109 (SEQ ID NO:144) pMM480 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:344 TG 110 (SEQ ID NO:145) 111 pMM481 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:345 TG P37829 Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence (SEQ ID NO:146) pMM482 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:346 TG 112 (SEQ ID NO:147) pMM483 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:347 TG 113 (SEQ ID NO:148) pMM484 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:348 TG 114 (SEQ ID NO:149) pMM485 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:349 TG 115 (SEQ ID NO:150) pMM464 ON-switch SEQ ID NO:219 SEQ ID NO:241 SEQ ID NO:13 SEQ ID NO:350 TG 116 (SEQ ID NO:151) pMM465 ON-switch SEQ ID NO:219 SEQ ID NO:242 SEQ ID NO:13 SEQ ID NO:351 TG 117 (SEQ ID NO:152) pMM466 ON-switch SEQ ID NO:219 SEQ ID NO:243 SEQ ID NO:13 SEQ ID NO:352 TG 118 (SEQ ID NO:153) pMM467 ON-switch SEQ ID NO:219 SEQ ID NO:244 SEQ ID NO:13 SEQ ID NO:353 TG 119 (SEQ ID NO:154) pMM468 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:350 TG 120 (SEQ ID NO:155) pMM469 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:351 TG 121 (SEQ ID NO:156) pMM470 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:352 TG 122 (SEQ ID NO:157) pMM471 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:354 TG 123 (SEQ ID NO:158) pMM472 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:227 TG 124 (SEQ ID NO:159) pMM473 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:356 TG 125 (SEQ ID NO:160) pMM474 ON-switch SEQ ID NO:219 SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:357 TG 126 (SEQ ID NO:161) pMM567 ON-switch SEQ ID NO:220 SEQ ID NO:242 SEQ ID NO:224 SEQ ID NO:352 TG 127 (SEQ ID NO:162) pMM568 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:352 TG 128 (SEQ ID NO:163) pMM569 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:227 TG 129 (SEQ ID NO:164) pMM570 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:357 TG 130 (SEQ ID NO:165) pMM591 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:358 TG 131 (SEQ ID NO:166) pMM592 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:359 TG 132 (SEQ ID NO:167) 133 pMM593 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:293 SEQ ID NO:227 TG P37829 Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence (SEQ ID NO:168) pMM594 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:294 SEQ ID NO:227 TG 134 (SEQ ID NO:169) pMM595 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:295 SEQ ID NO:227 TG 135 (SEQ ID NO:170) pMM596 ON-switch SEQ ID NO:220 SEQ ID NO:228 SEQ ID NO:224 SEQ ID NO:227 TG 136 (SEQ ID NO:171) pMM597 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:328 SEQ ID NO:227 TG 137 (SEQ ID NO:172) pMM598 ON-switch SEQ ID NO:220 SEQ ID NO:375 SEQ ID NO:296 SEQ ID NO:227 TG 138 (SEQ ID NO:173) pMM599 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:360 TG 139 (SEQ ID NO:174) pMM600 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:297 SEQ ID NO:227 TG 140 (SEQ ID NO:175) pMM601 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:298 SEQ ID NO:227 TG 141 (SEQ ID NO:176) pMM642 ON-switch SEQ ID NO:220 SEQ ID NO:246 SEQ ID NO:224 SEQ ID NO:227 TG 142 (SEQ ID NO:177) pMM643 ON-switch SEQ ID NO:220 SEQ ID NO:376 SEQ ID NO:224 SEQ ID NO:227 TG 143 (SEQ ID NO:178) pMM709 ON-switch SEQ ID NO:220 SEQ ID NO:247 SEQ ID NO:224 SEQ ID NO:227 TG 144 (SEQ ID NO:179) pMM644 ON-switch SEQ ID NO:220 SEQ ID NO:248 SEQ ID NO:224 SEQ ID NO:227 TG 145 (SEQ ID NO:180) pMM645 ON-switch SEQ ID NO:220 SEQ ID NO:249 SEQ ID NO:224 SEQ ID NO:227 TG 146 (SEQ ID NO:181) pMM646 ON-switch SEQ ID NO:220 SEQ ID NO:251 SEQ ID NO:224 SEQ ID NO:227 TG 147 (SEQ ID NO:182) pMM647 ON-switch SEQ ID NO:220 SEQ ID NO:250 SEQ ID NO:224 SEQ ID NO:227 TG 148 (SEQ ID NO:183) pMM648 ON-switch SEQ ID NO:220 SEQ ID NO:252 SEQ ID NO:224 SEQ ID NO:227 TG 149 (SEQ ID NO:184) pMM649 ON-switch SEQ ID NO:220 SEQ ID NO:253 SEQ ID NO:224 SEQ ID NO:227 TG 150 (SEQ ID NO:185) pMM650 ON-switch SEQ ID NO:220 SEQ ID NO:254 SEQ ID NO:224 SEQ ID NO:227 TG 151 (SEQ ID NO:186) pMM651 ON-switch SEQ ID NO:220 SEQ ID NO:255 SEQ ID NO:224 SEQ ID NO:227 TG 152 (SEQ ID NO:187) pMM634 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:361 TG 153 (SEQ ID NO:188) pMM711 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:362 TG 154 (SEQ ID NO:189) 155 pMM635 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:363 TG P37829 Row Switch Name Col. A Col. B Col. C Col. D Col. E 1st Nt Sequence 2nd Nt Sequence 3rd Nt Sequence 4th Nt Sequence 5th Nt Sequence (SEQ ID NO:190) pMM636 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:364 TG 156 (SEQ ID NO:191) pMM637 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:365 TG 157 (SEQ ID NO:192) pMM638 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:366 TG 158 (SEQ ID NO:193) pMM639 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:367 TG 159 (SEQ ID NO:194) pMM640 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:368 TG 160 (SEQ ID NO:195) pMM641 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:369 TG 161 (SEQ ID NO:196) pMM712 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:370 TG 162 (SEQ ID NO:197) pMM579 ON-switch SEQ ID NO:220 SEQ ID NO:10 SEQ ID NO:292 SEQ ID NO:227 TG 163 (SEQ ID NO:198) pMM618 ON-switch SEQ ID NO:237 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:227 TG 164 (SEQ ID NO:199) pMM619 ON-switch SEQ ID NO:236 SEQ ID NO:10 SEQ ID NO:224 SEQ ID NO:227 TG 165 (SEQ ID NO:200) *** The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. 5 The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described. Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with 10 reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference. Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the 15 inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used herein, a ‘peptide’ refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A ‘polypeptide’ is a 20 polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids. Reference herein to peptides, polypeptides and proteins also includes P37829 glycopeptides/glycopolypeptides/glycoproteins, lipopeptides/lipopolypeptides/lipoproteins, nucleopeptides/nucleopolypeptides/nucleoproteins, etc. As used herein, an amino acid sequence, or a region of a polypeptide, which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60%, e.g. one of at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the amino acid sequence of the amino acid sequence/polypeptide/region. An amino acid sequence/region/position of a polypeptide/amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence/region/position of a polypeptide/amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Söding, J.2005, Bioinformatics 21, 951-960). Similarly, a nucleotide sequence, or a region of a polynucleotide, which ‘corresponds’ to a specified reference nucleotide sequence or region of a polynucleotide has at least 60%, e.g. one of at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity to the amino acid sequence of the nucleotide sequence/polynucleotide/region. A polynucleotide/region/position of a polynucleotide/nucleotide sequence which ‘corresponds’ to a specified reference nucleotide sequence/region/position of a polynucleotide/nucleotide sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Söding, J.2005, Bioinformatics 21, 951-960). As used herein, an amino acid sequence (e.g. the amino acid sequence of a peptide/polypeptide/domain/region) which is ‘derived from’ a reference amino acid sequence (e.g. the amino acid sequence of a reference peptide/polypeptide/domain/region) comprises, or consists of, an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference amino acid sequence. Similarly, a nucleotide sequence (e.g. a nucleotide sequence of a polynucleotide) which is ‘derived from’ a reference nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference nucleotide sequence. It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and/or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment. Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated. P37829 Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with/on intact multi-cellular organisms. Brief Description of the Figures Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures. Figure 1. Schematic representation of the design of the In-frame ON-switch. The cassette comprising the in-frame switch is constructed using the exon 6-intron 6-exon 7-intron 7-exon 8 region of human SMN2 followed by the coding sequence of the gene of interest (GOI) downstream of exon 8. The translation start site is at the 5’ end of exon 6. Intron 6 is modified to reduce its length and exon 7 contains an insertion of the nucleotide A after position 48. The sequence of exon 8 is only the initial 23 nucleotides. In the absence of risdiplam splicing modifier, exon 7 is excluded resulting in the generation of a frameshift in the coding sequence of the GOI. Conversely, risdiplam treatment leads to exon 7 inclusion and the correct translation of the GOI open reading frame and its protein production. Star indicates the insertion of nucleotide “A” causing a frame-shift. Figure 2. Small molecule-mediated expression of GFP from the In-frame ON-switch 48h post-treatment with risdiplam analogue. FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the in-frame ON-switch pLS41. The graph shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. Figure 3. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pLS41 and pLS76 ON-switches. (A) Schematic representation indicating the changes in the modified (Mod) in-frame switch (i.e. pLS76) relative to the wildtype (WT) sequence of SMN2 intron 6 and exon 7. (B-D) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS41 or pLS76 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 3B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT) , or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows the results of analysis of the products of splicing by RT-PCR performed on HEK293 stably expressing pLS76, and treated with low (100nM) or high (1µM) concentrations of risdiplam, or DMSO. Figure 4. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pLS159 and pLS160 ON-switches, which have reduced intron 6 and intron 7 lengths relative to the pLS76 ON-switch. (A) Schematic representation of the differences between the pLS76, pLS159 and pLS160 ON-switches. (B-D) FACS-based measurements of GFP expression P37829 upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS76, pLS159, or pLS160 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 4B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 5. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pLS167 and pLS168 ON-switches, which have reduced exon 6 lengths relative to the pLS76 ON-switch. (A) Schematic representation of the differences between the pLS76, pLS167 and pLS168 ON-switches. The modified region is marked with a dashed box. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS76, pLS167, or pLS168 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 5B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 6. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pLS179 ON-switch, which combines the intron truncations of pLS159 and the exon 6 truncation of pLS168. (A) Schematic representation indicating the changes in the modified (Mod) in-frame switch (i.e. pLS76) relative to the wildtype (WT) sequence of SMN2 intron 6 and exon 7. (B-D) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS76, pLS159, pLS168 or pLS179 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 6B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows the results of analysis of the products of splicing by RT-PCR performed on HEK293 stably expressing pLS179, and treated with low (100nM) or high (1µM) concentrations of risdiplam, or DMSO. Figure 7. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM70, pMM71, pMM72, pMM73 and pMM112 ON-switches, which have reduced exon 8 lengths relative to the pLS179 ON-switch. (A) Schematic representation of the differences between the pLS179, pMM70, pMM71, pMM72, pMM73 and pMM112 ON-switches. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of P37829 risdiplam in HEK293 cells stably expressing the pLS179, pMM70, pMM71, pMM72, pMM73 or pMM112 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 7B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 8. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM130 ON-switch, in which the Kozak sequence and start codon present in pMM112 are removed. (A) Schematic representation of the differences between the pMM112 and pMM130 ON-switches. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS179, pMM112 or pMM130 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 8B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 9. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM143, pMM144, pMM145, pMM146 and pMM147 ON-switches, in which the ISS-N1 region of intron 7 is mutated to reduced leakiness. (A) Schematic representation of the differences between the pLS179, pMM143, pMM144, pMM145, pMM146 and pMM147 ON-switches. (B- D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS179, pMM143, pMM144, pMM145, pMM146 or pMM147 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 9B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 10. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM136, pMM137, pMM138, pMM139, pMM140, pMM141 and pMM142 ON-switches, in which the ESE2 region (marked in grey based on Sivaramakrishnan et al., Nat Commun 8, 1476 (2017)) of exon 7 is mutated to improve switch performance. (A) Schematic representation of the differences between the pLS179, pMM136, pMM137, pMM138, pMM139, pMM140, pMM141 and pMM142 ON-switches. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS179, pMM136, pMM137, pMM138, pMM139, pMM140, pMM141 or pMM142 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO- P37829 treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 10B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 11. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM151 and pMM152 ON-switches, which comprise deletions in exon 7. (A) Schematic representation of the differences between the pMM112, pMM151 and pMM152 ON- switches. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM112, pMM151 or pMM152 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 11B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 12. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM56, pMM59, pMM60, pMM61, pMM62 and pMM63 ON-switches, in which ATG codons in exon 6 are removed, and in which comprise modifications in exon 7. (A) Schematic representation of the differences between (Ai) pLS76 and pLS174, and (Aii) pLS174, pMM56, pMM59, pMM60, pMM61, pMM62 and pMM63 ON-switches. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS76, pMM56, pMM59, pMM60, pMM61, pMM62 and pMM63 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 12B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 13. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM108, pMM110, and pMM111 ON-switches, in which ATG codons in exon 6 are removed. (A) Schematic representation of the differences between the pLS76, pLS176, pMM59, pMM108, pMM110, and pMM111 ON-switches. Insertions relative to the sequence of pLS174 are shown in black and in bold, and mutations relative to the sequence of pLS174 are shown in black, italics and bold. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS176, pMM59, pMM108, pMM110, or pMM111 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 13B, as the total median GFP signal without normalization to DMSO control. (D) Shows the P37829 percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 14. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM242, pMM243, pMM244, pMM245, pMM246, and pMM247 ON- switches, in which ATG codons are introduced into exon 7. Insertions relative to the sequence of pLS174 are shown in black and in bold, and mutations relative to the sequence of pLS174 are shown in black, italics and bold. (A) Schematic representation of the differences between the pLS174, pMM59, pMM59, pMM242, pMM243, pMM244, pMM245, pMM246, and pMM247 ON-switches. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM242, pMM243, pMM244, pMM245, pMM246, or pMM247 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 14B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 15. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM123, pMM124, pMM125, pMM126, pMM127, pMM128 and pMM129 ON-switches, in which ATG codons are introduced into exon 7 downstream of ESE2. (A) Schematic representation of the differences between the pLS175, pMM123, pMM124, pMM125, pMM126, pMM127, pMM128 and pMM129 ON-switches. Insertions relative to the sequence of pLS175 are shown in black and in bold, and mutations relative to the sequence of pLS175 are shown in black, italics and bold. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM123, pMM124, pMM125, pMM126, pMM127, pMM128 or pMM129 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 15B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 16. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM201, pMM202, pMM203, pMM204, pMM205 and pMM206 ON- switches, in which ATG codons are introduced into exon 7 downstream of ESE2. (A) Schematic representation of the differences between the pLS175, pMM126, pMM201, pMM202, pMM203, pMM204, pMM205 and pMM206 ON-switches. Insertions relative to the comparator sequence are shown in black and in bold, and mutations relative to the comparator sequence are shown in black, italics and bold. (B- D). FACS-based measurements of GFP expression upon induction with increasing concentrations of P37829 risdiplam in HEK293 cells stably expressing the pMM126, pMM201, pMM202, pMM203, pMM204, pMM205 or pMM206 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 16B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 17. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM248, pMM249, pMM250 and pMM251 ON-switches, in which ATG codons are introduced into exon 7 downstream of ESE2. (A) Schematic representation of the differences between the pLS175, pMM129, pMM248, pMM249, pMM250 and pMM251 ON-switches. Insertions relative to the comparator sequence are shown in black and in bold, and mutations relative to the comparator sequence are shown in black, italics and bold. (B-D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM129, pMM248, pMM249, pMM250 or pMM251 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 17B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 18. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM252, pMM254, pMM255, pMM256 and pMM257 ON-switches, in which ATG codons are introduced into exon 7 downstream of ESE2. (A) Schematic representation of the differences between the pMM130, pMM252, pMM254, pMM255, pMM256 and pMM257 ON-switches. (B- D). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM126, pMM129, pMM130, pMM252, pMM254, pMM255, pMM256 or pMM257 constructs. (B) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the data of Figure 18B, as the total median GFP signal without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 19. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM198, pMM199 and pMM200 ON-switches, in which an ATG codon is introduced, spanning exons 7 and 8. (A) Schematic representation of the differences between the pLS76, pLS175, pMM198, pMM199 and pMM200 ON-switches. The start codon spanning exons 7 and 8 P37829 is shown in bold and italics. (B) Schematic representation of the predicted splicing of the split start codon ON-switches in the absence (DMSO control) or presence of risdiplam. (C-E). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM112, pMM198, pMM199 or pMM200 constructs. (C) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (D) Shows the data of Figure 19C, as the total median GFP signal without normalization to DMSO control. (E) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 20. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), for the pMM236, pMM237 and pMM238 ON-switches, in which ATG codons in exon 6 are removed, a weak Kozak sequence is introduced in exon 7, and an ATG codon is introduced in exon 8. (A) Schematic representation of the differences between the pLS76, pMM236, pMM237 and pMM238 ON-switches. (B) Schematic representation of the predicted splicing of the pMM236, pMM237 and pMM238 ON-switches in the absence (DMSO control) or presence of risdiplam. (C-E). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM112, pMM236, pMM237 or pMM238 constructs. (C) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (D) Shows the data of Figure 20C, as the total median GFP signal without normalization to DMSO control. (E) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 21. Analysis of constitutive reporter expression (i.e. in the absence of risdiplam), and risdiplam- induced suppression of reporter expression for the pMM193 and pMM194 OFF-switches. (A) Schematic representation of splicing of SMN2 exon 6 to exon 8-derived ON and OFF-switches. (B) Schematic representation of the differences between the pMM130 ON-switch and the pMM193 OFF-switch. (C) Schematic representation of the differences between the pMM193 and pMM194 OFF-switches. (D-F). FACS-based measurements of GFP expression upon treatment of HEK293 cells stably expressing the pMM130, pMM193, or pMM194 constructs with increasing concentrations of risdiplam. (D) Shows median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO- treated control cells, 48h after risdiplam treatment. (E) Shows the data of Figure 21D, as the total median GFP signal without normalization to DMSO control. (F) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells, at the highest concentration of risdiplam treatment (1µM). (G) Shows the level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 22. Analysis of risdiplam-induced reporter expression, and background reporter expression (i.e. in the absence of risdiplam), after replacing IRES-blasticidin sequence with BGH polyA signal for in-frame P37829 switches pMM259 (pMM112), pMM260 (pMM130), pMM262 (pLS168), pMM263 (pLS76), pMM272 (pMM198) and Monteys et al. Nature (2021) 596: 291-295 construct (pMM274). (A) Schematic representation of plasmid backbone modification. IRES-blasticidin-SV40 sequence was replaced with BGH polyA signal. In-frame switch sequences remain unmodified. (B) List of in-frame switches used for the experiment with their original numbers (C-E). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM259, pMM260, pMM262, pMM263, pMM272 and Monteys et al. Nature (2021) 596: 291-295 construct (pMM274) (C) Shows induction rate calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment, GFP-positive cells. (D) Shows the data of Figure 19C, expressed as the total median GFP signal without normalization to DMSO control. (E) Shows the percentage of GFP-positive (% GFP+) cells divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (G) Shows a fragment of Fig.22C with induction rate at 125 nM risdiplam concentration calculated as median GFP expression normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment, GFP-positive cells. Figure 23. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM259, pMM263, pMM273 and pMM274. (A) Schematic representation of the differences between the pMM259 and pMM273 ON-switches. (B) Schematic representation of the predicted splicing of the split start codon ON-switches in the absence (DMSO control) or presence of risdiplam. (C-E). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM259, pMM263, pMM273 and pMM274 constructs. (C) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (D) Shows the total median GFP signal of GFP positive cells without normalization to DMSO control. (E) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. Figure 24. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273, pMM362, pMM363, and pMM364. (A) Schematic representation of the differences between the pMM273, pMM362, pMM363, and pMM364 ON- switches. (B-F). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273, pMM362, pMM363, and pMM364 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or P37829 in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. Figure 25. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273, pMM358, pMM359, pMM360, and pMM361. (A) Schematic representation of the differences between the pMM273, pMM358, pMM359, pMM360, and pMM361 ON-switches. (B-F). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273, pMM358, pMM359, pMM360, and pMM361 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (G) Shows the results of analysis of the products of splicing by PCR performed on HEK293 stably expressing pMM273, pMM358, pMM359 and pMM360 and treated with increasing concentrations of risdiplam, or DMSO. Figure 26. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273 and pMM365 - pMM375. (A) Schematic representation of the differences between the pMM273 and pMM365 - pMM375 (B-G). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273 and pMM365 - pMM375 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). Figure 27. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273 and pMM376 - pMM387. (A) Schematic representation of the differences between the pMM273 and pMM376 - pMM387 (B-G). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 P37829 cells stably expressing the pMM273 and pMM376 - pMM387 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). Figure 28. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273, pMM436, pMM437, and pMM438. (A) Schematic representation of the differences between the pMM273, pMM436, pMM437, and pMM438 (B- F). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273, pMM436, pMM437, and pMM438 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. Figure 29. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273 and pMM477 - pMM485. (A) Schematic representation of the differences between the pMM273 and pMM477 - pMM485. (B-F). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273 and pMM477 - pMM485 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. P37829 Figure 30. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM273 and pMM464 - pMM474 (A) Schematic representation of the differences between the pMM273 and pMM464 - pMM474 as well as a table describing intron modifications in every construct. Intron sequences added to the constructs were taken from the SMN2 transcript (B-F). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273 and pMM464 - pMM474 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 250 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. Figure 31. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM466, pMM470, pMM472, pMM474 and pMM567 - pMM570 (A) Schematic representation of the differences between the pMM466, pMM470, pMM472, pMM474 and pMM567 - pMM570. Two modifications were introduced to pMM567 - pMM570, single A>C mutation in exon 6 to remove an ATG start site and TAA deletion in exon 7. (B-F) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM466, pMM470, pMM472, pMM474 and pMM567 - pMM570 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 62.5nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. Figure 32. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569 and pMM591 - pMM601. (A) Schematic representation of the differences between the pMM569 and pMM591 - pMM601. Two regions of pMM596 were modified: exon 7 region and intron 7 region. (B-G) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569 and pMM591 - pMM601 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% P37829 GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). Figure 33. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM642 - pMM651, and pMM709. (A) Schematic representation of the intron 6 deletion screen performed using pMM569 construct.5 nt step by step deletions spanning the whole intron 6 were introduced and tested. (B) Selected intron 6 mutations tested in constructs pMM642 - pMM651, and pMM709. (C-H) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM642 - pMM651, and pMM709. (C) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (D) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (E) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (G) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (H) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). Figure 34. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM634 - pMM641, pMM711 and pMM712. (A) Schematic representation of the intron 7 deletion screen performed using pMM569 construct.5 nt step by step deletions spanning the whole intron 7 were introduced and tested. (B) Selected intron 7 mutations tested in constructs pMM634 - pMM641, pMM711 and pMM712. (C-H) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM634 - pMM641, pMM711 and pMM712 constructs. (C) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (D) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (E) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). (F) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (G) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control P37829 cells, 48h after risdiplam treatment. (H) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). Figure 35. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM579, pMM595, and pMM596. (A) Schematic representation of the differences between the pMM569, pMM579, pMM595, and pMM596. Constructs pMM579, pMM595 and pMM596 were selected due to their low GFP expression in DMSO (low leakiness) (B-G) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM579, pMM595, and pMM596 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). Figure 36. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM618, and pMM619. (A) Schematic representation of the differences between the pMM569, pMM618, and pMM619. pMM618 has 13 nt deletion at the 5’ side of exon 6, while pMM619 has 30 nt deletion of exon 65’ side. (B-G) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM618, and pMM619 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). Figure 37. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569 switch with different promoters (pMM569, pMM581 - pMM584, pMM589, pMM590, and pMM562, and pMM619). (A) Schematic representation of the pMM569 switch and the list of all tested promoters. (B-F) FACS-based measurements of GFP P37829 expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM569, pMM581 - pMM584, pMM589, pMM590, and pMM562, and pMM619 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO- treated control cells, 48h after risdiplam treatment. (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). Figure 38. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM263, pMM274, pMM273, pMM359, and pMM569. (A) Schematic representation of the differences between the pMM263, pMM274, pMM273, pMM359, and pMM569. These constructs represent various iterations of the switch design. (B-G) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM263, pMM274, pMM273, pMM359, and pMM569 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP-positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 µM). (E) Shows the background level of total median GFP expression in wildtype HEK293 cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO- treated control cells, 48h after risdiplam treatment. (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 µM). Figure 39. Analysis of risdiplam-induced NanoLuc (NLuc) luciferase reporter expression, and background NLuc reporter expression (i.e. in the absence of risdiplam) for pMM568, pMM569, pMM570 and pMM579. (A) Schematic representation of the differences between the pMM568, pMM569, pMM570 and pMM579. (B-G) Luminescence-based measurements of NLuc luciferase expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM568, pMM569, pMM570 and pMM579 constructs. (B) Shows induction of NLuc expression calculated as NLuc signal expression at each concentration of risdiplam, normalized to NLuc signal in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total NLuc signal without normalization to DMSO control. (D) Shows the total NLuc signal for WT HEK293 cells as well as cells expressing indicated constructs, in the absence of risdiplam (DMSO treated). P37829 Figure 40. Risdiplam-mediated expression of Cas9 in HEK293T stable cell lines assessed by CD81 staining. (A) Schematic representation of the differences between the constructs pMM263, pMM274, pMM273, pMM359, and pMM569, representing various iterations of the switch design (B) Experimental setup. HEK293T with stably incorporated pMM263, pMM274, pMM273, pMM359, and pMM569 switches controlling Cas9 expression were nucleoporated with 3 different sgRNAs targeting CD81 locus, each at 150 nM concentration, seeded at 96-well plates and right after treated with various concentrations of risdiplam. After 72h cells were stained using APC-conjugated CD81 antibodies and analysed by FACS. (C) Percentage of CD81 positive cells in cells expressing pMM263 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam. (D) Percentage of CD81 positive cells in cells expressing pMM274 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam. (E) Percentage of CD81 positive cells in cells expressing pMM273 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam (F) Percentage of CD81 positive cells in cells expressing pMM359 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam. (G) Percentage of CD81 positive cells in cells expressing pMM569 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam. (H) Percentage of CD81 positive cells in cells expressing pMM596 switch in either non-treated cells or cells treated with sgRNA and increasing concentrations of risdiplam. Figure 41. Schematic representation of risdiplam-inducible switch application for CAR-T therapy. (A) Patient’s isolated T cells would be isolated and modified to introduce inducible CAR construct either alone, or combined with a standard CAR expressing construct. Next, cells would be infused into patients, who would activate CAR expression using risdiplam. The switch could be used either to express a single CAR receptor, or in combination with another CAR, to induce second CAR expression. Picture created with BioRender.com. Figure 42. Analysis of risdiplam-induced GFP reporter expression, and background GFP reporter expression (i.e. in the absence of risdiplam) for pMM569, pMM579, pMM595, and pMM596 in Jurkat leukemic T-cell immortalized cell line. (A) Schematic representation of the differences between the pMM569, pMM579, pMM595, and pMM596. Constructs pMM579, pMM595 and pMM596 were selected due to their low GFP expression in DMSO (low leakiness) (B-G) FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in Jurkat cells stably expressing the pMM569, pMM579, pMM595, and pMM596 constructs. (B) Shows induction of GFP expression calculated as median GFP expression at each concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (C) Shows the total median GFP signal of GFP-positive cells without normalization to DMSO control. (D) Shows the percentage of GFP- positive (% GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 µM). (E) Shows the background level of total median GFP expression in wildtype Jurkat cells (WT), or in cells expressing the indicated constructs, in the absence of risdiplam. (F) Shows induction of GFP expression calculated as median GFP expression at 62.5 nM concentration of risdiplam, normalized to median GFP expression in DMSO-treated control cells, 48h after risdiplam treatment. (G) Shows the percentage of GFP-positive (% GFP+) cells at DMSO divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 µM). P37829 Figure 43. Risdiplam-mediated expression of GFP in AAV-transduced neurons. (A) illustration of the in- frame switch constructs used in this experiment: pLS76 the original switch and pMM359 switch, the more optimized counterpart. (B) hESC-derived neurons were differentiated from the neuronal stem cell (NSC)- state for 28 days, transduced with AAV7.m8 containing the sequence illustrated in A. After three days of AAV-transduction, Risdiplam was added to the cell culture at varying concentrations as indicated. Medium was exchanged every 3 days with fresh addition of risdiplam and presented images were acquired 7 days after risdiplam treatment. Figure 44. Optimized in-frame switch pMM569 allows for controlled expression of MeCP2. (A) Schematic representation of the in-frame switch based on the sequence from pMM569. The gene of interest sequence of GFP was replaced by the MeCP2 coding sequence and cloned into piggybac plasmid backbone under CMV promoter. (B) H4 cells were used as wild type as a control for native MeCP2 expression in contrast to MeCP2-knockout (KO) H4 cells, which were generated as a single cell clone using CRISPR/Cas9-mediated knockout of MeCP2. H4-KO cells were transfected with pMM569, encoding for MeCP2 as a therapeutic protein. (C) Transfected H4-KO cells were treated with indicated concentrations of risdiplam for 24h and imaging was conducted using anti-MeCP2 antibody (cell signaling technology, cat#3456). Images were acquired and used to quantify the MeCP2 expression level in the respective groups. (D) Representative images are shown that were used to generate the quantification in C. Examples Example 1: Design and construction of the in-frame ON-switch The in-frame ON-switch comprises exon 6, exon 7, and a partial sequence of exon 8 of SMN2 (survival of motor neuron 2), along with intervening intronic regions (intron 6 and intron 7). GFP is used as a reporter gene. Exon 7 contains a single nucleotide insertion (A at position 48). This allows in-frame translation of GFP upon inclusion of exon 7 (in the presence of risdiplam) by inactivation of the translation termination codon at the 3’ end of exon 717. The entirety of exon 6 was used, while the downstream intronic region was reduced to 414 nucleotides (from its original length of 5769 nucleotides) comprising the first 202 and the last 212 nucleotides of intron 6. The entirety of intron 7 was used, followed by the first 23 nucleotides of exon 8 (Figure 1). A Kozak sequence (GCCACC) as well as an ATG codon were placed upstream of exon 6 for the initiation of translation. The gene expression was driven by the human cytomegalovirus (CMV) promoter. In the presence of increasing concentrations of the small molecule splicing modifier (SMSM), risdiplam, splicing occurs, causing inclusion of exon 7 in the final mRNA transcript, and in-frame translation of the GFP reporter gene. P37829 Example 2: Inducible reporter gene expression using the in-frame ON-switch in the presence of small molecule splice modifier (risdiplam analogue) Mammalian HEK293 cells were cultured and transfected as described in Example 23 with plasmids that encode either the in-frame, or frame-shifted gene cassette (see Figure 1 for schematic). Cells were seeded in 96-well plates at 10,000 cells/well and treated with varying amounts of risdiplam analogue (RO7021707-000-007). After 48h of incubation, cells were collected for flow-cytometry-based analysis using the CytoFLEX Flow Cytometer from Beckman Coulter (Figure 2). The fluorescence intensity of GFP (for in-frame switch) was measured and analysis performed using Flowjo software. Median GFP intensity of each of the treated conditions was normalized to DMSO-treated cells to result in a relative fold induction rate of reporter gene expression, informing on the performance (induction rate) of the switch. We measured increased GFP expression upon induction with the risdiplam analogue at nanomolar concentrations, which resulted in toxicity at high concentrations (>1.25 µM), leading to reduced GFP expression level (Figure 2). Example 3: Optimization of the in-frame switch: Reduction in leakiness (background expression). An improved version of the in-frame switch was generated by introducing two point mutations within the 3’ splice site of exon 7: acagGGT (WT) to ccagGAT (mutant), which has been shown in a previous study to promote skipping of exon 718 (Figure 3A). This construct was termed pLS76 and was used as a starting construct for further modifications as described below. In order to characterize the construct, HEK293 cells were transfected with WT (pLS41) and mutated (pLS76) constructs and treated with increasing amounts of risdiplam compound. The nucleotide sequences of the pLS41 and pLS76 ON-switches are shown in SEQ ID NOs:37 and 38, respectively. Fluorescence activated cell sorting (FACS)-based readouts revealed reduced background expression in the modified sequence (ccagGAT) compared to WT (acagGGT), with a comparable induction rate (Figure 3B-3D). Figure 3B, shows relative fold change in median GFP expression level normalized to DMSO control 48h after risdiplam treatment at the indicated concentrations. The total GFP signal is shown in Figure 3C, whereas Figure 3D represents the % of GFP- positive cells normalized to the condition with the highest risdiplam treatment. Untransfected, wildtype HEK293 cells serve thereby for gating out GFP-negative cells. This type of data presentation allows the demonstration of the increase in the number of GFP-positive cells rather than the GFP intensity upon induction with risdiplam. Next, we assessed the leakiness of the cells by comparing total GFP expression in cells treated with DMSO only (Figure 3E) and found lower background GFP expression level in the construct that contains the acagGGT> ccagGAT mutation. The splicing efficiency was finally analyzed by RT-PCR experiments showing partial (at 100 nM) or complete (at 1 µM) inclusion of exon 7 (Figure 3F) in the improved construct (pLS76). Example 4: Optimization of the in-frame switch: Reduction in intron length One of the main limitations of AAV-mediated gene therapy is the size constraint of the whole expression cassette of 4.7 kb. Thus, usage of small regulatory elements in AAV vectors is highly desirable. In order to optimize the size of the in-frame switch (pLS76), the length of the introns separating exon 6 and 7, and exon 7 and 8 were gradually reduced. P37829 In a first step, the central region of intron 6 was removed, leaving 102 nucleotides of the 5’ end and 162 nucleotides of the 3’ end of intron 6. Similarly, the central region of intron 7 was removed, leaving 101 nucleotides at the 5’ end and 151 nucleotides at the 3’ end. In both cases, only the central intronic regions were removed, in order to minimise the chances of disrupting splicing regulatory sequences, which are typically located in close proximity to 5’ and 3’ splice sites (E adjacent to their upstream and downstream flanking exons). This construct, termed pLS159, reduced the total length of the in-frame switch by 314 nucleotides. The length of the 5’ end and 3’ end of intron 6 and intron 7 were then further reduced to: intron 6 (72 nucleotides at 5’ end and 112 nucleotides at 3’ end); intron 7 (77 nucleotides at 5’ end and 100 nucleotides at 3’ end). This construct, termed pLS160, led to a total reduction of 492 nucleotides of the in-frame switch length. The nucleotide sequences of the pLS159 and pLS160 ON-switches are shown in SEQ ID NOs:39 and 40, respectively. The effect of the different modifications on the in-frame switch performance as assessed with FACS revealed that a moderate reduction in intron length by 314 nucleotides (pLS159) leads to a smaller construct with overall better performance. It was characterized by a higher fold change in GFP expression level upon induction with risdiplam relative to DMSO-treated cells (Figure 4B), an increase in total level of median GFP intensity (Figure 4C), as well as an increase in the number of GFP-positive cells (Figure 4D), while background expression level remained unchanged (Figure 4E). In contrast, extensive reduction in the length of introns by 492 nucleotides (pLS160) did not lead to increased GFP expression levels (Figure 4B-4D). Example 5: Optimization of the in-frame switch: Reduction in exon 6 length. As exonic splicing regulatory sequences are usually localized close to the 5’SS, the inventors investigated whether it was possible to further reduce the size of the in-frame switch, by gradually reducing its length from the 5’ end of exon 6. To do so, two variants of the switch were generated that were lacking 30 (pLS167) or 60 (pLS168) nucleotides of the 5’ end of exon 6 (illustrated in Figure 5A). Results showed increased overall expression of the reporter gene (GFP) as measured by FACS-based analysis. The nucleotide sequences of the pLS167 and pLS168 ON-switches are shown in SEQ ID NOs:41 and 42, respectively. In these experiments, HEK293 cells were stably transfected, followed by treatment with risdiplam at the indicated concentrations for 48(H)Original in-frame switch construct pLS76 served as a control. In comparison to pLS76, partial truncation of exon 6 by 30 bp (pLS167) significantly outperformed the original sequence, in terms of fold change increase (Figure 5B) as well as total increase in median GFP signal (Figure 5C). Although less pronounced than in pLS167, the pLS168 construct also showed increased GFP expression in comparison to pLS76 (Figure 5B-5C). Increased GFP expression from pLS167 and pLS168 constructs relative to pLS76 may be a consequence of the polypeptide added to GFP encoded by the pLS167 and pLS168 constructs. Increased GFP expression from pLS167 relative to pLS168 may be a consequence of pLS167 comprising a ‘G’ immediately after the Kozak and start codon sequences, instead of ‘A’ (as in the pLS168 construct). P37829 The number of GFP-positive cells in cultures expressing the original construct, pLS76, increased proportionally with increasing concentration of risdiplam. Similar observations were made with cells expressing pLS168 (Figure 5D). However, the number of GFP-positive cells in cultures expressing pLS167 was very high in cells treated with DMSO control, suggesting higher leakiness in these cells. Furthermore, absolute median GFP intensity between the various cells was compared, showing highest background expression level in cultures expressing pLS167 (Figure 5E). Therefore, for further optimization, pLS168 was chosen due to lower leakiness and improvement in the induction rate. Example 6: Combined modifications of reduced intron length and reduced exon length in one construct. Next, the modifications of pLS159 and pLS168 were combined into one construct. The minimal required intron length for optimal induction (pLS159) was combined with the minimal required length of exon 6 (pLS168). This combination resulted in construct pLS179 that contained a shorter sequence of exon 6 and a shorter sequence of intron 6 and intron 7. The nucleotide sequence of the pLS179 ON-switch is shown in SEQ ID NO:43. In total, the length of the switch was reduced from 1056 to 639 nucleotides (total reduction of 417 nucleotides) (Figure 6A). Furthermore, an ATG start codon present within the exon 6 sequence, downstream of the synthetic Kozak/Start codon was mutated (TACATGAGT>TACGGCAGT) in order to reduce alternative translation start sites. Similarly, 6 nucleotides at the 5’ end of the remaining exon 6 sequence (as modified in pLS168) were deleted: AGTATG, resulting in a shorter final sequence length of exon 6 of 45 bp. Two additional point mutations were introduced, A>G and T>C to improve the KOZAK sequence. To test these constructs, HEK293 were stably transfected. Cells were then treated with increasing amounts of risdiplam and GFP expression level was assessed using FACS-based approaches (Figure 6B-6E) or RT-PCR analysis (Figure 6F). The new construct that combined the exon 6 truncation with the intron 6 and intron 7 truncations resulted in an in-frame switch with significantly higher sensitivity to low nanomolar risdiplam concentrations. Results showed higher levels of GFP expression relative to DMSO control (Figure 6B), higher total GFP expression level (Figure 6C), as well as increased number of GFP-positive cells (Figure 6D). The latter is in line with the overall increase in background expression level of GFP (in DMSO-treated cells, Figure 6E). Furthermore, splicing efficiency was assessed by RT-PCR experiments. RNA was extracted from HEK293 cells stably transfected with pLS179 and treated with risdiplam at low (100nM) or high (1µM) concentrations. Results showed partial inclusion of exon 7 at low concentrations of risdiplam and full inclusion at high concentrations of risdiplam, correlating with increase in GFP signal (Figure 6F). In summary, results showed increased protein expression level upon appropriate truncation in the introns and exon 6 region. Example 7: Reduction in nucleotide sequence of exon 8. Based on the design of the in-frame switch and the position of the translation start site at the 5’ end of exon 6, the final amino acid sequence expressed using the in-frame switch is composed of the transgene of interest linked to a fragment of SMN2 exon 6, exon 7, and exon 8 due to inclusion of these exons after P37829 RNA splicing. This results in the generation of a tag at the N-terminal part of the expressed transgene, which might destabilize the protein or trigger an immune reaction. In order to reduce the tag length as much as possible, the length of the nucleotide sequence of exon 8 was reduced in a stepwise manner. As depicted in Figure 7, 5 different constructs were generated that were based on the optimized construct pLS179 (Figure 6) with varying lengths of exon 8 (starting construct pLS179: GAAATGCTGGCATAGAGCAGCAC; pMM70: GAAATGCTGGCATAGAG; pMM71: GAAATGCTGGC; pMM72: GAAATGCT; pMM73: GAAAT; and pMM112: GA). The first two nucleotides (GA) of exon 8 were kept unchanged in construct pMM112 in order to retain the 3’ splice site consensus motif. The nucleotide sequences of the pLS179, pMM70, pMM71, pMM72, pMM73 and pMM112 ON-switches are shown in SEQ ID NOs:43, 44, 45, 46, 47 and 22, respectively. HEK293 cells were stably transfected with these constructs, and treated with increasing amounts of risdiplam. FACS-based analysis showed that the reduction in exon 8 nucleotide sequence did not lead to significant changes in the relative (Figure 7B) or in the total (Figure 7C) GFP expression level in comparison to untruncated exon 8 (pLS179). Furthermore, no significant changes in the number of GFP- positive cells were observed (Figure 7D) and only a slight increase in the background expression level was seen (Figure 7E), suggesting that the truncations of exon 8 did not have a strong effect on the splicing-dependent GFP expression, while reducing the length of the tag by 6 amino acids (pMM112). Example 8: Reduction of the tag sequence by using ATG start site contained at the 3’ end of exon 6. Exon 6 inherently contains an ATG at the 3’ end (5’…TATTATATG’3). It was hypothesized that this ATG start site would be sufficient to drive translation and could potentially replace the synthetic Kozak/start site that had been introduced at the 5’ end of exon 6. A successful translation driven by an ATG in closer proximity to the transgene sequence will reduce the length of any amino acids that will be attached to the final transgene output due to exon inclusion. A new construct (pMM130) was generated based on pMM112, with the Kozak sequence/start site removed from the 5’ end of exon 6. The design of the constructs and their respective sequences are illustrated in Figure 8A. The nucleotide sequence of the pMM130 ON-switch is shown in SEQ ID NO:23. HEK293 cells were stably transfected with the constructs and analysed by FACS to assess relative (Figure 8B) and total (Figure 8C) GFP expression levels. The induction rate of the in-frame switch in response to varying concentrations of risdiplam could be maintained by the pMM130 construct that lacks a canonical Kozak (i.e. conforming to SEQ ID NO:33) and relies on the ATG site at the 3’ end of exon 6. In addition, no changes were observed in the number of GFP-positive cells as well as the background expression of GFP in DMSO-treated cells when comparing pMM130 to starting construct pMM112 or pLS179. With the modification in pMM130 that relies on ATG start site at the 3’ end of exon 6 the length of the tag added to the final transgene output could be reduced by 15 amino acids. In combination, the various different modifications reduced the length of the N-terminal tag from 64 amino acids in pLS76 to 20 amino acids in pMM130. In addition, the overall size of the switch was reduced from 1056 nucleotides for pLS76, to 618 nucleotides for pMM130. P37829 Example 9: Mutations in the Intronic Splicing Silencer-N1 (ISS-N1) region in order to reduce leakiness. ISS-N1 is a 15-nucleotide long motif located immediately downstream of the 5’ splice site of exon 7 of the SMN2 gene ((5’..GA|GTAAGTCTGCCAGCATTATGAAAGTGA..’3), ISS-N1 motif marked as bold). ISS- N1 was described as a major inhibitory element and a master checkpoint of SMN2 exon 7 splicing because its deletion fully restored exon 7 inclusion (Singh et al. RNA (2010) 16(6):1167-81). Modifications within the inhibitory region could help reduce background splicing events (leading to inclusion of exon 7 in the absence of the SMSM) driven by the native SMN2 sequence. Therefore, mutations were introduced within the ISS-N1 region that were found to lead to increased inhibition of exon 7 inclusion19. The modifications included nucleotide mutations: CAGCA>TCCTC (pMM143), CAGCA>AAGGC (pMM144), CACGA>TAGTC (pMM145); nucleotide insertions: TTT after position 10 of intron 7 (pMM146), and CCC after position 15 of intron 7 (pMM147). The starting construct for these modifications was pLS179. The nucleotide sequences of the pMM143, pMM144, pMM145, pMM146 and pMM147 ON-switches are shown in SEQ ID NOs:48, 49, 50, 51 and 52, respectively. The modified constructs were stably transfected into HEK293 cells. Risdiplam-dependent GFP expression was assessed using FACS-based readouts. In comparison to pLS179, modifications in the ISS-N1 region maintained comparable fold-change increase in relative GFP expression levels (Figure 9B), while total GFP expression levels were reduced (Figure 9C). Furthermore, results showed a reduction in the GFP-positive cells in DMSO-treated cells (Figure 9D) as well as reduced total median GFP intensity (Figure 9E). In conclusion, reduced leakiness results in reduced overall reporter gene expression, while relative reporter gene expression (to DMSO-treated cells) remains unchanged. Example 10: Mutations in the ESE2 region to improve switch performance The ESE2 region (AAAAAGAAAGGAA) was previously described as a risdiplam binding site15. To further optimize the in-frame switch performance by means of increasing induction rate, single point mutations were introduced, within the ESE2 region as highlighted in bold in Figure 10A. HEK293 cells were stably transfected with constructs carrying the respective mutations (pMM136-pMM142) and treated with increasing concentrations of risdiplam. The nucleotide sequences of the pMM136, pMM137, pMM138, pMM139, pMM140, pMM141 and pMM142 ON-switches are shown in SEQ ID NOs:53, 54, 55, 56, 58 and 59, respectively. No increase in the relative (Figure 10B) or total (Figure 10C) GFP expression level was observed. For the majority of the modifications (pMM136, pMM137, pMM138, pMM139, pMM142), increased GFP expression in DMSO-treated cells was seen, suggesting that the risdiplam-mediated splice regulation was hampered (Figure 10C). Similarly, the number of GFP-positive cells (Figure 10D) as well as the background expression level in DMSO-treated cells (Figure 10E) also indicated that the risdiplam- mediated splice regulation was impaired for the majority of modifications. However, two modifications (pMM140 and pMM141) showed comparable (pMM140) or a slightly lower (pMM141) leakiness in terms of both the number of GFP-positive cells (Figure 10D) and median GFP expression in DMSO (Figure 10E) compared to pLS179. In summary, the majority of indicated modifications within the ESE2 region do P37829 not lead to improved performance of the in-frame switch, with only the pMM141 construct causing slightly reduced leakiness. Example 11: Partial deletions in the exon 7 region do not lead to increased induction rate but cause slight reduction in leakiness. The risdiplam-mediated effect is dependent on exon 7 and was previously described to involve the ESE2 region as well as the 5’ splice site15. With the aim of reducing the length of the amino acid tag on the gene of interest, partial deletions in the exon 7 sequence were performed as described in Figure 11A (pMM151: deletion of 12bp, pMM152: deletion of 12bp and 9bp). The nucleotide sequences of the pMM151 and pMM152 ON-switches are shown in SEQ ID NOs:60 and 61, respectively. HEK293 cells were stably transfected with the original construct pMM112 and with the modified constructs pMM151 and pMM152 and treated with risdiplam at increasing concentrations. After 48h of treatment, FACS-based analysis was used to assess GFP intensity. The reduction in the exon 7 region resulted in lower induction levels as measured by median GFP signal relative to DMSO (Figure 11B) as well as the total median GFP signal (Figure 11C). In line with previous observations linking lower induction rate to lower background signal, results showed a lower number of cells expressing GFP in the absence of risdiplam (Figure 11D) and lower median GFP signal in DMSO (Figure 11E) in cells transfected with pMM151 and pMM152 in comparison to pMM112 (containing the complete exon 7 sequence). In conclusion, reduction in the exon 7 region reduces the leakiness of the in-frame switch. Example 12: Introducing translation start site within exon 7 The current construct design leads to the generation of a residual amino acid chain that is fused to the N- terminus of the gene of interest due to the positioning of the translation start site (Kozak and ATG) at the 5’ end of exon 6. With the aim of reducing the tag length, the Kozak and ATG start site at exon 6 were removed and all alternative ATGs within the exon 6 sequence were mutated as described in Figure 12A(i) (ATG>GGC). This construct was named pLS174 and served as the starting construct to introduce Kozak and ATG start sites within the exon 7 sequence. The nucleotide sequence of the pLS174 ON-switch is shown in SEQ ID NO:62. As described in Figure 12A(ii), 6 different constructs were generated, in which a Kozak/ATG site was created by single point mutations or nucleotide insertions. The first construct (pMM56) contained mutations downstream of the ESE2 region to recreate a Kozak and ATG start site (GCTCACAT- - >GCCACCATG), construct pMM59 contained an insertion of Kozak sequence and mutation of the sequence to resemble ATG upstream of the ESE2 region (------AGA > GCCACCATG), pMM60 was modified by 2 point mutations and an insertion (AT------- > GCCACCATG), pMM61 contained mutations and a single nucleotide insertion to create a Kozak-like sequence that has previously been shown to enhance translation initiation site20, (T-ATAAG > CAACATG), pMM62 contained mutations and a single nucleotide insertion (AATATAA-G > GCCACCATG), pMM63 contained mutations and 2 nucleotide insertions (TT—CCTTA > GCCACCATG). The nucleotide sequences of the pMM56, pMM59, pMM60, pMM61 and pMM62 ON-switches are shown in SEQ ID NOs:63, 24, 64, 65, 66 and 67, respectively. P37829 As previously described, HEK293 cells were stably transfected with the respective constructs and blasticidin-selected cell pool was treated with increasing amounts of risdiplam as indicated in Figure 12B- 12D. The original construct pLS76 (Figure 3) was used as a control construct. Using FACS-based measurements, pMM56 and pMM60-63 were found to result in high levels of GFP-positive cells and constitutive GFP expression in the absence of risdiplam (Figure 12C–12E) leading to lack of control of transgene expression with risdiplam (Figure 12B). In contrast, cells expressing pMM59 showed an increase in the GFP induction rate to much higher level than cells expression pLS76 (Figure 12B), as well as an increase in the total median GFP signal with increasing risdiplam concentrations (Figure 12C). The total GFP-positive cells in pMM59 were significantly higher than in pLS76, but to a lower extent than pMM56 and pMM61-63 (Figure 12D). Furthermore, the background expression of GFP in the absence of risdiplam was only slightly higher in pMM59 in comparison to pLS76, however, significantly lower than in pMM56 and pMM61-63. In pMM59, the Kozak/ATG translation start site is provided upstream of ESE2, whereas in pMM56 and pMM60-63 the Kozak/ATG translation start site is provided downstream of ESE2. The presented data suggest that pMM59 with a Kozak sequence/start site introduced upstream of the ESE2 region provides very high induction rate as well as low leakiness and can be used to reduce the size of the tag on the final transgene output. Additionally, pMM59 represents a new in-frame switch design, where the translation start site is inside the inducible exon itself. Example 13: Modifications in the 5’ splice site of exon 6 in order to remove the ATG start site. Alternative ATG translation start sites within exon 6 were mutated in pLS176 as indicated in Figure 13A and highlighted in bold. The previous construct pMM59 carried a modification at the 5’ splice site of exon 6 from ATG to CTG. With the aim of testing various modifications and their impact on the 5’ splice site usage of exon 6, 3 additional constructs to pMM59 were generated, in which the ATG sequence at the 3’ end of exon 6 was mutated to CAG (pMM108), GTG (pMM110), TTG (pMM111) in comparison to CTG (pMM59). The nucleotide sequences of the pMM108, pMM110 and pMM111 ON-switches are shown in SEQ ID NOs:69, 70 and 71, respectively. HEK293 cells were stably transfected with pMM59, pMM108, pMM110 and pMM11 and treated with different concentrations of risdiplam for 48h. FACS-based analysis on the median GFP expression relative to DMSO (Figure 13B) as well as the total GFP expression (Figure 13C) revealed no major differences between pMM108 and pMM59 in-frame switches, leading to a conclusion that both CTG and CAG can be used in the 5’ splice site. However, when mutating the 3’ end of exon 6 to TTG (pMM111) or GTG (pMM110), the GFP induction rate is reduced. In line with previous results showing that lower leakiness follows lower induction rate, results showed reduced total number of GFP-positive cells (Figure 13D) as well as reduced median GFP expression in DMSO-treated cells stably expressing pMM110 and pMM111 (Figure 13E). For the following modifications, pMM59 was used as a starting construct. Example 14: Introduction of translation start site upstream of the ESE2 sequence in exon 7. Based on the previous results using pMM59, a construct in which Kozak and ATG start site were positioned upstream of the ESE2 sequence by point mutations and insertions, various constructs that contain Kozak and ATG at different positions upstream of the ESE2 region were generated, as illustrated in Figure 14A. Constructs were generated containing: Kozak-ATG (GCCACCATG, in pMM59 and P37829 pMM242); Kozak-like/ATG (GCACCATG in pMM243 and pMM244); another Kozak-like/ATG sequence (CACCATG, pMM245); only ATG insertion (pMM246); and ATG insertion within the ESE2 region (pMM247). In addition, pMM244, pMM245, and pMM246 contained a C>G mutation that was placed downstream of the inserted ATG. The nucleotide sequences of the pMM242, pMM243, pMM244, pMM245, pMM246 and pMM247 ON-switches are shown in SEQ ID NOs:72, 73, 74, 75, 76 and 77, respectively. Results showed risdiplam-regulated induction only in the constructs pMM243 and pMM247, whereas all the other constructs with the described modifications did not show any risdiplam-dependent GFP expression as measured using FACS (Figure 14B-14C). Instead, results showed a high number of GFP positive cells (Figure 14D) as well as high median GFP expression (Figure 14E) in DMSO-treated cells, indicating very high leakiness and lack of regulation through these mutations (pMM242, pMM244, pMM245, pMM246). There are several important conclusions from the experiment. Firstly, a comparison between pMM59 and pMM242 shows that moving the Kozak sequence by merely 3 nucleotides leads to constitutive GFP expression in pMM242 compared to pMM59. Furthermore, comparison between pMM243 and pMM244, which differ only by a single point mutation (C to G), shows that the region around ESE2 is highly sensitive to changes. Thus, it is unexpected that the insertion of ATG in the middle of ESE2 sequence leads to a fully functional switch, with very desirable properties (low leakiness and high induction rate). This observation suggest that the current understanding of the role and sequence requirements of ESE2 might not be complete. Example 15: Introduction of translation start site within exon 7 and downstream of the ESE2 sequence. Different outcomes have been seen from different modifications and positioning of the translation start site in relation to the ESE2 region. Therefore, it was decided to further evaluate the effect of positioning the ATG start site downstream of the ESE2 sequence. pLS175 was used as the starting construct that contained mutations of ATG start site (indicated in bold in Figure 15A) as well as ATG>CAG mutation in the 5’ end of exon 6. From this starting construct, a wide variety of constructs were generated: pMM123 (insertion of T at position 53 of exon 7), pMM124 (insertion of G at position 49 of exon 7, a mutation A>G at position 50), pMM125 (insertion of AT at position 31 of exon 7, a single point mutation of T>G at position 32 to resemble ATG, and an insertion of G at position 34 of exon 7), pMM126 (insertion of G at position 40 of exon 7), pMM127 (insertion of A at position 43 and two point mutations AA>GG at position 42), pMM128 (insertion of AT at position 54 of exon 7), and pMM129 (deletion of AT at position 47 and mutation of A>G at position 51). The nucleotide sequences of the pLS175, pMM123, pMM124, pMM125, pMM126, pMM127, pMM128 and pMM129 ON-switches are shown in SEQ ID NOs:78, 79, 80, 81, 82, 83, 84 and 85, respectively. Results showed risdiplam-dependent increase in relative GFP induction rate (Figure 15B) as well as increase in total level of median GFP (Figure 15C) in the construct pMM126. All the other constructs were less sensitive in the induction rate, possibly due to high leakiness as shown by measuring the total GFP- positive cells as well as total median GFP intensity in DMSO-treated cells (Figure 15D-15E) as seen in pMM123, pMM125, pMM127, and pMM128. The two constructs pMM124 and pMM129 also showed low P37829 levels of leakiness (Figure 15E), however, no induction was seen in pMM124 and relatively low induction in pMM129. In the case of pMM124, the lack of induction was due to the formation a cryptic splice site (GT) splicing to exon 8, as described previously (WO 2009/151546 A2). Example 16: Additional modifications to pMM126 in order to improve performance. Following up on the successful insertion of an ATG site downstream of the ESE2 sequence in pMM126, pMM126 was further modified in order to increase the induction rate by generating a stronger translation start site. Six new constructs (illustrated in Figure 16A) were designed as follows: pMM201 (mutation T>G), pMM202 (C>A, and T>G), pMM203 (generation of a novel ATG start site: G>A and C>G, T>C), pMM204 (GT>TC, C>A, and insertion of TG to reconstitute ATG), pMM205 (deletion of G at position 40, insertion of TG at position 46), pMM206 (deletion of G at position 40, insertion of ATGGA at position 45). The nucleotide sequences of the pMM201, pMM202, pMM203, pMM204, pMM205 and pMM206 ON- switches are shown in SEQ ID NOs:86, 87, 88, 89, 90 and 91, respectively. The described constructs were transfected into HEK293 cells for stable expression and treated at increasing risdiplam concentrations for 48(H)Although FACS-based measurements showed that introducing the modifications described in Figure 16A did not result in improved GFP induction rate (Figure 16B) or total GFP median expression level (Figure 16C), constructs pMM204 and pMM205 showed reduced leakiness compared to pMM126 (lower median GFP signal in DMSO) with a comparable induction rate. Moreover, results showed an increased number of GFP-positive cells in the DMSO-treated cells, stably expressing pMM201, pMM202 and pMM206 (Figure 16C-16E). Interestingly, single or double nucleotide mutations in constructs pMM201 and pMM202 lead to constitutive GFP expression, while bigger changes as in pMM203 had much smaller impact, suggesting again that the region downstream of ESE2 is very sensitive to modifications. Example 17: Engineering of a stronger translation start site in pMM129 As described in Figure 15, pMM129 did show improved performance in terms of low leakiness and still contained acceptable induction rate, although to a significantly lower extent than pMM126. Nevertheless, as pMM129 has significantly reduced tag size compared to pMM126, the sequence context of the ATG start site was modified in order to create a strong translation start site, with the aim of increasing the GFP expression rate in the presence of risdiplam. Based on the work by Noderer et al.20, the following mutations and/or insertions were introduced: pMM248 (insertion of TAA at position 49, G>T mutation at position 57), pMM249 (T>A at position 44), pMM250 (T>G at position 44), pMM251 (T>C mutation at position 42, deletion of TT at position 43). The nucleotide sequences of the pMM248, pMM249, pMM250, and pMM251 ON-switches are shown in SEQ ID NOs:92, 93, 94 and 95, respectively. The above-described constructs (illustrated in Figure 17A) were transfected into HEK293 cells for stable expression and treated at increasing risdiplam concentrations for 48h. No improved performance was observed through the introduction of these modifications, i.e. no increase in GFP induction rate (Figure 17B), increase in total level of median GFP including the DMSO-treated control (Figure 17C-17E), suggesting that the introduced modifications led to increased leakiness of the constructs and no improvements in risdiplam-controlled GFP expression. Again, increase in leakiness in constructs pMM249 P37829 and pMM250, having just single point mutations compared to pMM129, shows the sensitivity of this region to any changes. Example 18: Implementing learnings on translation start site (Kozak/ATG) within exon 7 on the short in-frame switch pMM130 Next, it was decided to apply the learnings from moving the translation start site to exon 7 on the shortest version of the in-frame switch, pMM130 (Figure 8). As illustrated in Figure 18A, the ATG site in construct pMM130 was firstly removed to create pMM252, with all ATG sites mutated. The modifications of the previously described pMM126, pMM128, and pMM129 were added to pMM252, resulting in pMM254, pMM255, and pMM256, respectively. One additional modification was added resulting in the construct pMM257, which contained an ATG start site by insertion of a G at position 49. The nucleotide sequences of the pMM252, pMM254, pMM255, pMM256 and pMM257 ON-switches are shown in SEQ ID NOs:96, 97, 98, 99 and 100, respectively. These modifications result in a significantly reduced length of the amino acid tag to the transgene of interest (see table “Tag length” in Figure 18A). HEK293 cells were transfected with the indicated constructs and selected for stable expression under blasticidin selection marker. Cells were treated with varying concentrations of risdiplam for 48h before GFP expression was measured by FACS. Results showed a general increase in leakiness from each of the modifications that were introduced into the shorter in-frame switch pMM130. pMM254 (short in-frame switch with translation start site in exon 7) showed higher leakiness and hence lower induction rate than the control construct pMM126 (long in- frame switch with translation start site in exon 7). Similar observations were made with pMM255 and its control construct pMM128, as well as pMM256 and its control construct pMM129. These observations were true for GFP induction rate (Figure 18B), total level of median GFP (Figure 18C), as well as the number of GFP-positive cells (Figure 18D). The effect of increased background expression level (GFP expression in DMSO-treated cells) is further shown in Figure 18E. However, important to note is that despite the increased leakiness, the newly modified constructs result in a shorter tag length (see Figure 18A). Finally, pMM257 did not lead to increase in leakiness; however, induction rate was slightly reduced (Figure 18A-18E). Example 19: Engineering of splice-induced formation of ATG translation start site between exon 7 and exon 8 All the previously described constructs had a short peptide added to the expressed transgene. In order to remove the tag entirely, we generated a split-ATG approach that was modified to contain an A at the 3’ end of exon 7 and a TG at the 5’ end of exon 8. With no risdiplam added, splicing occurs between exon 6 and exon 8, and no translation start site is formed. Upon treatment with risdiplam, exon 7 is spliced in, forming the ATG site together with exon 8 and driving translation of the gene of interest. No additional tag is added in this case (Figure 19B). In order to achieve this, constructs were designed based on pLS175, a construct in which alternative ATG start sites in exon 6 were mutated (Figure 19A). As the construct pLS175 contains 23 nucleotide long exon 8 sequence, an 8 amino acid tag still remains. However, as demonstrated in the Figure 7, exon 8 can be truncated to just 2 nucleotides, thus completely removing the tag. As described in Figure 19A, 3 constructs were generated with different mutations: pMM198 contained P37829 a mutation GA>TG in the 5’ end of exon 8, pMM199 and pMM200 contained the same mutation as pMM198 but were additionally modified in the intron 7 region in order to increase the strength of polypyrimidine tract with the aim of increasing splicing efficiency between exon 7 and exon 8 and consequently compensating for the generation of a weak splice site by mutating the 5’ end of exon 8 from GA (consensus) to TG (non-optimal splice site). The described constructs were transfected into HEK293 cells for stable expression and treated with risdiplam at increasing concentrations for 48h before they were assessed for GFP expression by FACS-based measurements. pMM112, the short improved in- frame switch was used as a control construct to compare the performance of the split-ATG constructs. The nucleotide sequences of the pMM198, pMM199 and pMM200 ON-switches are shown in SEQ ID NOs:25, 101 and 102, respectively. Results showed that all the split-ATG-based constructs (pMM198-pMM200) showed sensitivity to risdiplam treatment as seen by induced GFP expression relative to DMSO control (Figure 19C), as well as the total level of median GFP expression (Figure 19D), although to a lower extent than pMM112. The increase in the number of GFP-positive cells upon increase in the concentration of risdiplam was also measured (Figure 19E). The leakiness of the split-ATG switch was slightly higher than in pMM112 (Figure 19F), however, the modifications that were introduced in intron 7 did not lead to significant changes in the GFP expression level (Figure 19C-19E). In conclusion, the in-frame switch can be successfully engineered to form an ATG translation start site dependent on exon 7 inclusion without compromising the risdiplam-mediated regulation of GFP expression. Example 20: Introducing ATG start site in exon 8 In another attempt to reduce the length of the amino acid tag fused the gene of interest, the placement of the ATG start site at the 5’ end of exon 8 sequence was investigated. To test this approach, pLS76 was used as a starting construct. The Kozak and ATG sites were removed from the 5’ end of exon 6 and alternative ATG start sites within exon 6 were mutated (Figure 20A). Additionally, the 3’ end of exon 6 was mutated in each of the modified constructs as follows: pMM236 (ATG>CAG), pMM237 (ATG>TAG), pMM238 (ATG>TTG). The mutations in combination with the upstream sequence result in a suboptimal Kozak-like sequence. The exon 7 sequence remained as described for pLS76. In order to introduce a translation start site within exon 8, a mutation was introduced at the 5’ end of exon 8 in pMM236-pMM238 (G-A>ATG) as illustrated in Figure 20A. The risdiplam-mediated gene expression control is retained by the presence of a Kozak-like sequence within the native sequence of exon 7 (highlighted in bold in Figure 20B). The nucleotide sequences of the pMM236, pMM237 and pMM238 ON switches are shown in SEQ ID NOs:103, 104 and 105, respectively. The presented concept relies on the presence of risdiplam to allow inclusion of exon 7 and hence the introduction of an efficient Kozak-like sequence contained within exon 7 (TAAGGA) to enhance translation driven by the downstream ATG start codon present in exon 8 (at the 5’ end of pMM236- pMM238). In the absence of risdiplam, only a weak Kozak-like sequence (provided by the mutations at the 3’ end of exon 6, see Figure 20A) will be present and the translation start will not be enhanced. The presented design was tested in HEK293 cells that were stably expressing pMM236-pMM238 and compared to the improved in-frame switch pMM112. Cells were treated with increasing amounts of P37829 risdiplam for 48 h, followed by FACS-based analysis. Results showed that GFP expression by the present constructs can be induced with increasing amounts of risdiplam as shown by assessment of the induction rate (Figure 20C) as well as the total median expression level of GFP (Figure 20D), suggesting that the weak/strong Kozak-like concept in combination with exon 7 inclusion allows a certain control over GFP expression level. However, high GFP expression levels were also measured in the uninduced, DMSO- treated cells as shown by the total cell number of GFP-positive cells (Figure 20E) as well as the median GFP signal (Figure 20F). As hypothesized, the weaker the Kozak-like sequence in exon 8, the lower the GFP expression in DMSO and higher the induction rate. In summary, the Kozak sequence-based switch does allow control over gene expression, however, the background expression is elevated. Example 21: Engineering a SMN2-based OFF-switch Inducible gene expression systems could not only be developed as an ON-switch to turn on gene expression but also as an OFF-switch to inhibit gene expression. In this context, a risdiplam-inducible OFF-switch was developed that would respond to various concentrations of risdiplam by suppressing transgene translation. The design strategy of the presented ON-switch was used and a construct was engineered to allow inhibition of translation by introducing a single nucleotide change (Figure 21A). To create an OFF-switch, the design was based on the pMM130 construct having reduced intron length (intron 6 and 7), reduced exon length (exon 6 and 8), as well as reduced transgene tag size (by using the ATG codon positioned at the 3’ end of exon 6 as the translation start site) (Figure 21B). Presence of ATG at the 3’ end of exon 6 allows for complete tag removal in the OFF-switch. pMM130 contains only 2 nucleotides of exon 8 (GA); by adding an additional nucleotide (G) downstream of the sequence (Figure 21B), a frame-shift in the transgene sequence (positioned downstream of exon 8) can be introduced upon inclusion of exon 7 by risdiplam, giving rise to an OFF-switch. In contrast, in the absence of risdiplam (exclusion of exon 7), the transgene can be correctly translated. In order to reduce the length of a peptide produced in the OFF state and drive transcript degradation by nonsense mediated decay mechanism, the presented OFF-switch (pMM193, Figure 21B) was further modified by introducing premature termination codon in exon 7. To do that, a nucleotide deletion at position 19 of exon 7 was created (TCAAAAA>TCAAAA-), (Figure 21C), which will lead to the generation of a premature STOP codon downstream of the mutation within exon 7. In the presence of risdiplam, exon 7 will be included within the mature RNA transcript and with the presence of the premature STOP codon, the translation of the downstream-encoded transgene will be interrupted and transcript degraded (Figure 21C, pMM194). The nucleotide sequences of the pMM193 and pMM194 OFF-switches are shown in SEQ ID NOs:28 and 29, respectively. As for previous designs, HEK293 cells were used, stably expressing the constructs pMM193, pMM194 and pMM130 as a control, cells were treated for 48h with increasing amounts of risdiplam and FACS used to measure changes in GFP expression as a response to risdiplam treatment. As shown in Figure 21D-21E, GFP expression was significantly reduced in pMM193-, and pMM194-transfected cells with increasing concentrations of risdiplam, while cells transduced with pMM130 (the ON-switch) showed increased GFP expression level. The number of GFP-positive cells in the cells expressing the OFF- P37829 (pMM193, pMM194) or the ON-switch (pMM130) was also measured. A complete reduction in GFP- positive cells in the population that carries the OFF-switch was not observed (Figure 21F). It is possible that incubation of risdiplam for 48h is not sufficient to measure complete reduction of the GFP due to the enhanced GFP version used in these assays. Total GFP expression level in untreated cells was also measured. As expected, GFP expression is very high in pMM193 and pMM194-transfected cells in the absence of risdiplam (Figure 21G). Example 22: Constructs lacking selection antibiotic resistance gene All previously tested constructs had IRES-blasticidin-SV40 sequence added after the GFP encoding sequence leading to expression of antibiotic resistance gene (blasticidin), which was used for stable cell line selection. However, as the in-frame switch is intended to be used without antibiotic selection in AAV viral vectors, the inventors decided to replace the whole IRES-blasticidin-SV40 fragment with BGH polyA (Figure 22A). Five different constructs representing various modifications of the in-frame switch were selected: pMM263 (pLS76) SMN2 in-frame switch, pMM262 (pLS168) in-frame switch with 60 nucleotide reduction of exon 6, pMM259 (NB pMM112 is the same construct as pMM259) improved in-frame switch combining truncated introns, exon 6 and exon 8, pMM260 (pMM13) improved in-frame switch with reduced tag sequence and pMM272 (pMM198) split ATG in-frame switch. In addition, a previously published ON-switch was added for comparison pMM274 (Monteys et al. Nature (2021) 596: 291-295) (Figure 22B). The described constructs were transfected into HEK293 cells for stable expression and treated with risdiplam at increasing concentrations for 48 h before they were assessed for GFP expression by FACS-based measurements. The nucleotide sequences of the pMM263, pMM262, pMM259, pMM260, pMM272, pMM274 switches are shown in SEQ ID NOs: 38, 42, 22, 23, 25 and 107, respectively. Results showed that all the BGH polyA constructs exhibited comparable induction rates to their IRES- blasticidin-SV40 counterparts (Figure 22C), however with increased median GFP signal and GFP leakiness in DMSO, which is most likely caused by increased transcript stability driven by removal of the long 3’UTR comprised of IRES-blasticidin and usage of a stronger polyA signal sequence (BGH vs SV40) (Figure 22D to 22F). Importantly, a comparison of the in-frame switches of the present disclosure with those of the previously published switch (Monteys et al. Nature (2021) 596: 291-295) shows that the present in-frame switches have significantly higher (up to 13x higher, when compared pMM259 vs pMM274) induction rates at all low risdiplam concentrations (Figure 22G). Example 23: Generation of short split ATG switch [pMM273] Despite significantly smaller size and better induction rate compared to pMM263 and pMM274 switches, pMM259 still has a short peptide ‘tag’ added to the expressed transgene. In order to remove the tag entirely, we employed a split-ATG approach, as in Example 19, wherein the 5’ end of exon 8 was modified to contain ‘TG’ instead of ‘GA’, to create pMM273 (Figure 23A). With no risdiplam added, splicing occurs between exon 6 and exon 8, and no translation start site is formed. Upon treatment with risdiplam, exon 7 is spliced in, forming the ATG site together with exon 8 and driving translation of the gene of interest. No additional tag is added in this case (Figure 23B). In order to achieve this, all remaining ATG start sites were removed in construct pMM263 and exon 8 was mutated to TG resulting in P37829 the construct pMM273 (Figure 23A) . As the construct pMM273 contains only a 2 nucleotide-long exon 8, which forms an active ATG start site together with the exon 7, there is no amino acid tag added. The described constructs were transfected into HEK293 cells for stable expression and treated with risdiplam at increasing concentrations for 48h before they were assessed for GFP expression by FACS-based measurements. pMM274, pMM263 and pMM259 were used as control constructs to compare the performance of the split-ATG constructs. The nucleotide sequence of the pMM273 ON-switch is shown in SEQ ID NO:111. The results show that split-ATG-based construct pMM273 showed sensitivity to risdiplam treatment as seen by induced GFP expression relative to DMSO control (Figure 23C), as well as the total level of median GFP expression (Figure 23D), although to a lesser extent than pMM259. The increase in the number of GFP-positive cells upon increase in the concentration of risdiplam was also measured (Figure 23E), however both pMM259 and pMM273 have almost 100% GFP positive cells in DMSO. Although almost all the cells express GFP without risdiplam treatment, the median GFP expression in DMSO of the split-ATG switch pMM273 was slightly lower compared to pMM259 (Figure 23F). In conclusion, the in- frame switch can be successfully engineered to form an ATG translation start site dependent on exon 7 inclusion, without compromising the risdiplam-mediated regulation of GFP expression. Example 24: Modifications of exon 75’splice site sequences in pMM273 Based on the results obtained with pMM273, we investigated the effect of modifications within the 5’ splice site (5’ss) within the exon 7 sequence. Three new constructs were generated with the modifications indicated in Figure 24: pMM362 (G>A at position -3 in exon 7, a deletion of nucleotide C at position +7 of intron 7; and a mutation T>G at position +8 of intron 7), pMM363 (A>G and G>T at positions -5 and -3, respectively, and TC>AT mutation within intron 7), and pMM364 (C>G mutation at position +7 within intron 7). HEK293 cells were stably transfected with each one the constructs or pMM273 (as a control), and treated with the indicated risdiplam concentrations for 48h. The modification of pMM362 disrupted splicing regulation and rendered the switch constitutively active without dependency on the presence or absence of risdiplam (Figure 24 A-C). By contrast, pMM363 and pMM364 clearly showed reduced leakiness in comparison to pMM273 (Figure 24E). However, the level of expression of the transgene was reduced from these constructs compared to pMM273 (Figure 24C). Furthermore, the total expression of the transgene at low risdiplam concentrations was significantly higher for pMM364 than it was for pMM363 (Figure 24C), indicating that the modifications in pMM364 outperformed pMM273 and could be used for further optimization. Example 25: ESE2 region deletions in pMM273 to reduce leakiness in DMSO In order to further reduce the background expression in the absence of risdiplam, we hypothesized that deletion of the ESE2 region and adjacent sequence within the exon 7 region would lead to reduced risdiplam-independent inclusion of exon 7. In addition, two of the mutations evaluated (in constructs pMM358 and pMM359) have previously been shown to reduce background SMN2 splicing levels (Ishigami et al.22). We designed four different constructs (Figure 25A) based on the original construct pMM273 having the original ESE2 sequence (AAAAAGAGGAA): pMM358 (AAAAAGAGGAA>GAAGGA), pMM359 (complete deletion of ESE2 sequence), pMM360 (complete deletion of ESE2 sequence, and P37829 deletion of the downstream nucleotides GGTG), and pMM361 (complete deletion of the ESE2 sequence, deletion of the upstream nucleotides AAAATC, and deletion of the downstream nucleotides GGTGC). Our results revealed a dramatic reduction in median GFP leakiness for all the constructs harboring ESE2 deletions (pMM358-pMM361), as well as reduced number of GFP-positive cells in untreated cells compared to pMM273 (Figure 25D-E). However, only constructs pMM358 and pMM359 were able to respond to risdiplam at concentrations below 500 nM (Figure 25A), suggesting that the strong truncation of exon 7 as designed in pMM360 and pMM361 disrupts splicing. This was further confirmed by PCR amplification of the constructs, which revealed reduced splicing efficiency upon risdiplam treatment for constructs pMM358 and pMM359, but no exon 7 inclusion for construct pMM360 (Figure 25G). Lower basal splicing of constructs pMM358 and pMM359 resulted in lower leakiness, but also lower median GFP expression levels upon risdiplam induction (Figure 25B). Despite lower median GFP expression (Figure 25C), construct pMM359 shows a significantly better induction rate (Figure 25A, 25F) and lower leakiness (Figure 25E) compared to pMM273, making it a better-suited ON-switch for applications requiring low leakiness. Example 26: Testing ESE2 region deletions in pMM273 Based on the results obtained by removing the ESE2 sequence (Example 25), we hypothesized that a gradual reduction of the ESE2 region and adjacent sequences could result in a construct with reduced leakiness levels that maintains high expression and induction rate. We investigated the effect of deleting the ESE2 region and the adjacent sequences, in a step-by-step manner. First, we designed 11 constructs where the ESE2 region (AAAAAGAAGGAAGG, underlined hereafter) region was either partially or completely removed (Figure 26A). pMM365 has almost complete ESE2 region deletion (- AAAAAGAAGGAAG) and pMM366 has complete ESE2 region deletion (-AAAAAGAAGGAAGG). For pMM367, pMM372, pMM373, pMM374, the ESE2 region and downstream nucleotides were also deleted: pMM367 (-AAAAAGAAGGAAGGT), pMM372 (-AAAAAGAAGGAAGGTGC), pMM373 (- AAAAAGAAGGAAGGTGCTC), pMM374 (-AAAAAGAAGGAAGGTGCTCAC). In Example 25, removal of GGTG sequence in pMM360 resulted in a complete loss of splicing as compared to pMM359. Thus, in pMM375, we introduced the same deletion as in pMM374 (-AAAAAGAAGGAAGGTGCTCAC), but additionally mutated 4 downstream nucleotides from ATTC to GGTG. Finally, we also tested only partial ESE2 region deletions in pMM368 (-AAAAAGAAGG), pMM369 (-AAAAAGAAG), pMM370 (-AAAAAGAA) and pMM371 (-AAAAA). HEK293 cells, stably transfected with one of the various different constructs or pMM273 (as a control), and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP signal in response to risdiplam for most of the constructs (Figure 26B-C, 26F), with the exception of the constructs having large deletions (pMM372, pMM373, pMM374), where induction was low and further decreased with increasing size of the deletion. Interestingly, pMM375 – which has the same deletion as inactive pMM374, but with an additional ATTC> GGTG mutation downstream of the ESE2 region – had completely rescued risdiplam induction of transgene expression, which was higher than pMM273 (Figure 26F). These data suggest that the GGTG sequence has an important regulatory role in the construct's splicing and response to risdiplam. Median GFP expression in DMSO (leakiness) was substantially decreased in all of the constructs tested, except P37829 for pMM369, pMM370 and pMM371 (Figure 26E). However, all constructs resulted in more than 95% GFP+ positive cells in DMSO (Figure 26G). Step by step deletions of the ESE2 sequence allowed us to see a striking difference between constructs pMM369 (-AAAAAGAAG, high leakiness, approx.3x reduced compared to pMM273) and pMM368 (-AAAAAGAAGG, very low leakiness, approx.10x reduced compared to pMM273), where deletion of one additional nucleotide (G) dramatically reduced leakiness (Figure 26E). Introducing gradual deletions to the ESE2 region, we identified several mutations that led to lower median GFP leakiness, but maintained high induction rate, such as pMM368, pMM369, pMM375. Example 27: Testing ESE2 region deletions in pMM273 Based on the results obtained in the gradual ESE2 region deletions (Example 26), we tested several other deletions, where the ESE2 (AAAAAGAAGGAAGG, underlined hereafter) region was either partially or completely removed, while keeping the GGTG sequence intact and instead focusing on deletions of the region upstream of the ESE2 region (Figure 27A). In pMM376, pMM377 and pMM378 we tested complete ESE2 region deletion combined with additional upstream deletions: pMM376 (- TCAAAAAGAAGGAA), pMM377 (-AAAATCAAAAAGAAGGAA) and pMM378 (- CAAAATCAAAAAGAAGGAA). For pMM379, the ESE2 region was only partially deleted (- ACAAAATCAAAA). pMM380 tested deletion in a downstream region (-CTCACATTC), while pMM381 combined pMM379 (-ACAAAATCAAAA) and pMM380 deletions (-CTCACATTC). Finally, pMM382 deleted the majority of SMN2 exon 7 (-ACAAAATCAAAAAGAAGGAAGGTGCTCACATTC), while pMM386 (-AAA) and pMM387 (-A) tested small ESE2 region deletions not tested in Example 26. HEK293 cells, stably transfected with one of the various different constructs or pMM273 (as a control), and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP expression in response to risdiplam for most of the constructs (Figure 27B-C, 27F), except pMM382 (which has a large deletion, leading to no splicing) and pMM386 (where (AAA) deletion led to constitutive splicing of the construct). Constructs pMM376, pMM377, pMM379, pMM380 and pMM381 showed increased induction rate compared to pMM273 at 250 nM concentration of risdiplam (Figure 27F). However these constructs displayed reduced median GFP signal compared to pMM273 (Figure 27C). Relative to pMM273, median GFP expression in DMSO (leakiness) was decreased the most for constructs pMM376, pMM378, pMM381 and pMM382, with a modest decrease in pMM377 and pMM379, and no change or upregulation for pMM380, pMM386 and pMM387 (Figure 27E). However, all the constructs resulted in more than 95% GFP+ positive cells in DMSO (Figure 27G). Compared to pMM273, despite lower median GFP expression, construct pMM381 shows significantly lower leakiness and higher induction rate, making it a better-suited ON-switch for applications requiring low leakiness. Example 28: Testing exon 7 deletions in pMM273 Based on the results obtained after GGTG deletion combined with the ESE2 region deletion (Examples 25 and 26), the GGTG sequence appears to have a regulatory role in exon 7 splicing. We designed three mutations to test its role in isolation from the ESE2 region sequence: pMM436 harboring partial ESE2 P37829 (AGAAGG) deletion and partial GGTG (TGCT) deletion, pMM437 where GGTG was deleted and pMM438 where GGTGCT sequence was deleted (Figure 28A). HEK293 cells, stably transfected with one of the various different constructs or pMM273 (as a control), and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP signal in response to risdiplam for all the constructs, except pMM436 which was inducible only at higher risdiplam concentrations and showed very low median GFP expression (Figure 28B-C, 28F). Deletion of GGTG (pMM437) or GGTGCT (pMM438) resulted in significantly lower leakiness compared to pMM273 (Figure 28E), as well as higher induction rate at risdiplam concentrations above 125 nM (Figure 28A). The percentage of GFP+ cells relative to pMM273 was lower for pMM436, but comparable for pMM437 and pMM438 (Figure 28D). Lower median GFP leakiness in DMSO, translated to lower median expression upon induction for pMM437 and pMM438 making them better-suited for applications requiring low leakiness and lower total transgene expression. Example 29: Intron 7 deletions in pMM273 In a recently-published study, Recinos et al.23 identified a region of SMN2 intron 7 [334, 358] as being important for SMN2 exon 7 splicing enhancement. In order to test whether deletion of this region can decrease exon 7 incorporation in DMSO (leakiness) we designed multiple deletions of the region (pMM477-pMM483) using the pMM273 ON-switch as a starting point (Figure 29A). In addition, we introduced a single point A>G mutation in construct pMM484 and randomized mutation GCACTGTACACTCTGACATATGAA>TGAATTAGGAGGGGAGGATTCATT in pMM485 (Figure 29A). HEK293 cells, stably transfected with one of the various different constructs or pMM273 (as a control), and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP expression in response to risdiplam, although to various extents, for all the constructs (Figure 29B-C, 29F). pMM485 yielded very high GFP expression levels in DMSO (Figure 29E) and displayed a low induction rate (Figure29B). Compared to pMM273 – except pMM482 – all the remaining constructs had comparable leakiness or higher leakiness (Figure 29E), comparable induction at 250 nM risdiplam (Figure 29F) and greater percentage of GFP+ cells in DMSO (Figure 29D). Only construct pMM482 showed moderately (approx.50%) decrease leakiness and comparable induction rate (Figure29A, 29F) relative to pMM273. In conclusion, we could not confirm any significant role of the previously described intron 7 region on exon 7 basal splicing in our splice switch. Example 30: Intron length testing in pMM273 Intron length may influence regulation and efficiency of splicing. In order to test whether SMN2 exon 7 inclusion without risdiplam treatment could be further reduced, we designed 11 different constructs (pMM464-pMM474) based on pMM273, where we introduced back previously deleted (Example 4) intronic sequences. Fragments were introduced in the middle of each intron, the length of each fragment and the total intron length are summarised in table shown in Figure 30A. HEK293 cells, stably transfected with one of the various different constructs or pMM273 (as a control), and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP P37829 expression levels. Results showed increased GFP signal in response to risdiplam for all the constructs (Figure 30B-C, 30F). Although the percentage of GFP+ cells was comparable for all the constructs (Figure 30D), the median GFP signal was significantly reduced for all the tested constructs compared to pMM273, with the highest GFP signal reduction in pMM466, pMM470, pMM472 and pMM474 (Figure 30E). Similarly, the induction rate was increased relative to pMM273 for all the tested constructs at all risdiplam concentrations (Figure 30B). At 250 nM concentration of risdiplam, constructs pMM466, pMM470, pMM472 and pMM474 showed the highest induction (Figure 30F) and were selected for further evaluation. Example 31: Exon 6 and 7 mutations in the improved constructs We further modified constructs selected in the Example 30 (pMM466, pMM470, pMM472, pMM474) by mutating a remaining ATG site in exon 6 (ATG>CTG) and deleting a TAA sequence in exon 7 (Figure 31A). The TAA sequence was identified to increase induction rate in a screen where every single, two, three, four or five nucleotides of exon 7 were deleted in construct pMM273 and analyzed by FACS (results not shown). HEK293 cells, stably transfected with one of the various different constructs and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP expression in response to risdiplam for all the constructs (Figure 30B-C, 30F). While the percentage of GFP+ positive cells in DMSO was slightly decreased in all modified constructs (pMM567-pMM570) (Figure31D), the median GFP expression was comparable (Figure 31E). However, deletion of TAA sequence significantly increased induction rate at low risdiplam concentrations (Figure 31B), with approx.8-fold induction for pMM567-570 compared to 6-fold for the constructs from Example 30 (Figure 31F). pMM569 was selected as a particularly promising construct for further evaluation, as it combined low leakiness with high induction rate at low risdiplam concentrations. Example 32: Exon 6 and 7 mutations in pMM569 We next ran a randomized mutation screen to identify novel mutations decreasing pMM569 leakiness and potentially improving the induction rate.10 constructs, pMM591 and pMM593 to pMM601 were selected for further evaluation (Figure 32A). In addition, we tested one of the previous intron 7 mutation constructs (pMM482, Example 29). HEK293 cells, stably transfected with one of the various different constructs, and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP signal in response to risdiplam for the majority of constructs, except pMM593 and pMM597, which exhibited only a minimal GFP expression induction (Figure 32B-C, 32F). Constructs pMM594 and pMM599 had significantly improved GFP expression induction at increasing risdiplam concentrations relative to pMM569 (Figure 32B), with an approximate 50% increase at 62.5 nM risdiplam (Figure 32F). However, like pMM600, pMM594 was found to be significantly more leaky in DMSO, both in terms of the percentage of GFP-expressing cells (up to 3x more GFP+ cells) (Figure 32G) and median GFP expression (Figure 32E). Constructs pMM593, pMM595, pMM596, pMM597 and pMM601 had P37829 decreased percentage of GFP+ cells in DMSO compared to pMM569, but only pMM595, pMM596 and pMM601 had an acceptable induction rate at 62.5 nM of risdiplam (Figure 32F). To summarize, we identified pMM599 (C>A mutation in intron 7) as promising construct for increasing induction rate, without increasing leakiness. We also identified the pMM595 (TTTTAGAC>ATAATTAA mutation at position 3-10 of exon 7), pMM596 (CCTT deletion at position 257-260 of intron 6) and pMM601 (AAT>GCC at position 46 of exon 7) mutations as being variations leading to reduced leakiness levels compared to pMM569. Example 33: Intron 6 deletion screen in pMM569 While Example 30 suggests that increasing intron length significantly reduces leakiness, we wanted to identify any remaining intronic sequences regulating SMN2 exon 7 splicing and to this end we performed a whole intron 6 deletion screen, with 5-nt long non-overlapping deletions (Figure 33A). We next selected 11 intron 6 deletion constructs (pMM642-651, pMM709) for further evaluation (Figure 33B). HEK293 cells, stably transfected with one of the various different constructs, and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP signal in response to risdiplam for all the constructs (Figure 33C-D, 33G). Constructs pMM642, pMM645, pMM649 and pMM651 showed increased induction rate at all risdiplam concentrations (Figure 33C), with approximately 25-30% higher induction for pMM642, pMM649 and pMM651 and 2-fold higher for pMM645 at 62.5 nM risdiplam concentration. Leakiness measured as the percentage of GFP+ positive cells in DMSO (Figure 33H) and median GFP expression in DMSO (Figure 33F) was slightly increased or comparable for all the tested constructs, with the exception of increased median GFP expression for pMM650 (Figure 33F). Overall, mutations pMM642, pMM645, pMM649 and pMM651 showed slightly better induction rate with a comparable leakiness levels to pMM569. Example 34: Intron 7 deletion screen in pMM569 We next performed a whole intron 7 deletion screen, with 5-nt long non-overlapping deletions (Figure 34A). We next selected 10 intron 7 deletion constructs (pMM634-641, pMM711, pMM712) for further evaluation (Figure 34B). HEK293 cells, stably transfected with one of the various different constructs, and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP signal in response to risdiplam for all the constructs (Figure 34C-D, 34G). However, no construct showed increased induction rate compared to pMM569 (Figure 34C). The only exception was pMM635 at 62.5 nM risdiplam (Figure 34G), however that was an outlier, as it was not consistent across concentrations (Figure 34C). Leakiness measured as the percentage of GFP+ positive cells in DMSO (Figure 33H) and median GFP expression in DMSO (Figure 33F) was slightly decreased for deletions pMM636, pMM637 and pMM638, while increased for pMM639 and pMM711, with pMM711 being the most leaky construct. P37829 Example 35: Comparison of leakiness-reducing mutations in pMM569 In order to design a construct tailored for application where low leakiness levels are essential, we combined pMM569 switch with selected mutations shown to reduce leakiness. We selected three mutations: pMM579 having ESE2 region deletion (as in pMM359), pMM595 with mutated exon 7 and pMM596 with intron 6 deletion (Figure 35A). HEK293 cells, stably transfected with one of the various different constructs, and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP signal in response to risdiplam for all the constructs (Figure 35B-C, 35F). Although none of the constructs displayed reduced median GFP expression compared to pMM569 (Figure 35E), pMM579 and pMM596 significantly reduced the percentage of GFP+ cells compared to pMM569 (Figure 35G). While the leakiness of pMM579 and pMM596 was similar, the induction rate and median GFP expression was significantly better for pMM596 (Figure 35B-C), with approximately 5-fold induction at 62.5 nM of risdiplam for pMM596, compared to 2-fold for pMM579 (Figure 35F). Design pMM596 was selected as a promising low leakiness variant of pMM569. Example 36: Exon 6 deletions in pMM569 To further reduce the size of the pMM569 ON-switch, we tested two exon 6 deletions: from 45 nt in pMM569 to 30 nt in pMM618 and 15 nt in pMM619 (Figure 36A). HEK293 cells, stably transfected with one of the various different constructs, and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Both induction rate and median GFP signal were comparable for all three constructs at all risdiplam concentrations (Figure 36B-C), with 8-12-fold induction at 62.5 nM risdiplam for all constructs. A slight increase in the percentage of GFP+ cells (Figure 36D, G) as well as median GFP signal in DMSO (Figure 36E) for pMM618 and pMM619 revealed a modest increase in leakiness with a shorter exon 6. To summarize, both exon 6 deletions combine reduced size with similar performance to pMM569. Example 37: Testing mammalian promoters with pMM569 All previous experiments were performed using a construct employing a CMV promoter. In order to test performance of the risdiplam-inducible switch with other commonly-used promoters we combined pMM569 design with 7 other promoters: pMM581 (full length (FL) CAG promoter), pMM582 (hEF1a promoter), pMM583 (hUBiC promoter), pMM584 (RSV promoter), pMM589 (TK promoter), pMM590 (PGK promoter) and pMM562 (minimal CAG promoter) (Figure 37A). HEK293 cells, stably transfected with one of the various different constructs, and treated with indicated risdiplam concentrations for 48h, were analyzed by FACS to measure GFP expression levels. Results showed increased GFP signal in response to risdiplam for all the constructs, except pMM589 (TK promoter) and pMM590 (PGK promoter), which showed a very modest increase only at the highest risdiplam concentrations investigated (Figure 37B-C). At 62.5 nM risdiplam concentration, constructs pMM582 (hEF1a) and pMM652 (minimal CAG) had comparable induction rate to pMM569 (CMV), while pMM581 (FL-CAG), pMM583 (hUbiC), pMM584 (RSV), although still inducible, were 2-3-fold lower P37829 (Figure 37F). Construct pMM581 (FL-CAG promoter) showed very high leakiness measured both by median GFP expression and % of GFP+ cells (Figure39E, G), while minimal CAG promoter (pMM652) and hEF1a (pMM582) leakiness was comparable to CMV. Constructs pMM583 (hUbiC) and pMM584 (RSV) had reduced % of GFP+ cells, while constructs pMM589 (TK) and pMM590 (PGK) had almost no GFP+ cells in DMSO (Figure 31G). It is important to note that although constructs pMM589 (TK) and pMM590 (PGK) had very low induction, they both reached 100% GFP+ cells at 500 nM of risdiplam meaning that the splice switch was still active, only it was masked by low GFP expression levels. Example 38: Comparison of various iterations of risdiplam-regulated splice switch Based on previous data generated with various iterations of in-frame-based, risdiplam-regulated splice switches, we performed an experiment comparing the original in-frame splice switch variant based on sequence described in pLS76 (pMM263), a construct based on a splice switch published by Monteys et al. Nature (2021) 596: 291-295 (pMM274), the split ATG short switch (pMM273), a pMM273-based switch with a truncation within the ESE2 region, as well as the split ATG switch (pMM569) (Figure 38A). We used HEK293 cells to transfect the constructs described above in order to compare their performance side-by-side. pMM569 showed superior performance in terms of induction rate compared to published constructs as well as various versions of the improved in-frame switch (Figure 38B). At low risdiplam concentration (62.5 nM), pMM569 showed approx.17-fold induction rate compared to 7-fold for pMM273 and 4-fold for pMM359. While the total level of GFP expression remained unchanged between pMM273, pMM359 and pMM569 at the highest risdiplam concentrations, pMM569 had significantly higher median GFP expression at lower risdiplam concentrations compared to pMM359 (Figure 38C). Background expression, i.e. in the absence of risdiplam, exon 7 inclusion and hence median GFP expression was highest in construct pMM273, whereas all other constructs were comparable (Figure 38E). At the same time, the percentage of GFP positive cells was lowest for the least inducible constructs (pMM263 and pMM274) and highest for pMM273, while pMM359 and pMM569 had approximately 50% GFP-positive cells in DMSO (Figure 38D, 38G). While the pMM569 switch achieved almost 100% GFP-positive cells at 15 nM risdiplam, the starting construct (pMM263) as well as the published (pMM274) required much higher risdiplam concentrations. In conclusion, we identified pMM569 as an ON-switch construct that outperformed other variations in terms of induction rate, and having background GFP expression levels comparable to the least leaky constructs tested. Example 39: Risdiplam-regulated NanoLuciferase (NLuc) expression In order to test the applicability of the optimized in-frame switch, we assessed whether a different reporter gene can be controlled similarity to GFP by risdiplam. Therefore, we used NanoLuc (NLuc) as a reporter and cloned it into several variations of the switch selected as well performing in Example 31: pMM568, pMM569 (front-runner, reduced length of intron 7 in comparison to pMM568), pMM570 (reduced length of intron 7 in comparison to pMM568), and pMM579 (low leaky version of pMM569, additional deletion of the ESE2 region sequence), (Figure 39A). HEK293 cells stably transfected with one of the various different constructs were treated with indicated risdiplam concentrations for 48h and cells were lysed and analyzed for NLuc expression (Figure 39B-D). We observed increased induction rate in NLuc expression in all of the constructs, however, the deletion of the ESE2 region sequence in pMM612 (switch pMM579 with NLuc as a reporter) resulted in reduced induction rates at low nM concentrations of risdiplam in P37829 comparison to the other switch variants (Figure 39B), in line with reduced total median GFP signal (Figure 39C). However, we did observe slight reduction in the background expression level in construct pMM611 (switch pMM570 with NLuc as a reporter). Example 40: Risdiplam-regulated Cas9 expression for gene editing applications As a potential application for regulated gene expression systems, we used Cas9 as a protein of interest under the control of risdiplam. We used the same switch designs as in Example 38, and replaced the GFP reporter sequence with a sequence that encodes for Cas9 protein (Figure 40A). We introduced one additional construct (pMM596), which has a deletion within the intron 6 sequence of TCCT as illustrated in Figure 40A. This construct has been shown previously (Example 32) to have lower background expression level, a criteria required for optimal regulation of Cas9 protein expression. In order to test the performance of the various versions of the in-frame switch, we stably transfected HEK293 cells with the above-mentioned constructs using piggybac method. Having established stable HEK293T cell lines, we have used nucleofection-mediated delivery of 3 different sgRNA (UUGGCUUCCUGGGCUGCUA, GCAGCCCUCCACUCCCAUGG, GGCGCUGUCAUGAUGUUCGU) that target the CD81 gene, encoding for a cell-surface protein. Risdiplam was added at the indicated concentrations and cells were incubated for 72h before they were stained with anti-CD81 antibody to assess reduction of expression of the protein upon Cas9 induction with risdiplam using FACS-based analysis (Figure 40B). Cells that were not transfected with sgRNA mix served as a negative control. In all of the constructs described in Figure 40A, we observed a risdiplam dose-dependent reduction in CD81 expression (Figure 40B-H), with a maximum decrease of 50% for the previously published construct (pMM274) (Figure 40D) and up to 12% for pMM596 (Figure 40H). However, we also observed risdiplam-independent reduction of CD81 expression, driven by leaky Cas9 expression, in particular in constructs based on pMM263 (Figure 40C), pMM273 (Figure 40E), pMM359 (Figure 40F), and pMM569 (Figure 40G). However, the risdiplam- independend reduction of CD81 expression was significantly lower in the construct having the pMM596 ON-switch (Figure 40H), reaching levels observed for the previously published sequence pMM274 (Figure 40D). In conclusion, the improved ON-switch sequence pMM596, with the significantly reduced leakiness profile, is a promising candidate to be used as a regulation system for inducible Cas9 expression. Example 41: Risdiplam-regulated CAR expression in CAR-T therapy A potential therapeutic application for small molecule-regulated gene expression system is illustrated in Figure 41, which is based on the hypothesis that risdiplam could be used to induce the expression of chimeric antigen receptors (CARs) in T cells. An option for regulated expression of CARs in T cells could translate into huge benefits for patients, as regulated CAR expression could increase safety of the therapy, allowing some of the side effects observed in the CAR-T therapy space to be overcome, e.g. strong immune reaction and the well-established cytokine storm effect. Furthemore, it could allow for using more aggressive constructs, or using second inducible CAR-expressing constructs to prevent development of cancer resistance during CAR-T therapy. In order to assess the suitability of the risdiplam-inducible ON-switch for such applications, we investigated the switch in a more relevant cellular model, namely Jurkat cells, an immortalized cell line P37829 derived from leukemic T-cells. In order to test the in-frame switch in these cells, we used pMM569 and introduced some mutations in order to test the effect on the leakiness in these cells. We introduced a deletion in the ESE2 region (pMM579), a modification that was previously shown to reduce background expression, pMM595 with the indicated mutations highlighted with bold letters within the exon 7 sequence in Figure 42A, and pMM596 (deletion of TCCT within the intron 6 sequence) – see Figure 42A). Jurkat cells were stably transfected with these constructs and GFP was measured using FACS-based readouts 48h after addition of risdiplam at the indicated concentrations (Figure 42B-G). Similar to our observations in HEK293 cells, we observed high induction rates and low leakiness for the pMM596 switch compared to the other constructs (Figure 42B, 42E-G). In comparison, pMM595 showed higher leakiness (Figure 42E, 42G), although the induction rate was not reduced (Figure 42B, 42E-G), as we have observed that the total level of median GFP signal (Figure 42C) as well as the number of GFP expressing cells (Figure 42D) was high accordingly. In conclusion, we have established that the in-frame, risdiplam-inducible ON-switch performs to a similar standard in Jurkat cells, a T-cell model with relevance for testing CAR-T cells and we propose the use of this switch regulated by risdiplam as a therapeutic approach to regulate CAR expression in patients. Example 42: Risdiplam-regulated expression of GFP in AAV-transduced neurons. The in-frame switch constructs pLS76 and its optimized counterpart pMM359 (Figure 43A) were incorporated into an adeno-associated virus (AAV) vector genome, subsequently packaged into AAV vectors with the AAV2.7m8 capsid, and employed to transduce human embryonic stem cell (hESC)- derived neurons at a multiplicity of infection (MOI) of 20,000 genome-containing vector particles per cell. Prior to AAV infection, neurons underwent a differentiation period of 21 days. At three days following AAV transduction, varying concentrations of risdiplam were added to the cultures, with DMSO serving as the negative control. The incucyte live cell imaging analysis system was utilized to assess the expression of the reporter gene GFP in neurons transduced with AAV vectors containing the different in-frame switches, providing insights into both transduction efficiency and the functionality of the in-frame switches within the AAV context. Consistent with our findings with cell lines transfected with the ON-switch constructs, induction of GFP expression was observed with the AAV genome containing the pLS76 switch as depicted in Figure 43B. Notably, the GFP expression mediated by the optimized pMM359 sequence was significantly enhanced at lower concentrations of risdiplam. These results indicate that the performance of the in-frame switch is directly transferable to an AAV vectored context, confirming the compatibility of the in-frame switch with AAV-based gene delivery applications. Example 43: Risdiplam-regulated expression of MeCP2 protein We next sought to confirm that the in-frame switches characterised herein are relevant for the risdiplam- inducible expression of other proteins of interest, e.g. therapeutically-relevant proteins. The GFP reporter in the pMM569 switch was substituted with the MeCP2 coding sequence (as depicted in Figure 44A). We utilized MeCP2-knockout H4 cells as a model system, which were transfected with the piggyBac system to introduce the MeCP2-expressing plasmid controlled by the in-frame switch. Wildtype H4 cells, expressing endogenous levels of MeCP2 and non-transfected MeCP2-knockout cells served as controls (shown in Figure 44B). The Operetta imaging system was employed to detect MeCP2 expression, with P37829 cells stained using an anti-MeCP2 antibody and DAPI to visualize nuclei. Following transfection, cells were selected with puromycin to isolate cells stably-expressing the constructs, which were then exposed to various concentrations of risdiplam for 24 hours (illustrated in Figures 44C-D). In the MeCP2-knockout cells, no protein expression was observed in the absence of risdiplam; however, MeCP2 expression was restored and enhanced in a risdiplam concentration-dependent manner when controlled by the in-frame switch (Figures 44C-D). These findings demonstrate that the pMM569 in-frame switch is not only effective for regulating reporter gene (GFP) expression, but is also capable of modulating the expression of therapeutically-relevant proteins of interest, such as MeCP2. The switch could potentially be used to regulate MeCP2 expression for the treatment of Rett Syndrome. Example 44: Materials and Methods Cell culture: HEK293 [American Type Culture Collection (ATCC): CRL-1573] and H4 [ATCC: HTB-148] cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; cat. no. #97068-085, lot no. #065K19, VWR) and 1% (v/v) penicillin/streptomycin solution (Sigma-Aldrich) later referred to as DMEM Complete Medium (DMEM). Jurkat cells (#BA131204, Hoffmann-La Roche, Nutley, NJ) were cultured in RPMI 1640 Medium with GlutaMAX™ Supplement (#61870-036, Gibco) supplemented with 10% FBS (#A3160402, Gibco) and 1% Penicillin-Streptomycin (10,000 U/mL) (#15140122, Gibco). Cells were maintained at 0.5-1.5 x 106 cells/ml. In order to select the antibiotic resistant cells, 0.8 - 1 μg/ml of puromycin (#A1113803, Gibco) or 20 µg/mL blasticidin (Invitrogen, cat. no. #A1113902) was added to the media. Cells were cultivated in standard cell culture conditions, at 37°C in a humidified atmosphere containing 5% CO2. Neuronal cell culture and differentiation: Human embryonic stem cell (hESC)-derived neural progenitor cells were cultured in laminin (Cat#MSPPLN521-05, VWR)-coated plates in Maintenance medium: DMEM/F12 medium (Cat#31331028, ThermoFischer), mixed at a 1:1 ratio with Neurobasalmedium (Cat# 321103049, ThermoFischer), supplemented with B27 (Cat#12587010), M2 (Cat317502048, ThermoFischer), b- Mercaptoethanol (Cat#31350010, ThermoFischer), and Penicillin/Streptomycin (Cat#15140122, ThermoFischer). Cells were cultured and expanded in this Medium and then transferred to predifferentiation medium for 1 week: Above-descrived maintenance medium, supplemented with sonic hedgehog (Cat#100-45, Peprotech), FGF8b (Cat3100-25, Peprotech), and Aa2-P (Cat#A8960, SigmaAldrich). After seven days of culture in pre-differentiation medium, cells were then transferred to differentiation medium (BGAA), which is composed of maintenance medium as described above, supplemented with BDNF (Cat# AF-450-02, Peprotech), GDNF (Cat3 AF-450-10, Peprotech), Aa2-P (Cat#A8960, SigmaAldrich), cAMP (Cat# D009, BioLog), and Laminin (Cat#11243217001, SigmaAldrich). Cells were cultured in this medium for a total of 21 days before transduction with AAVs, medium was thereafter exchanged every 3 days. Transfection: HEK293 and H4 cells were transfected using the piggyBac transposase system and Lipofectamine 2000 (Invitrogen, cat. no. #11668019) used according to manufacturer’s instructions.0.35 × 106 cells were seeded per well in a 6-well plate 24 hours (h) before transfection. Cells were then co-transfected with a P37829 PiggyBac helper plasmid encoding for the transposase enzyme and the donor plasmid that contains the gene of interest under the control of the various splice switch versions at a 3:1 ratio (donor:helper plasmid).24h post-transfection, cells underwent blasticidin (Invitrogen, cat. no. #A1113902) selection for 10 days at the concentration of 20 µg/mL or puromycin (Gibco, #A1113803) selection for 5 days at the concentration of 0.8 µg/mL After the selection, cells were washed with DPBS, trypsinized with Trypsin- EDTA (0.05%), phenol red (#25300054, Gibco), resuspended in culture media and seeded on 96-well plates (50000 cells/well). After 24h cells were treated with varying concentrations of risdiplam or DMSO as a negative control. Nucleofection: Nucleofection of HEK293 and Jurkat cells was performed using 4D-Nucleofector® X Unit (#AAF-1003X, Lonza) according to the manufacturer's protocol. In order to nucleofect Jurkat cells ,1 million cells per reaction was collected, washed once with DPBS, no calcium, no magnesium (#14190144, Gibco), spun down and resuspended in 100 μL of SE Cell Line NucleofectorTM Solution (#V4XC-1024, Lonza). Resuspended cells were mixed with 2 μg of the donor plasmid that contains the gene of interest under the control of the various splice switch versions and 0.65 μg of PiggyBac encoding plasmid (3:1 ratio of donor:helper plasmids) and transferred to NucleocuvetteTM Vessel. Vessels were transferred to 4D- Nucleofector® X Unit and nucleoporated using the CL-120 program. Subsequently, 400 μL of culture media (RPMI 1640) was added to the vessels and cells were incubated for 5 min. at RT. Next, the whole volume was transferred to 12-well plates and an additional 0.5 mL of fresh media was added. Cells were incubated for 48h after which they were selected with puromycin (0.8 μg/ml) for 5 days. After the selection, cells were washed with DPBS, resuspended in culture media, seeded on 96-well plates (60000 cells/well), and treated with varying concentrations of risdiplam or DMSO as a negative control. For HEK293 nucleoporation with sgRNAs, 1 million HEK293 cells stably expressing splice-switch inducible Cas9 enzyme was collected, washed with DPBS, spun down and resuspended in 100 μL of SF Cell Line NucleofectorTM Solution (#V4XC-2032, Lonza) containing three sgRNAs at 150 nM concentration (sgRNA1: UUGGCUUCCUGGGCUGCUA, sgRNA2: GCAGCCCUCCACUCCCAUGG, sgRNA3: GGCGCUGUCAUGAUGUUCGU). Next, cells were transferred to NucleocuvetteTM Vessels and nucleoporated in 4D-Nucleofector® X Unit using CM-130 program. Subsequently, 400 μL of culture media (DMEM High-glucose (#11965092, Gibco) was added to the vessels and cells were incubated for 5 min. at RT. Finally, cells were resuspended in culture media, seeded on 96-well plates and treated with varying concentrations of risdiplam or DMSO as a negative control. AAV transduction: For transducing neuronal cells with AAVs vectoring GFP transgene under the control of a risdiplam- inducible ON-switch (Example 42), AAVs were produced using standard production protocols. HEK293T cells were cultured in DMEM supplemented with 10% FBS until they reached 50 to 70% confluency. The cells were then triple transfected with pHelper, pAAV ITR-expression, and pAAV Rep-Cap plasmids using PEI reagent in 1x cellFactory5. After 72 hours post-transfection, AAV particles were harvested from the cells. The crude lysate was prepared by lysing the cell pellet with 0.1% Triton and digesting with Benzonase. Primary capture of AAV particles was performed using Poros CaptureSelect AAV-X P37829 (ThermoFisher). A secondary purification step was conducted using iodixanol gradient centrifugation to enrich for full capsids. For quality control, vector genome titer was determined by performing qPCR with ITR-specific primers (ITR_qPCR_F: CGGCCTCAGTGAGCGA, qPCR_R: ggaacccctagtgatggagtt). The purity of the final product was assessed via 10% SDS-PAGE using Stain technology (Bio-Rad). A purity level of more than 95% was confirmed if only VP1, VP2, and VP3 proteins were detectable. Endotoxin levels were measured using the Endosafe-nexgen-PTS Spectrometer from Charles River Laboratories, with all constructs showing endotoxin levels below the detection limit. We selected the capsid AAV2.7m8 as these capsids have previously shown to transduce neurons (data not shown). The multiplicity of infection was titrated and the viral genome of 20.000 genomic copies per cell showed balanced transduction efficiency versus cell survival. Six hours after AAV transduction, medium was removed and fresh BGAA medium was added to the cells. Cells were allowed to rest for 3 days before adding risdiplam at varying concentrations. GFP expression was monitored using Incucyte Live Imaging device and images were acquired for 7 days. Every 3 days, fresh BGAA medium was exchanged alongside risdiplam. Cloning of the constructs All the tested constructs were ordered and cloned using Gibson cloning at GenScript. Assessment of splicing efficiency Flow Cytometry Reporter gene expression of either EGFP or mCHERRY in the presence of varying amounts of inducer compound (risdiplam (2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(4,7-diazaspiro[2.5]octan-7-yl)-4H- pyrido[1,2-a]pyrimidin-4-one) or risdiplam analogue ((R)-7-(hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)-2-(2- methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one)) was monitored by measuring the intensity of the fluorescent proteins using flow cytometry. Therefore, cells were washed with PBS, trypsinized and resuspended in autoMACS Running Buffer (Miltenyi Biotec, cat. No. #130-091-221). The analysis was done using CytoFLEX LX flow cytometer (Beckman Coulter). The measurement was analysed using FlowJo software (version 10.8.1) and exported to CSV file and later analysed in Excel (Microsoft). Fold changes in reporter gene expression were calculated based on the median fluorescence intensity measured in the compound-treated cells relative to DMSO-treated cells. RNA isolation HEK293 cells with stably incorporated splice constructs were seeded on 6-well plates (0.5 x 106 cells/well) and treated with DMSO or risdiplam for 24(H)After 24h cells were washed with PBS (-/-), trypsynised, resuspended in DMEM CM and transferred to 1.5 mL Eppendorf tubes. Next, cells were centrifuged at 300 x g for 5 min at room temperature, washed once with PBS (-/-) and cell pellets were frozen at -80°C. RNA was isolated using RNeasy Mini Kit (Qiagen, cat. no. #74104) according to manufacturer’s protocol. After the isolation RNA concentration was measured and all samples were diluted with UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen, cat. no. #10977015) to the same P37829 concentration. The isolated RNA was stored at -80°C and subsequently used for cDNA synthesis by reverse transcriptase (RT)-PCR. cDNA synthesis 2 µg of the isolated total RNA were used for the cDNA synthesis using SuperScript IV VILO (Invitrogen, cat. no. #18090050) according to manufacturer’s protocol. In short, RNA was either mixed with SuperScript VILO reverse transcriptase (RT) (+RT) or without RT (–RT), 4x master mix and water in 0.2 mL 8-Strip Non-Flex PCR Tubes (Starlab, cat. no. #I1402-3700) to the final volume of 20 µL. Tubes were incubated at 25°C for 10 min, followed by 50°C for 30 min and 80°C for 5 min. Samples were diluted to 100 µL with UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen, cat. no. #10977015) to the final cDNA concentration of 20 ng/µL and stored at -20°C. PCR Synthetized cDNA was used for PCRs to analyze splicing efficiency of the tested constructs. PCRs were set up using 10 µL of Q5 Hot Start HF 2x Master Mix (NEB, cat. No. #M0494L), 1 µL forward (MM16, TTGACCTCCATAGAAGACACC) and 1 µL reverse primer (MM40, GCAGATGAACTTCAGGGTCAG) to the final concentration of 0.5 µM each, 6 µL H2O and 2 µL cDNA to the total volume of 20 µl in 0.2 mL 8- Strip Non-Flex PCR Tubes. PCR thermocycling conditions were as follows: 98°C for 1 min; 15 cycles of (98°C for 15 seconds, 64°C for 15 seconds, 72°C for 30 seconds); 72°C for 2 min; 4°C hold. PCR products were resolved for 20 min on 2% E-Gel™ 48-well Agarose Gels (Invitrogen, cat. no. #G820802) with E-Gel 50 bp DNA Ladder (Invitrogen, cat. no. #10488099) and imaged using E-Gel Power Snap Electrophoresis System (G8300). Immunocytochemistry staining In order to stain for cells expression MeCP2 protein (Example 43), H4 cells were transfected with a knockout in MeCP2 with plasmid containing the in-frame switch according to sequence pMM569 and the MeCP2 sequence (plasmid pMM586) using piggybac system according to described protocol. Cells were selected for stable expressers using puromycin. The stable cell pool was cultured in DMEM, supplemented with FBS and P/S and induced with varying concentrations of risdiplam as indicated or DMSO as a negative control. After 24h, cells were used for staining by a washing step with PBS, followed by fixation in paraformaldehyde (4%) for 15min at room temperature (RT) and an additional washing step with PBS. Fixed cells were then treated with PBS, supplemented with 0.3% Triton X-100 for 15 min at RT, washed with PBS and then incubated for 1 hour at RT with a blocking solution, composed of 10% goat serum (G9023) and 0.3% Triton X-100 in PBS. In a next step, cells were incubated with anti-MeCP2 antibody (Cat#3456, Cell Signaling Technologies) at a dilution rate of 1:200 in blocking solution overnight at 4C before 3 washing steps followed with PBS. A secondary antibody (Goat anti-Rabbit, Cat# A32733, ThermoFischer) was then used at a 1:1000 dilution and incubated for 1 hour at RT, followed by 3 washing steps with PBS. To visualize the nucleus, cells were incubated with DAPI at a 1:1000 for 5min, washed with PBS and images were acquired using the Operetta CLS High-Content Analysis System. NanoLuc (NLuc) measurement NLuc measurements (Example 39) were performed using Nano-Glo® Luciferase Assay System (Promega, #N1120) according to the manufacturer protocol. In short, Nano-Glo reagent was prepared at P37829 room temperature by combining one volume of Nano-Glo® Luciferase Assay Substrate with 50 volumes of Nano-Glo® Luciferase Assay Buffer. Next, the reagent was added to each measured well using a volume of reagent equal to that of the cell culture media in each well (100 μL per one well of 96-well plate). Plates were incubated for 3 minutes and measured using PHERAstar FSX Microplate Reader (BMG Labtech). Cas9 experiments CD81 staining: HEK293 cells were nucleoporated with CD81-targeting sgRNAs as described in the nucleofection section hereinabove, seeded on 96-well plates and treated with varying concentrations of risdiplam or DMSO as a negative control for 3 days. To analyse the amount of CD81+ on cell surface, cells were first detached with 40 μL of Trypsin-EDTA (0.05%), phenol red (#25300054, Gibco) and next resuspended in the additional volume of 100 μL DBPS to a total volume of 140 μL. Cells were transferred to fresh 96-well Microplates, PP, U-bottom, 0,3 ml (#701330, Brand), spun down and washed once with DPBS. Next, cells were resuspended in 50 μL of 1:10 mix of APC-conjugated CD81 (#561958, BD) and DBPS and incubated for 15 min. at 4˚C. Cells were spun down (180xg, 3 min.), the staining solution was removed, and cells were washed three times with DPBS. Finally, cells were resuspended in DBPS and analyzed by flow cytometry using CytoFLEX LX Flow Cytometer (BeckmanCoulter). The populations of CD81+ cells were defined using CD81-stained HEK293 WT cells (no sgRNA). The measurement was analysed using FlowJo software (version 10.8.1), exported to CSV file and later analysed in Excel (Microsoft). References 1. Lombardi, L. M., Baker, S.(A)& Zoghbi,(H)Y. MECP2 disorders: from the clinic to mice and back. J Clin Invest 125, 2914-2923, doi:10.1172/JCI78167 (2015). 2. Sceniak, M. P. et al. Mechanisms of Functional Hypoconnectivity in the Medial Prefrontal Cortex of Mecp2 Null Mice. Cereb Cortex 26, 1938-1956, doi:10.1093/cercor/bhv002 (2016). 3. Tillotson, R. & Bird,(A)The Molecular Basis of MeCP2 Function in the Brain. J Mol Biol, doi:10.1016/j.jmb.2019.10.004 (2019). 4. Tillotson, R. et al. Radically truncated MeCP2 rescues Rett syndrome-like neurological defects. Nature 550, 398-401, doi:10.1038/nature24058 (2017). 5. Gadalla, K. K. et al. Improved survival and reduced phenotypic severity following AAV9/MECP2 gene transfer to neonatal and juvenile male Mecp2 knockout mice. Mol Ther 21, 18-30, doi:10.1038/mt.2012.200 (2013). 6. Sinnett, S.(E)et al. Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery. Mol Ther Methods Clin Dev 5, 106-115, doi:10.1016/j.omtm.2017.04.006 (2017). P37829 7. Garg, S. K. et al. Systemic delivery of MeCP2 rescues behavioral and cellular deficits in female mouse models of Rett syndrome. J Neurosci 33, 13612-13620, doi:10.1523/JNEUROSCI.1854-13.2013 (2013). 8. Matagne, V. et al. A codon-optimized Mecp2 transgene corrects breathing deficits and improves survival in a mouse model of Rett syndrome. Neurobiol Dis 99, 1-11, doi:10.1016/j.nbd.2016.12.009 (2017). 9. Van Esch,(H)in GeneReviews((R)) (eds M. P. Adam et al.) (1993). 10. Gadalla, K. K., Bailey, M.(E)& Cobb, S. R. MeCP2 and Rett syndrome: reversibility and potential avenues for therapy. Biochem J 439, 1-14, doi:10.1042/BJ20110648 (2011). 11. Ginhoux,(F)et al. HLA-A*0201-restricted cytolytic responses to the rtTA transactivator dominant and cryptic epitopes compromise transgene expression induced by the tetracycline on system. Mol Ther 10, 279-289, doi:10.1016/j.ymthe.2004.05.012 (2004). 12. Hoyng, S.(A)et al. Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve. Gene Ther 21, 549-557, doi:10.1038/gt.2014.22 (2014). 13. Tickner, Z. J. & Farzan, M. Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors. Pharmaceuticals (Basel) 14, doi:10.3390/ph14060554 (2021). 14. Karagiannis, P., Fujita, Y. & Saito,(H)RNA-based gene circuits for cell regulation. Proc Jpn Acad Ser B Phys Biol Sci 92, 412-422, doi:10.2183/pjab.92.412 (2016). 15. Sivaramakrishnan, M. et al. Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers. Nat Commun 8, 1476, doi:10.1038/s41467-017-01559-4 (2017). 16. Campagne, S. et al. Structural basis of a small molecule targeting RNA for a specific splicing correction. Nat Chem Biol 15, 1191-1198, doi:10.1038/s41589-019-0384-5 (2019). 17 Zhang, M. L., Lorson,(C)L., Androphy,(E)J. & Zhou, J. An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA. Gene Ther 8, 1532-1538, doi:10.1038/sj.gt.3301550 (2001). 18. Cho, S. et al.3' Splice site sequences of spinal muscular atrophy related SMN2 pre-mRNA include enhancers for nearby exons. ScientificWorldJournal 2014, 617842, doi:10.1155/2014/617842 (2014). 19. Gao, Y. et al. Systematic characterization of short intronic splicing-regulatory elements in SMN2 pre-mRNA. Nucleic Acids Res 50, 731-749, doi:10.1093/nar/gkab1280 (2022). 20. Noderer, W. L. et al. Quantitative analysis of mammalian translation initiation sites by FACS-seq. Mol Syst Biol 10, 748, doi:10.15252/msb.20145136 (2014). P37829 21. Naryshkin, N.(A)et al. Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy. Science 345, 688-693, doi:10.1126/science.1250127 (2014). 22. Ishigami, Y. et al., Nature Communications 15: 1880, doi: 10.1038/s41467-024-46090-5 (2024). 23. Recinos, Y. et al., bioRxiv [Preprint].2023 Aug 21:2023.08.21.554109. doi: 10.1101/2023.08.21.554109.

Claims

P37829 Claims: 1. A polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:222, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:378 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:226; (iv) a fourth nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:380 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end; (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘GA’, ‘TG’ or ‘TT’, or (b) comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:19, wherein the nucleotide sequence comprises ‘GA’, ‘TG’ or ‘TT’ at the positions corresponding to positions 1 and 2 of SEQ ID NO:19, or (c) encoding a polypeptide of interest, and comprising ‘GA’, ‘TG’ or ‘TT’ at positions 1 and 2; and (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a) or (v)(b), a sixth nucleotide sequence encoding a polypeptide of interest; wherein the polynucleotide comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. 2. The polynucleotide according to claim 1, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. 3. The polynucleotide according to claim 1 or claim 2, wherein the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:222. 4. The polynucleotide according to any one of claims 1 to 3, wherein the first nucleotide sequence comprises, or consists of, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:106, SEQ ID NO:219 or SEQ ID NO:220. 5. The polynucleotide according to any one of claims 1 to 4, wherein the second nucleotide sequence comprises SEQ ID NO:7 at its 5’ end, and comprises SEQ ID NO:8 or SEQ ID NO:377 at its 3’ end. 6. The polynucleotide according to any one of claims 1 to 5, wherein the second nucleotide sequence consists of fewer than 500 nucleotides. P37829 7. The polynucleotide according to any one of claims 1 to 6, wherein the second nucleotide sequence comprises, or consists of, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:228. 8. The polynucleotide according to any one of claims 1 to 7, wherein the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:226. 9. The polynucleotide according to any one of claims 1 to 8, wherein the third nucleotide sequence consists of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:223 or SEQ ID NO:224. 10. The polynucleotide according to any one of claims 1 to 9, wherein the fourth nucleotide sequence comprises SEQ ID NO:15 or SEQ ID NO:379 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end. 11. The polynucleotide according to any one of claims 1 to 10, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides. 12. The polynucleotide according to any one of claims 1 to 11, wherein the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:227 or SEQ ID NO:340. 13. The polynucleotide according to any one of claims 1 to 12, wherein the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’ or ‘TG’, or (b) comprises, or consists of, a nucleotide sequence according to SEQ ID NO:19. 14. The polynucleotide according to any one of claims 1 to 13, wherein the fifth nucleotide sequence: (a) consists of the dinucleotide ‘GA’ or ‘TG’, or (b) comprises, or consists of, SEQ ID NO:20 or SEQ ID NO:21. 15. The polynucleotide according to any one of claims 1 to 14, wherein the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:164, SEQ ID NO:171, SEQ ID NO:116, SEQ ID NO:114, SEQ ID NO:111, SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:22. 16. The polynucleotide according to any one of claims 1 to 15, wherein the polynucleotide further comprises a promoter sequence 5’ to the start codon. 17. The polynucleotide according to any one of claims 1 to 16, wherein the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a polypeptide of interest. 18. The polynucleotide according to any one of claims 1 to 17, wherein the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. 19. A vector comprising a polynucleotide according to any one of claims 1 to 18. P37829 20. The vector according to claim 19, wherein the vector is an adeno-associated virus (AAV) vector. 21. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 18, or a vector according to claim 19 or claim 20, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. 22. A cell comprising a polynucleotide according to any one of claims 1 to 18, or a vector according to claim 19 or claim 20. 23. The cell according to claim 22, wherein the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 24. A method for modifying a cell to express a polypeptide of interest, comprising: (i) introducing into a cell a polynucleotide according to any one of claims 1 to 18, or a vector according to claim 19 or claim 20; and (ii) subsequently contacting the cell with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 25. A method for expressing a polypeptide of interest in a cell, comprising contacting a cell according to claim 22 with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 26. A polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or claim 20, or a pharmaceutical composition according to claim 21, for use in a method of medical treatment or prophylaxis. 27. A polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or claim 20, or a pharmaceutical composition according to claim 21, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 28. Use of a polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or claim 20, or a pharmaceutical composition according to claim 21, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 29. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject a polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or claim 20, or a pharmaceutical composition according to claim 21. P37829 30. The polynucleotide, vector, or pharmaceutical composition for use according to claim 27, the use according to claim 28, or the method according to claim 29, wherein treating or preventing the disease or condition further comprises administering to the subject a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 31. The polynucleotide, vector, or pharmaceutical composition for use according to claim 27 or claim 30, the use according to claim 28 or claim 30, or the method according to claim 29 or claim 30, wherein the disease or condition is a disease or condition characterised by deficiency of the polypeptide of interest. 32. A kit, comprising: (i) a polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or claim 20, or a pharmaceutical composition according to claim 21; and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 33. A polynucleotide comprising in 5’ to 3’ order: (i) a first nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:1, wherein the first nucleotide sequence does not comprise SEQ ID NO:2; (ii) a second nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:7 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:8 at its 3’ end, and consisting of fewer than 1044 nucleotides; (iii) a third nucleotide sequence consisting of a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:26, wherein the third nucleotide sequence comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:12; (iv) a fourth nucleotide sequence, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:15 at its 5’ end, and comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:16 at its 3’ end; (v) a fifth nucleotide sequence: (a) consisting of the trinucleotide ‘GAG’ or (b) encoding a polypeptide of interest, and comprising ‘GAG’ at positions 1 to 3; and (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a), a sixth nucleotide sequence encoding a polypeptide of interest; wherein the polynucleotide comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. 34. The polynucleotide according to claim 33, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. 35. The polynucleotide according to claim 33 or claim 34, wherein the first nucleotide sequence comprises a nucleotide sequence according to SEQ ID NO:1. P37829 36. The polynucleotide according to any one of claims 33 to 35, wherein the first nucleotide sequence comprises, or consists of, SEQ ID NO:3. 37. The polynucleotide according to any one of claims 33 to 36, wherein the second nucleotide sequence comprises SEQ ID NO:7 at its 5’ end, and comprises SEQ ID NO:8 at its 3’ end. 38. The polynucleotide according to any one of claims 33 to 37, wherein the second nucleotide sequence consists of fewer than 500 nucleotides. 39. The polynucleotide according to any one of claims 33 to 38, wherein the second nucleotide sequence comprises, or consists of, SEQ ID NO:10. 40. The polynucleotide according to any one of claims 33 to 39, wherein the third nucleotide sequence consists of a nucleotide sequence according to SEQ ID NO:26. 41. The polynucleotide according to any one of claims 33 to 40, wherein the third nucleotide sequence consists of SEQ ID NO:13 or SEQ ID NO:27. 42. The polynucleotide according to any one of claims 33 to 41, wherein the fourth nucleotide sequence comprises SEQ ID NO:15 at its 5’ end, and comprises SEQ ID NO:16 at its 3’ end. 43. The polynucleotide according to any one of claims 33 to 42, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides. 44. The polynucleotide according to any one of claims 33 to 43, wherein the fourth nucleotide sequence comprises, or consists of, SEQ ID NO:17. 45. The polynucleotide according to any one of claims 33 to 44, wherein the polynucleotide comprises at least 80% sequence identity to SEQ ID NO:28 or SEQ ID NO:29. 46. The polynucleotide according to any one of claims 33 to 45, wherein the polynucleotide further comprises a promoter sequence 5’ to the start codon. 47. The polynucleotide according to any one of claims 33 to 46, wherein the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a polypeptide of interest. 48. The polynucleotide according to any one of claims 33 to 47, wherein the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. 49. A vector comprising a polynucleotide according to any one of claims 33 to 48. 50. The vector according to claim 49, wherein the vector is an adeno-associated virus (AAV) vector. P37829 51. A pharmaceutical composition comprising a polynucleotide according to any one of claims 33 to 48, or a vector according to claim 49 or claim 50, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. 52. A cell comprising a polynucleotide according to any one of claims 33 to 48, or a vector according to claim 49 or claim 50. 53. The cell according to claim 52, wherein the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 54. A method for modifying a cell to express a polypeptide of interest, comprising introducing into a cell a polynucleotide according to any one of claims 33 to 48, or a vector according to claim 49 or claim 50. 55. A method for inhibiting expression of a polypeptide of interest in a cell, comprising contacting a cell according to claim 52 with a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam. 56. A polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51, for use in a method of medical treatment or prophylaxis. 57. A polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 58. Use of a polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51, in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest. 59. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the polypeptide of interest, comprising administering to a subject a polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51. 60. The polynucleotide, vector, or pharmaceutical composition for use according to claim 57, the use according to claim 58, or the method according to claim 59, wherein the disease or condition is a disease or condition characterised by deficiency of the polypeptide of interest. P37829 61. A kit, comprising: (i) a polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51; and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.
PCT/EP2024/065689 2023-06-09 2024-06-07 Inducible gene expression system Ceased WO2024251925A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202480046722.2A CN121488042A (en) 2023-06-09 2024-06-07 Inducible gene expression system
EP24732224.1A EP4724585A1 (en) 2023-06-09 2024-06-07 Inducible gene expression system
AU2024286618A AU2024286618A1 (en) 2023-06-09 2024-06-07 Inducible gene expression system
KR1020257043252A KR20260022333A (en) 2023-06-09 2024-06-07 Inducible gene expression system

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23178358 2023-06-09
EP23178358.0 2023-06-09

Publications (1)

Publication Number Publication Date
WO2024251925A1 true WO2024251925A1 (en) 2024-12-12

Family

ID=86896093

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/065689 Ceased WO2024251925A1 (en) 2023-06-09 2024-06-07 Inducible gene expression system

Country Status (5)

Country Link
EP (1) EP4724585A1 (en)
KR (1) KR20260022333A (en)
CN (1) CN121488042A (en)
AU (1) AU2024286618A1 (en)
WO (1) WO2024251925A1 (en)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009151546A2 (en) 2008-05-27 2009-12-17 Ptc Therapeutics, Inc. Methods for treating spinal muscular atrophy
WO2013112788A1 (en) * 2012-01-26 2013-08-01 Ptc Therapeutics, Inc. Compounds for treating spinal muscular atrophy
JP2015504057A (en) * 2011-12-30 2015-02-05 ピーティーシー セラピューティクス， インコーポレイテッド Compounds for treating spinal muscular atrophy
US8986935B2 (en) * 2008-08-13 2015-03-24 Ptc Therapeutics, Inc. Methods for treating spinal muscular atrophy
JP2015509956A (en) * 2012-03-01 2015-04-02 ピーティーシー セラピューティクス， インコーポレイテッド Compounds for treating spinal muscular atrophy
JP2015517988A (en) * 2012-03-23 2015-06-25 ピーティーシー セラピューティクス， インコーポレイテッド Compounds for treating spinal muscular atrophy
WO2015173181A1 (en) 2014-05-15 2015-11-19 F. Hoffmann-La Roche Ag Compounds for treating spinal muscular atrophy
JP2017122097A (en) * 2012-02-10 2017-07-13 ピーティーシー セラピューティクス， インコーポレイテッド Compounds for treating spinal muscular atrophy
WO2021163556A1 (en) 2020-02-12 2021-08-19 The Children's Hospital Of Philadelphia Compositions and methods for inducible alternative splicing regulation of gene expression
WO2022204471A1 (en) 2021-03-26 2022-09-29 Ptc Therapeutics Inc. Regulation of transgene expression using a small molecule inducible splicing switch

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009151546A2 (en) 2008-05-27 2009-12-17 Ptc Therapeutics, Inc. Methods for treating spinal muscular atrophy
US8986935B2 (en) * 2008-08-13 2015-03-24 Ptc Therapeutics, Inc. Methods for treating spinal muscular atrophy
JP2015504057A (en) * 2011-12-30 2015-02-05 ピーティーシー セラピューティクス， インコーポレイテッド Compounds for treating spinal muscular atrophy
WO2013112788A1 (en) * 2012-01-26 2013-08-01 Ptc Therapeutics, Inc. Compounds for treating spinal muscular atrophy
JP2017122097A (en) * 2012-02-10 2017-07-13 ピーティーシー セラピューティクス， インコーポレイテッド Compounds for treating spinal muscular atrophy
JP2015509956A (en) * 2012-03-01 2015-04-02 ピーティーシー セラピューティクス， インコーポレイテッド Compounds for treating spinal muscular atrophy
JP2015517988A (en) * 2012-03-23 2015-06-25 ピーティーシー セラピューティクス， インコーポレイテッド Compounds for treating spinal muscular atrophy
WO2015173181A1 (en) 2014-05-15 2015-11-19 F. Hoffmann-La Roche Ag Compounds for treating spinal muscular atrophy
WO2021163556A1 (en) 2020-02-12 2021-08-19 The Children's Hospital Of Philadelphia Compositions and methods for inducible alternative splicing regulation of gene expression
WO2022204471A1 (en) 2021-03-26 2022-09-29 Ptc Therapeutics Inc. Regulation of transgene expression using a small molecule inducible splicing switch

Non-Patent Citations (60)

* Cited by examiner, † Cited by third party
Title
"Remington's `The Science and Practice of Pharmacy", 2020, ACADEMIC PRESS
ALICIA J. ANGELBELLO ET AL: "Small molecule targeting of RNA structures in neurological disorders", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 1471, no. 1, 9 April 2019 (2019-04-09), US, pages 57 - 71, XP055767614, ISSN: 0077-8923, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785366/> [retrieved on 20240820], DOI: 10.1111/nyas.14051 *
BENIZRI ET AL., BIOCONJUG CHEM., vol. 30, no. 2, 2019, pages 366 - 383
BÜNINGSRIVASTAVA, MOLECULAR THERAPY: METHODS & CLINICAL DEVELOPMENT, vol. 12, 2019, pages 248 - 265
CAMPAGNE, S. ET AL.: "Structural basis of a small molecule targeting RNA for a specific splicing correction.", NAT CHEM BIOL, vol. 15, 2019, pages 1191 - 1198, XP036927808, DOI: 10.1038/s41589-019-0384-5
CHEN ET AL., MOL THER METHODS CLIN DEV., vol. 3, 2016, pages 16023
CHO, S. ET AL.: "3' Splice site sequences of spinal muscular atrophy related SMN2 pre-mRNA include enhancers for nearby exons.", SCIENTIFICWORLDJOURNAL, 2014, pages 617842
FU ET AL., BIOCONJUG CHEM., vol. 25, no. 9, 2014, pages 1602 - 1608
GADALLA, K. K. ET AL.: "Improved survival and reduced phenotypic severity following AAV9/MECP2 gene transfer to neonatal and juvenile male Mecp2 knockout mice", MOL THER, vol. 21, 2013, pages 18 - 30, XP055323577, DOI: 10.1038/mt.2012.200
GADALLA, K. K.BAILEY, M.(ECOBB, S. R.: "MeCP2 and Rett syndrome: reversibility and potential avenues for therapy.", BIOCHEM J, vol. 439, 2011, pages 1 - 14
GAO, Y. ET AL.: "Systematic characterization of short intronic splicing-regulatory elements in SMN2 pre-mRNA.", NUCLEIC ACIDS RES, vol. 50, 2022, pages 731 - 749
GARG, S. K. ET AL.: "Systemic delivery of MeCP2 rescues behavioral and cellular deficits in female mouse models of Rett syndrome.", J NEUROSCI, vol. 33, 2013, pages 13612 - 13620, XP055475145, DOI: 10.1523/JNEUROSCI.1854-13.2013
GINHOUX,(F ET AL.: "HLA-A*0201-restricted cytolytic responses to the rtTA transactivator dominant and cryptic epitopes compromise transgene expression induced by the tetracycline on system.", MOL THER, vol. 10, 2004, pages 279 - 289, XP004660565, DOI: 10.1016/j.ymthe.2004.05.012
HASANE RATNI ET AL: "Discovery of Risdiplam, a Selective Survival of Motor Neuron-2 ( SMN2 ) Gene Splicing Modifier for the Treatment of Spinal Muscular Atrophy (SMA)", JOURNAL OF MEDICINAL CHEMISTRY, vol. 61, no. 15, 25 July 2018 (2018-07-25), US, pages 6501 - 6517, XP055610353, ISSN: 0022-2623, Retrieved from the Internet <URL:https://pubs.acs.org/doi/10.1021/acs.jmedchem.8b00741> [retrieved on 20240820], DOI: 10.1021/acs.jmedchem.8b00741 *
HAVESHASTINGS, NUCLEIC ACIDS RES., vol. 44, no. 14, 2016, pages 6549 - 6563
HOYNG, S.(A ET AL.: "Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve.", GENE THER, vol. 21, 2014, pages 549 - 557, XP037771085, DOI: 10.1038/gt.2014.22
HU ET AL., SIG. TRANSDUC. TAR. THER., vol. 5, 2020, pages 101
ISHIGAMI, Y. ET AL., NATURE COMMUNICATIONS, vol. 15, 2024, pages 1880
KARAGIANNIS, P.FUJITA, Y.SAITO,(H: "RNA-based gene circuits for cell regulation.", PROC JPN ACAD SER B PHYS BIOL SCI, vol. 92, 2016, pages 412 - 422, XP055601170, DOI: 10.2183/pjab.92.412
KATOHSTANDLEY, MOLECULAR BIOLOGY AND EVOLUTION, vol. 30, no. 4, 2013, pages 772 - 780
KIM ET AL., PLOS ONE, vol. 6, 2011, pages e18556
KLETZL HEIDEMARIE ET AL: "The oral splicing modifier RG7800 increases full lengthsurvival of motor neuron 2mRNA and survival of motor neuron protein: Results from trials in healthy adults and patients with spinal muscular atrophy", NEUROMUSCULAR DISORDERS, ELSEVIER LTD, GB, vol. 29, no. 1, 30 October 2018 (2018-10-30), pages 21 - 29, XP085595139, ISSN: 0960-8966, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S096089661830539X?via%3Dihub> [retrieved on 20240820], DOI: 10.1016/J.NMD.2018.10.001 *
KOH ET AL., MOLECULAR THERAPY - NUCLEIC ACIDS, vol. 2, 2013, pages e114
LASSMANNSONNHAMMER, BMC BIOINFORMATICS, vol. 6, 2005, pages 298
LI ET AL., INT. J. MOL. SCI., vol. 16, 2015, pages 19518 - 19536
LIDA ET AL., BIOMED RES INT., vol. 2013, 2013, pages 974819
LISAMULSKI, NAT. REV. GENET., vol. 12, 2020, pages 255 - 272
LOMBARDI, L. M.BAKER, S.(AZOGHBI,(H)Y: "MECP2 disorders: from the clinic to mice and back", J CLIN INVEST, vol. 125, 2015, pages 2914 - 2923
MANASWINI SIVARAMAKRISHNAN ET AL: "Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers", NATURE COMMUNICATIONS, vol. 8, no. 1, 14 November 2017 (2017-11-14), XP055520527, Retrieved from the Internet <URL:https://www.nature.com/articles/s41467-017-01559-4> [retrieved on 20240820], DOI: 10.1038/s41467-017-01559-4 *
MATAGNE, V. ET AL.: "A codon-optimized Mecp2 transgene corrects breathing deficits and improves survival in a mouse model of Rett syndrome", NEUROBIOL DIS, vol. 99, 2017, pages 1 - 11
MAUS ET AL., ANNU REV IMMUNOL, vol. 32, 2014, pages 189 - 225
MAUS ET AL., ANNU REV IMMUNOL., vol. 32, 2014, pages 189 - 225
MCCARTY, MOL THER., vol. 16, no. 10, 2008, pages 1648 - 56
MONTEYS ALEX MAS ET AL: "Regulated control of gene therapies by drug-induced splicing", NATURE,, vol. 596, no. 7871, 28 July 2021 (2021-07-28), pages 291 - 295, XP037535482, Retrieved from the Internet <URL:https://www.nature.com/articles/s41586-021-03770-2> [retrieved on 20240820], DOI: 10.1038/S41586-021-03770-2 *
MONTEYS ET AL., NATURE, vol. 596, 2021, pages 291 - 295
NARYSHKIN, N.(A ET AL.: "Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy", SCIENCE, vol. 345, 2014, pages 688 - 693, XP055809115, DOI: 10.1126/science.1250127
NODERER, W. L. ET AL.: "Quantitative analysis of mammalian translation initiation sites by FACS-seq", MOL SYST BIOL, vol. 10, 2014, pages 748
NOTREDAME ET AL., J. MOL. BIOL., vol. 302, 2000, pages 205 - 217
PALACIOS DANIEL S.: "Drug Hunting at the Nexus of Medicinal Chemistry and Chemical Biology and the Discovery of Novel Therapeutic Modalities", JOURNAL OF MEDICINAL CHEMISTRY, vol. 65, no. 20, 7 October 2022 (2022-10-07), US, pages 13594 - 13613, XP093064426, ISSN: 0022-2623, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/acs.jmedchem.2c01491> [retrieved on 20240820], DOI: 10.1021/acs.jmedchem.2c01491 *
RAMDAS SITHARA ET AL: "New treatments in spinal muscular atrophy: an overview of currently available data", EXPERT OPIN PHARMACOTHER, vol. 21, no. 3, 24 January 2020 (2020-01-24), London, UK, pages 307 - 315, XP055891962, ISSN: 1465-6566, Retrieved from the Internet <URL:https://www.tandfonline.com/doi/pdf/10.1080/14656566.2019.1704732?needAccess=true> [retrieved on 20240820], DOI: 10.1080/14656566.2019.1704732 *
RECINOS, Y. ET AL., , BIORXIV [PREPRINT]., 21 August 2023 (2023-08-21)
RYDING ET AL., JOURNAL OF ENDOCRINOLOGY, vol. 171, 2001, pages 1 - 14
SCENIAK, M. P. ET AL.: "Mechanisms of Functional Hypoconnectivity in the Medial Prefrontal Cortex of Mecp2 Null Mice.", CEREB CORTEX, vol. 26, 2016, pages 1938 - 1956
SCHNEIDER-POETSCH ET AL., , THE JOURNAL OF ANTIBIOTICS, vol. 74, 2021, pages 603 - 616
SCHNEIDER-POETSCH TILMAN ET AL: "Splicing modulators: on the way from nature to clinic", THE JOURNAL OF ANTIBIOTICS, vol. 74, no. 10, 3 August 2021 (2021-08-03), UK / JP, pages 603 - 616, XP093196285, ISSN: 0021-8820, Retrieved from the Internet <URL:https://www.nature.com/articles/s41429-021-00450-1> [retrieved on 20240820], DOI: 10.1038/s41429-021-00450-1 *
SIMMONSALBEROLA-ILA, METHODS MOL BIOL., vol. 1323, 2016, pages 99 - 108
SINGH ET AL., RNA, vol. 16, no. 6, 2010, pages 1167 - 81
SINNETT, S.(E ET AL.: "Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery", MOL THER METHODS CLIN DEV, vol. 5, 2017, pages 106 - 115, XP055534097, DOI: 10.1016/j.omtm.2017.04.006
SIVARAMAKRISHNAN, M. ET AL.: "Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers.", NAT COMMUN, vol. 8, 2017, pages 1476, XP055520527, DOI: 10.1038/s41467-017-01559-4
SÖDING, J., BIOINFORMATICS, vol. 21, 2005, pages 951 - 960
TANG ET AL., MOLECULES, vol. 26, no. 8, 2021, pages 2263
TICKNER, Z. J.FARZAN, M: "Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors", PHARMACEUTICALS (BASEL, 2021, pages 14
TILLOTSON, R. ET AL.: "Radically truncated MeCP2 rescues Rett syndrome-like neurological defects.", NATURE, vol. 550, 2017, pages 398 - 401, XP055473489, DOI: 10.1038/nature24058
TILLOTSON, R.BIRD,(A: "The Molecular Basis of MeCP2 Function in the Brain.", J MOL BIOL, 2019
VAN ESCH,(H ET AL.: "GeneReviews((R", 1993
WANG ET AL., NAT. REV. DRUG DISCOV, vol. 18, 2019, pages 358 - 378
WANG ET AL., NAT. REV. DRUG DISCOV., vol. 18, 2019, pages 358 - 378
WANGRIVIÈRE, MOL THER ONCOLYTICS., vol. 3, 2016, pages 16015
ZHANG ET AL., GENE THER., vol. 8, 2001, pages 1532 - 1538
ZHANG, M. L.LORSON,(C)L.ANDROPHY,(E)J.ZHOU, J.: "An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA", GENE THER, vol. 8, 2001, pages 1532 - 1538, XP037773718, DOI: 10.1038/sj.gt.3301550

Also Published As

Publication number Publication date
EP4724585A1 (en) 2026-04-15
AU2024286618A1 (en) 2025-10-30
CN121488042A (en) 2026-02-06
KR20260022333A (en) 2026-02-19

Similar Documents

Publication Publication Date Title
KR102699584B1 (en) Control of gene expression by aptamer-mediated regulation of alternative splicing
JP7416451B2 (en) Targeted nuclear RNA cleavage and polyadenylation by CRISPR-Cas
JP2022084810A (en) Gene therapy for retinitis pigmentosa
CA2792696C (en) Modified u7 snrnas for treatment of neuromuscular diseases
JP2021502058A (en) Compositions and methods for editing RNA
US20220133768A1 (en) Crispr/rna-guided nuclease-related methods and compositions for treating rho-associated autosomal-dominant retinitis pigmentosa (adrp)
TW202411426A (en) Engineered class 2 type v crispr systems
US12391920B2 (en) Models of tauopathy
JP2026034639A (en) Adeno-associated virus vectors for the treatment of Rett syndrome
CA3008264A1 (en) Gene therapy for ocular disorders
CA3136004A1 (en) Gene therapies for lysosomal disorders
AU2017227776A1 (en) AAV vectors for treatment of dominant retinitis pigmentosa
US20220010314A1 (en) Rnai induced reduction of ataxin-3 for the treatment of spinocerebellar ataxia type 3
US20100303776A1 (en) Methods and compositions for regulated expression of multiple nucleic acids
Strings-Ufombah et al. BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy
KR20180117630A (en) Regulation of Gene Expression by Utter-Modulated Polyadenylation
US20240002863A1 (en) Synthetic site-specific rna editing entities
JP2021101713A (en) Treatment of myotonic dystrophy
EP4724585A1 (en) Inducible gene expression system
US20240207448A1 (en) Crispr/rna-guided nuclease-related methods and compositions for treating rho-associated autosomal-dominant retinitis pigmentosa (adrp)
WO2024078345A1 (en) Snrna nucleic acid molecule and application thereof
JP2025508694A (en) Therapeutic agents for treating polyQ diseases
US20250381303A1 (en) Crispr interference therapeutics for c9orf72 repeat expansion disease
WO2025202080A1 (en) Acta1 expression control elements
KR20260046119A (en) Uses of bGH-SV40L serial PolyA to enhance introduced gene expression during unidirectional gene insertion

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24732224

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: AU2024286618

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2024286618

Country of ref document: AU

Date of ref document: 20240607

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2025571590

Country of ref document: JP

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112025026490

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 2025571590

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2025139054

Country of ref document: RU

Ref document number: 2024732224

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024732224

Country of ref document: EP

Effective date: 20260109

ENP Entry into the national phase

Ref document number: 2024732224

Country of ref document: EP

Effective date: 20260109

ENP Entry into the national phase

Ref document number: 2024732224

Country of ref document: EP

Effective date: 20260109

ENP Entry into the national phase

Ref document number: 2024732224

Country of ref document: EP

Effective date: 20260109

ENP Entry into the national phase

Ref document number: 2024732224

Country of ref document: EP

Effective date: 20260109

WWP Wipo information: published in national office

Ref document number: 2024732224

Country of ref document: EP