WO2024251925A1 - Système d'expression génique inductible - Google Patents

Système d'expression génique inductible Download PDF

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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
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Prior art keywords
nucleotide sequence
seq
polynucleotide
sequence
polypeptide
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PCT/EP2024/065689
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Inventor
Martin Ebeling
Ravi Jagasia
Mateusz Piotr MENDEL
Filip ROUDNICKY
Lina SCHUKUR
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F Hoffmann La Roche AG
Hoffmann La Roche Inc
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F Hoffmann La Roche AG
Hoffmann La Roche Inc
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Priority to CN202480046722.2A priority Critical patent/CN121488042A/zh
Priority to EP24732224.1A priority patent/EP4724585A1/fr
Priority to AU2024286618A priority patent/AU2024286618A1/en
Priority to KR1020257043252A priority patent/KR20260022333A/ko
Publication of WO2024251925A1 publication Critical patent/WO2024251925A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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).

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Abstract

La présente invention concerne les domaines de la biologie moléculaire et de la technologie des acides nucléiques. La présente invention concerne également la thérapie et la prophylaxie de maladies.
PCT/EP2024/065689 2023-06-09 2024-06-07 Système d'expression génique inductible Ceased WO2024251925A1 (fr)

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