WO2024258925A1 - Vecteurs aav-cftr et leurs procédés d'utilisation - Google Patents

Vecteurs aav-cftr et leurs procédés d'utilisation Download PDF

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WO2024258925A1
WO2024258925A1 PCT/US2024/033546 US2024033546W WO2024258925A1 WO 2024258925 A1 WO2024258925 A1 WO 2024258925A1 US 2024033546 W US2024033546 W US 2024033546W WO 2024258925 A1 WO2024258925 A1 WO 2024258925A1
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cftr
sequence
seq
nucleic acid
aav
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Yutaka Maeda
John BREWINGTON
Alicia OSTMANN
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Cincinnati Childrens Hospital Medical Center
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Cincinnati Childrens Hospital Medical Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors

Definitions

  • a Sequence Listing submitted as an XML text file via EFS-WEB is hereby incorporated by reference.
  • the name of the XML file for the Sequence Listing is 2022- 1201c_SequenceList.xml, the date of the creation of the XML file is June 11, 2024, and the size of the XML file is 43,051 bytes.
  • Cystic Fibrosis is a lethal genetic disease resulting in multiorgan dysfunction that is caused by loss of function for the anion channel CFTR.
  • CFTR is a large gene, 1480 amino acids in length, a defect in which is the cause of cystic fibrosis (CF).
  • HEMT highly effective CFTR modulator therapy
  • elexacaftor/tezacaftor/ivacaftor has significantly improved the care of many individuals with CF, about 10% of patients with CF carry CFTR mutations which are ineligible for these drugs.
  • portions of the CFTR gene e.g., the N-terminus or part of the R domain
  • portions of the CFTR gene have previously been deleted, allowing the shortened CFTR gene to be packaged into AAV2 or AAV5.
  • AAV2, AAV5, or AAV6.2 attempts have been made to deliver split CFTR mRNA using AAV2, AAV5, or AAV6.2, enabling spliceosome-mediated RNA trans- splicing and restoration of CFTR function, but were not validated in vivo.
  • paired nucleic acid construct systems for the delivery of a cystic fibrosis transmembrane conductance regulator (CFTR) protein to an individual in need thereof.
  • the two-part system may comprise a first construct comprising a first portion of a CFTR gene fused to intein-N cDNA (CFTR-N-inteinN) and a second construct comprising a second portion of a CFTR gene fused to intein-C cDNA (inteinC-CFTR-C).
  • methods of treating an individual having cystic fibrosis (CF) comprising administration of the paired nucleic acid construct systems.
  • FIG. 1 depicts a schematic of intein technology to deliver CFTR to CF airway using AAV.
  • FIG. 2A depicts a schematic for the construct for an AAV6 carrying split CFTRs fused with inteins.
  • This vector (N+C; Dual AAV6) restores the physiological functions of CFTR in F508del homozygous primary human bronchial epithelial (F508del HBE) cells.
  • FIG. 2B shown is immunofluorescence images indicating that Flag-tagged N-terminus portion of CFTR attached to intein N (intN) is co-expressed with HA-tagged C-terminus portion of CFTR attached to intein C (intC) in 293T cells.
  • FIG. 2C shown are western blots indicating split CFTRs and recombined CFTR are detected by Flag or HA antibody. N-terminus portion is detected by Flag or CFTR antibody, and C-terminus portion is detected by HA antibody in 293T human embryonic kidney cells 3 days after infection with AAV6 carrying CFTR-N-intN and intC- CFTR-C.
  • FIG. 2D CFTR activity was assessed in Ussing chambers. F508dcl primary HBE cells grown at air- liquid interface were mounted in Ussing chambers and short-circuit current was measured under voltage clamp conditions.
  • FIGS. 3A-3C demonstrate that AAV6.2FF (a variant of AAV6 serotype) carrying split CFTRs (N+C; dAAV) restores the physiological function of CFTR in primary nasal epithelial cells from people with CF (PwCF) who are insensitive to CFTR modulators.
  • FIG. 3A Nasal epithelial cells from PwCF who are insensitive to CFTR modulators were expanded in submerged culture in vitro, infected with AAV6.2FF carrying split CFTRs, and subsequently transferred to air-liquid interface culture.
  • 3C CFTR-dependent short-circuit current in nasal cells homozygous for c850dup (3B) or 1525-1G>A (C) CFTR, which are insensitive to Elexacaftor/Tezacaftor/Ivacaftor.
  • Cells treated with dual AAVs demonstrated significant improvements in CFTR-specific current (cAMP + VX-770) compared to untreated cells.
  • FIG. 4 depicts in vitro analysis using human CF primary airway cells.
  • In vitro analysis can be conducted 1) using nasal or bronchial airway cells isolated from CF patients, including patients who do not respond to CF modulators (e.g., elexacaftor/tezacaftor/ivacaftor). 2) isolated cells can be cultured in monolayer at air-liquid interface (ALT). 3) cultured differentiated nasal epithelial cells can be infected with AAV carrying split CFTRs. 4) CFTR function can be assessed in AAV infected cells (1) using Ussing Chambers (B). Mucocilliary clearance in the infected cells can be assessed (C&D).
  • CF modulators e.g., elexacaftor/tezacaftor/ivacaftor
  • ALT air-liquid interface
  • 3) cultured differentiated nasal epithelial cells can be infected with AAV carrying split CFTRs.
  • CFTR function can
  • Immunofluorescence staining can be conducted to assess which cell types express recombined split CFTRs delivered by AAV (A&E). Swelling test, indicating CFTR function can also be conducted using spheroid culture for additional cells (F).
  • FIGS. 5A-5D depict in vivo mouse analysis that can be conducted using a CF- relevant model (ENaCP) mice that produce abundant mucus in the airways.
  • FIG. 5A shown is a construct that was used to create ENaCP transgenic mice. Rat CCSP promoter is active in club/secretory airway cells in mice. Mucus stained with Alcian blue was detected in trachea of the mouse. Shown image was obtained from a sacrificed ENaCP transgenic mouse three days after birth. Trachea region of the mouse was harvested and fixed by 4% paraformaldehyde. Fixed tissue was embedded in paraffin. Paraffin section was used for Alcian blue staining.
  • FIG. 5A shown is a construct that was used to create ENaCP transgenic mice. Rat CCSP promoter is active in club/secretory airway cells in mice. Mucus stained with Alcian blue was detected in trachea of the mouse. Shown image was obtained from a sacrificed ENaCP transgenic mouse three days after birth
  • FIG. 5B ENaCP transgenic mice whose airway filled with mucus can be intratracheally infected with AAV carrying split CFTRs (a dual AAV). Months (up to a year) after infection mouse lungs can be harvested for assessing the expression of recombined CFTR by immunohistochemistry and western blotting.
  • FIG. 5C GFP expression in airways transduced intratracheally by AAV6.2 (a variant of AAV6 serotype) was higher than that by AAV6 in ENaCP transgenic mice. Black dots indicate GFP staining. Two months old ENaCP transgenic mice were intratracheally infected with AAV6 or AAV6.2 carrying GFP driven by CMV.
  • FIG. 5D GFP protein expression from left lungs of the indicated mice with or without transduction of AAV6.2 carrying GFP was assessed by western blotting.
  • AAV6.2 carrying GFP was intratracheally injected into the indicated mice.
  • One week after injection left lungs were harvested, and protein extracted.
  • Western blotting (IB) was performed using extracts with GFP (Cell Signaling Technology, cat# 2956) and Actin (MilliporeSigma, cat# A2066) antibodies.
  • FIG. 6 depicts a cellular response in human nasal epithelial (HNE) cultures from an individual with cystic fibrosis (CF) without CFTR modulator access.
  • the left panel shows representative short circuit current tracings, with vehicle-treated cells in solid lines and dual AAV (dAAV)-treated cells in dashed lines.
  • the right panel indicates the patient’s CFTR genotype and shows aggregate tracing data for CFTR function under control, modulator-treated, and dAAV-treated conditions.
  • CFTR function >10% of wild-type is easily achieved.
  • n 4 inserts. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001 by one-way ANOVA with Dunnet’s multiple comparisons test against control samples.
  • FIG. 7 depicts a cellular response in human nasal epithelial (HNE) cultures from an individual with cystic fibrosis (CF) without CFTR modulator access.
  • the left panel shows representative short circuit current tracings, with vehicle-treated cells in solid lines and dual AAV (dAAV)-treated cells in dashed lines.
  • the right panel indicates the patient’s CFTR genotype and shows aggregate tracing data for CFTR function under control, modulator-treated, and dA AV-treated conditions.
  • CFTR function >10% of wild-type is easily achieved.
  • n 4 inserts. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001 by one-way ANOVA with Dunnet’s multiple comparisons test against control samples.
  • FIG. 8 depicts a cellular response in human nasal epithelial (HNE) cultures from an individual with cystic fibrosis (CF) without CFTR modulator access.
  • the left panel shows representative short circuit current tracings, with vehicle-treated cells in solid lines and dual AAV (dAAV)-treated cells in dashed lines.
  • the right panel indicates the patient’s CFTR genotype and shows aggregate tracing data for CFTR function under control, modulator-treated, and dAAV-treated conditions.
  • CFTR function >10% of wild-type is easily achieved.
  • n 4 inserts. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001 by one-way ANOVA with Dunnet’s multiple comparisons test against control samples.
  • FIG. 9 depicts a cellular response in human nasal epithelial (HNE) cultures from an individual with cystic fibrosis (CF) without CFTR modulator access.
  • the left panel shows representative short circuit current tracings, with vehicle-treated cells in solid lines and dual AAV (dAAV)-treated cells in dashed lines.
  • the right panel indicates the patient’s CFTR genotype and shows aggregate tracing data for CFTR function under control, modulator-treated, and dAAV-treated conditions.
  • CFTR function >10% of wild-type is easily achieved.
  • n 4 inserts. **p ⁇ 0.01 ; ***p ⁇ 0.001 ; ****p ⁇ 0.0001 by one-way ANOVA with Dunnet’s multiple comparisons test against control samples.
  • FIG. 10 depicts a cellular response in human nasal epithelial (HNE) cultures from an individual with cystic fibrosis (CF) without CFTR modulator access.
  • HNE human nasal epithelial
  • the left panel shows representative short circuit current tracings, with vehicle-treated cells in solid lines and dual AAV (dAAV)-treated cells in dashed lines.
  • the right panel indicates the patient’s CFTR genotype and shows aggregate tracing data for CFTR function under control, modulator-treated, and dAAV-treated conditions.
  • CFTR function >10% of wild-type is easily achieved.
  • n 4 inserts. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001 by one-way ANOVA with Dunnet’s multiple comparisons test against control samples.
  • FIG. 11 depicts a cellular response in human nasal epithelial (HNE) cultures from an individual with cystic fibrosis (CF) without CFTR modulator access.
  • the left panel shows representative short circuit current tracings, with vehicle-treated cells in solid lines and dual AAV (dAAV)-trcatcd cells in dashed lines.
  • the right panel indicates the patient’s CFTR genotype and shows aggregate tracing data for CFTR function under control, modulator-treated, and dAAV-treated conditions. A statistically significant response to dAAV is observed.
  • n 4 inserts. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001 by one-way ANOVA with Dunnet’s multiple comparisons test against control samples.
  • the methods may comprise, consist of, or consist essentially of the elements of the compositions and/or methods as described herein, as well as any additional or optional element described herein or otherwise useful in the manufacture or use of human CF R-Rhodothermus marinus intein constructs.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • the terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.
  • AAV-delivered Cas9 half-genes are transcribed, translated, and the resulting proteins recombined by intein-mediated protein ligation to form a complete Cas9 protein. 15,16
  • This strategy circumvents the size limitation of AAV, and so can be adapted to deliver CFTR (4,440 bp).
  • the N-terminal portion of CFTR cDNA is fused to intein-N cDNA, and the C-terminal portion is fused to intein-C cDNA, and each are packaged into separate AAVs.
  • each gene produces the coded half-protein, and the two portions of CFTR protein are recombined in the cells by intein-N and intein-C fusion to produce intact full-length CFTR (without insertions or deletions). Because the intcins only combine in sequence, this approach ensures correct alignment of the CFTR halves, unlike traditional concatemerization approaches. 17 While this approach has previously been proposed by Zhu et al., 18 19 to Applicant’s knowledge, actual AAVs have not yet been made and tested in CF patient cells. Of note, a dual- AAV approach has recently been proven to be successful for AAV-mediated delivery of a large otoferlin (OTOF) gene to correct hearing loss clinically. 20
  • OTOF otoferlin
  • the delivery system employs the coding sequence of a CFTR gene divided into at least two CFTR gene fragments, wherein the at least two fragments total the entirety of the wild-type CFTR gene, and wherein the at least two CFTR gene fragments can be expressed and joined to form a functional CFTR protein.
  • the disclosed compositions comprise a CFTR gene fragment that is attached to (flanked by) an N terminal region or C terminal region of an intein sequence.
  • the CFTR gene can then be delivered in two parts to a cell or tissue, upon which the gene is expressed and the two gene products form a functional CFTR protein.
  • the CFTR fragments can be delivered using two adeno-associated virus (AAV) constructs as described herein or otherwise known in the art.
  • AAV adeno-associated virus
  • the C-terminal and N-terminal regions of intein bring the two CFTR gene fragments together in vivo to form a functional CFTR protein.
  • administration of the intein-CFTR fragment constructs allows for delivery of a fully intact and functional CFTR protein directly to the tissue of the individual, for example via airway epithelial cells.
  • a paired nucleic acid construct system for delivery of a human cystic fibrosis transmembrane conductance regulator (CFTR) protein to an individual in need thereof.
  • the system may comprise, for example,: a first construct comprising a first portion (N-terminus) of a human CFTR gene fused to Rhodothermus marinas intein-N cDNA (CFTR-N-inteinN); and a second construct comprising a second portion (C-terminus) of the human CFTR gene fused to Rhodothermus marin s intein-C cDNA (inteinC-CFTR-C); wherein the first portion of the CFTR gene comprises the N terminal portion of the CFTR gene and the second portion of the CFTR gene comprises the carboxy terminal portion of the CFTR gene.
  • the first portion of the CFTR gene encodes for a protein having at least 90%, or at least 95% sequence identity to SEQ ID NO: 6, and the second portion of the CFTR gene encodes for a protein having at least 90%, or at least 95% sequence identity to SEQ ID NO: 8, wherein the first and second protein encoded by the first and second portions of the CFTR gene can be joined to form a complete CFTR protein sequence having wild-type CFTR functionality. That is, the first portion of the CFTR gene and the second portion of the CFTR gene are capable of being fused and expressing a functional CFTR protein.
  • the functional CFTR protein comprises a CFTR R domain.
  • the R domain is defined as amino acids 590-831, encoded by exon 13, which span the region between the C-terminal boundary of the first nucleotide binding fold and the second transmembrane domain, and which is phosphorylated to allow the channel to open.
  • the paired nucleic acid construct system further comprises a first AAV vector operatively linked to the first construct; and a second AAV vector operatively linked to the second construct.
  • first AAV vector and/or the second AAV vector may be the same vector. In aspects, the first AAV vector and/or the second AAV vector may be different AAV vectors. Exemplary AAV vectors are described herein.
  • a method of using the disclosed paired nucleic acid construct system comprising administering the paired nucleic acid construct system as described herein, to the individual.
  • the method may comprise contacting the paired nucleic acid construct system with an airway epithelial cell of the individual.
  • the administration of the paired nucleic acid construct system provides, restores, or improves CFTR activity in an airway cell of the individual.
  • the individual being treated may be one having a class 1 mutation.
  • the individual may have one more mutations selected from G542X, R553X, and W1282X, with reference to the CFTR gene.
  • the individual may have one or both of a F508 deletion and a 2184delA mutation.
  • the individual may be one who does not respond to a CFTR modulator drug.
  • respond is meant that the individual does not show improvement, or significant improvement, in response to the CFTR modulator drug.
  • the CFTR modulator drug is TRIKAFTA (elexacaftor/tezacaftor/ivacaftor).
  • the paired nucleic acids of the system may be administered via methods described herein.
  • the paired nucleic acids of the system may be administered simultaneously, or sequentially.
  • the paired nucleic acids of the system may be administered every three months, or every six months, or every nine months, or once a year.
  • the administration is selected from intranasal delivery, pulmonary delivery, and/or both intranasal and pulmonary delivery.
  • a cell, or plurality of cells that express the paired nucleic acid construct system as described herein.
  • Inteins are genetic elements transcribed and translated within a host protein from which they self-excise similarly to a protein intron, without leaving amino acid modifications in the final protein product.
  • An intein is a segment of a protein that is able to excise itself and join the remaining portions (the exteins) with a peptide bond in a process known as protein splicing.
  • an N-Intein is an intein fragment located at the N-terminus of (and fused with) a first protein fragment (e.g., a first CFTR fragment) and a C-Intein is an intein fragment located at the C- terminus of (and fused with) the second protein fragment (e.g., a second CFTR fragment), wherein upon expression of the two fragments, the two intein fragments undergo protein trans splicing and are joined to form a full intein, and the two fragments are joined (e.g., the two CFTR fragments), wherein when the two polypeptides form a full length protein, the full length protein is reconstituted (e.g., a full-length CFTR protein, having wild type CFTR functional activity).
  • the first intein sequence may be an N-intein sequence and the second intein sequence may be a C-Intein sequence, wherein the N-Intein and the C-Intein may be derived from the same
  • Exemplary inteins include an intein from the DnaE gene (e.g., DNA polymerase III subunit alpha) from cyanobacteria including Nostoc punctiforme (Npu) Synechocystis sp. PCC6803 (Ssp), Fischerella sp. PCC 9605, Scytonema tolypothrichoides, Cyanobacteria bacterium SW_9_47_5, Nodularia spumigena, Nostoc flagelliforme, Crocosphaera watsonii WH 8502, Chroococcidiopsis cubana CCALA 043, Trichodesmium erythraeum.
  • the intein is that of Rhodothermus marinus, for example, an intein from Rhodothermus marinus DnaB. 15,16
  • the N-intein is that of Rhodothermus marinus DnaB and has a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 1, wherein SEQ ID NO: 1 is
  • the IntN sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 2, wherein SEQ ID NO: 2 is
  • the Intein C is that of Rhodothermus marinus DnaB and has a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 3, wherein SEQ ID NO: 3 is
  • the Intein C (IntC) sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 4, wherein SEQ ID NO: 4 is
  • MAAACPELRQLAQSDVYWDPIVSIEPDGVEEVFDLTVPGPHNFVANDIIAHNS SEQ ID NO: 4
  • compositions and methods may be used for delivery of the CFTR protein to a cell, particularly to a cell of an individual having cystic fibrosis (CF).
  • the CFTR gene encodes for the CFTR protein, composed of 1,480 amino acids, which functions as a channel across the membrane of cells.
  • the CFTR coding sequence may be split into two portions.
  • the N-intein coding sequence is fused in frame with the sequence coding for the N-tcrminal portion of the CFTR sequence
  • the C-lntcin coding sequence is fused in frame with the sequence coding for the C-terminal portion of the CFTR sequence.
  • the inteins undergo autocatalytic excision and form a ligated extein, e.g. the reconstituted CFTR protein.
  • the disclosed systems and methods employ a paired nucleic acid delivery system.
  • the paired nucleic acid delivery system employs a first CFTR fragment, the first CFTR fragment being capable of expressing a gene product (protein/polypeptide) which can be joined with a second gene product to form a functional CFTR protein.
  • the N-terminal portion of the CFTR may comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 5), as follows.
  • the CFTR-N sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 6, wherein SEQ ID NO: 6 is MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRE LASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAI YLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSL LSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGR MMMKYRDQRAGKI
  • the second CFTR fragment of the paired nucleic acid delivery system is a CFTR fragment capable of expressing a gene product (protein/polypeptide) which can be joined with the first gene product to form a functional CFTR protein.
  • the C-terminal portion of the CFTR comprises a sequence having at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 7, wherein SEQ ID NO: 7is
  • the CFTR-C sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 8, wherein SEQ ID NO: 8 is SIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHS RNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQ APMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVP VIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKA LNLHTAN
  • a vector system is also described, in which the vector system may be used to express the coding sequence of the first and second CFTR gene fragment in a cell, for example an in vivo cell of a human tissue, for example lung tissue of an individual who has cystic fibrosis (CF).
  • the cell is a cell of a human tissue, for example lung tissue of an individual who has CF, and who is not responsive to drug therapy, wherein the drug therapy is the administration of CF modulators (e.g., elexacaftor/tezacaftor/ivacaftor).
  • the vector system may comprise two vectors, each vector comprising a portion of the CFTR coding sequence flanked by an intein sequence as described above, wherein the 5' end of the coding sequence is flanked at the 3' terminus by the sequence of an N-intein, and the 3' end of the coding sequence of the gene of interest is flanked by the sequence of a C-Intein, such that when both vectors are expressed in a cell, two fusion proteins are produced and the full length CFTR is generated as a result of a spontaneous trans- splicing reaction.
  • the disclosed vectors and systems concern a virus or virion comprising a polynucleotide, expression construct, or vector construct as described herein.
  • the virus or virion is an AAV virus.
  • Adeno-associated virus AAV
  • AAV Adeno-associated virus
  • Methods for preparing viruses and virions comprising a heterologous polynucleotide or construct are known in the art.
  • cells can be coinfected or transfected with adenovirus or polynucleotide constructs comprising adenovirus genes suitable for AAV helper function. Examples of materials and methods are described, for example, in U.S.
  • An AAV virus or AAV vector useful for administration of the paired nucleic acid delivery system can be of any AAV serotype, including, but not limited to, serotype AAV serotype 1 (AAVl), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAVl 1), or AAV serotype 12 (AAV12), or any other serotype as known to one of ordinary skill in the viral arts.
  • AAV serotype 1 AAVl
  • AAV2 AAV serotype 2
  • AAV3 AAV-3
  • AAV serotype 4 AAV4
  • AAV serotype 5 AAV5
  • AAV serotype 6 A
  • vector system may include the AAV backbone, an inverted terminal repeat, a promoter sequence, the CFTR sequence, an intein sequence, and one or more linker sequences as described herein.
  • AAV-mediated delivery of other large-sized genes using inteins has been performed in preclinical models.
  • 20 ’ 24 Both ciliated and non-ciliated airway epithelial cells of different species, including human, mouse and pig, have been shown to be efficiently transduced by apical infection with AAV5, AAV6, AAV6.2, AAV6.2FF and AAV2H22 in vitro and in vivo.
  • RNA-seq technology has led to identification of diverse cell types in airway epithelia, including ionocytes, basal, tuft, secretory, goblet and ciliated cells. 29,30 Ionocytes and secretory cells have been particularly highlighted due to a high-level expression of CFTR in those cells. 30,31
  • the AAV vector is AAV6.
  • the AAV vector is AAV6.2, an AAV6 F129E point mutant of AAV6.
  • the AAV vector is AAV6.2FF, which is a triple AAV6 mutant containing F129E, Y445F, and Y731F mutations.
  • the AAV6.2FF has improved transduction of lung epithelial cells, including airway epithelial cells, as compared to AAV6.
  • AAV6.2FF is understood by one of ordinary skill in the art and is described in, for example, A Novel Triple-Mutant AAV6 Capsid Induces Rapid and Potent Transgene Expression in the Muscle and Respiratory Tract of Mice, van Eieshout, Eaura P. el al., Molecular Therapy Methods & Clinical Development, Volume 9, 323 - 329 and Thomas, S.P., Spinelli, M.M., Rghei, A.D. et al. Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF. BMC Biotechnol 24, 22 (2024). https://doi.org/10.1186/sl2896- 024-00853-6. Promoters
  • the constructs described herein may comprise a promoter.
  • Promoters may be ubiquitous, artificial, or tissue specific promoters, including fragments and variants thereof retaining a transcription promoter activity.
  • Exemplary promoters are photoreceptor-specific promoters including photoreceptor- specific human G protein-coupled receptor kinase 1 (GRK1), Interphotoreceptor retinoid binding protein promoter (IRBP), Rhodopsin promoter (RHO), vitelliform macular’ dystrophy 2 promoter (VMD2), Rhodopsin kinase promoter (RK);
  • Further exemplary promoters are muscle- specific promoters including MCK, MYODI; liver-specific promoters including thyroxine binding globulin (TBG), hybrid liver- specific promoter (HLP); neuron- specific promoters including hSYNl, CaMKIla; kidney-specific promoters including Ksp- cadherinl6, NKCC2.
  • Ubiquitous promoters include ubiquitous cytomegalovirus (CMV) and short CMV promoters
  • Further exemplary promoters include GRK1, TBG, CaMKIla, Ksp- cadherinl6, native gene promoters, cytomegalovirus (CMV) promoter (KF853603.1, bp 149- 735), chimeric CMV/chicken beta-actin promoter (CBA) and the truncated form of CBA (smCBA) promoter, Rhodopsin promoter (NG_009115, bp 4205-5010), Interphotoreceptor retinoid binding protein promoter (NG_029718.1, bp 4777-5011), vitelliform macular dystrophy 2 promoter (NG_009033.1, bp 4870-5470), PR-specific human G protein-coupled receptor kinase 1 (hGRKl; AY327580.1 bpl793-2087 or bp 1793-1991)
  • the promoter is the SMVP promoter.
  • the SMVP promoter is generated by fusing the SV40 enhancer-CMV-promoter-chimeric intron.
  • the SMVP promoter sequence may comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 9, wherein SEQ ID NO: 9 is
  • Promoters can be incorporated into a construct using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in a vector of the invention. In aspects, the promoter can be positioned about the same distance from the transcription start site as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity.
  • a transcription staid site is typically included in the 5' construct but not in the 3' construct. In further aspects, a transcription start site may be included in the 3' construct upstream of the degradation signal.
  • polynucleotides and polypeptides of the subject invention encompasses those specifically exemplified herein, as well as any natural variants thereof, as well as any variants which can be created artificially, so long as those variants retain the desired functional activity.
  • polypeptides which have the same amino acid sequences of a polypeptide exemplified herein except for amino acid substitutions, additions, or deletions within the sequence of the polypeptide, as long as these variant polypeptides retain substantially the same relevant functional activity as the polypeptides specifically exemplified herein.
  • conservative amino acid substitutions within a polypeptide which do not affect the function of the polypeptide would be within the scope of the subject invention.
  • the polypeptides disclosed herein should be understood to include variants and fragments, as discussed above, of the specifically exemplified sequences.
  • the subject invention further includes nucleotide sequences which encode the polypeptides disclosed herein.
  • nucleotide sequences can be readily constructed by those skilled in the art having the knowledge of the protein and amino acid sequences which are presented herein. As would be appreciated by one skilled in the art, the degeneracy of the genetic code enables the artisan to construct a variety of nucleotide sequences that encode a particular polypeptide or protein. The choice of a particular nucleotide sequence could depend, for example, upon the codon usage of a particular expression system or host cell. Polypeptides having substitution of amino acids other than those specifically exemplified in the subject polypeptides are also contemplated within the scope of the present invention.
  • non-natural amino acids can be substituted for the amino acids of a polypeptide of the invention, so long as the polypeptide having substituted amino acids retains substantially the same activity as the polypeptide in which amino acids have not been substituted.
  • non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4- diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, 2-amino butyric acid, y- amino butyric acid, s-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyiic acid, 3- amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, r- butylglycine, r-butylalanine
  • Non-natural amino acids also include amino acids having derivatized side groups.
  • any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form.
  • Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby a polypeptide having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the polypeptide having the substitution still retains substantially the same biological activity as a polypeptide that does not have the substitution.
  • Table 1 provides a listing of examples of amino acids belonging to each class.
  • polynucleotides which have the same nucleotide sequences of a polynucleotide exemplified herein except for nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, as long as these variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides specifically exemplified herein (e.g., they encode a protein having the same amino acid sequence or the same functional activity as encoded by the exemplified polynucleotide).
  • the polynucleotides disclosed herein should be understood to include variants and fragments, as discussed above, of the specifically exemplified sequences.
  • the subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences of the invention so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis, T. et al, 1982).
  • Polynucleotides described herein can also be defined in terms of more particular identity and/or similarity ranges with those exemplified herein.
  • the sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%.
  • the identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or greater as compared to a sequence exemplified herein.
  • the first vector and/or the second vector further comprise a 5'-terminal repeat (5'-TR) nucleotide sequence and a 3'-terminal repeat (3'-TR) nucleotide sequence, preferably the 5'-TR is a 5'-inverted terminal repeat (5'-ITR) nucleotide sequence and the 3'-TR is a 3'-inverted terminal repeat (3'-ITR) nucleotide sequence.
  • 5'-TR is a 5'-inverted terminal repeat (5'-ITR) nucleotide sequence
  • 3'-TR is a 3'-inverted terminal repeat (3'-ITR) nucleotide sequence.
  • the first vector and/or the second vector further comprise a polyadenylation signal nucleotide sequence.
  • the coding sequence is split into the first portion and the second portion at a position consisting of a nucleophile amino acid which does not fall within a structural domain or a functional domain of the encoded protein product, wherein the nucleophile amino acid is selected from serine, threonine, or cysteine.
  • At least one of the first vector and the second vector further comprise at least one enhancer or regulatory nucleotide sequence, operably linked to the coding sequence.
  • a first construct comprising a first CFTR sequence and a first intein is disclosed.
  • An exemplary first construct is the “Flag- CFTR-N-intN” construct. It should be noted, however, that the “flag” portion is optional and may be omitted for clinical administration to an individual.
  • the Flag-CFTR-N-intN for use in the disclosed compositions and methods may be made by employing routine molecular biology techniques as understood in the art and as described in Chew WL, Tabebordbar M, Cheng JK, et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods. 2016; 13( 1O):868- 874.
  • the human CFTR N-terminal lobe may be fused with the Rhodothermus marinus N-split-intcin and the human CFTR C-tcrminal lobe with the Rhodothermus marinus C-split-intein.
  • the component sequences for manufacture of the Flag- CFTR-N-intN construct and delivery vehicle are described below.
  • the Flag-CFTR-N-intN construct may comprise a tag.
  • the tag may be a flag sequence.
  • the flag sequence can serve as a marker for the detection of the CFTR protein following its delivery to a cell or an individual.
  • the flag component is included for experimental applications.
  • the flag component is incorporated for therapeutic applications.
  • the construct is designed without the inclusion of a flag.
  • a suitable flag sequence is a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 10 , wherein SEQ ID NO: 10 is
  • the Flag sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to
  • the human CFTR-N sequence of the construct comprises a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 5, wherein SEQ ID NO: 5 is
  • TTGATGATATGGAG (CFTR-N, SEQ ID NO: 5).
  • the CFTR-N sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 6, wherein SEQ ID NO: 6 is
  • Rhodothermus marinus Intein-N (intN) sequence is a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 1, wherein SEQ ID NO: 1 is
  • the IntN sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 2, wherein SEQ ID NO: 2 is
  • any of the above sequences may be inserted into an AAV vector for delivery to a cell or an individual.
  • the sequences are operatively linked.
  • each of the sequences are operatively linked via a linker sequence as described herein.
  • the CFTR fragment and intein are directly connected, and are not connected via a linker sequence.
  • the CFTR-N and Int-N are directly connected, and are not connected via a linker sequence.
  • the CFTR-C and Int-C are directly connected, and are not connected via a linker sequence.
  • the above sequences may be inserted into an AAV vector for delivery.
  • the full sequence of the Flag-CFTR-N-intN is a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 12, wherein SEQ ID NO: 12 is ATG GAC TAC AAA GAC CAT GAC GGT GAT TAT AAA GAT CAT GAC ATC GAT TAC AAG GAT GAC GAT GAC AAG CTT ATG CAG AGG TCG CCT CTG GAA AAG GCC AGC GTT GTC TCC AAA CTT TTT TTC AGC TGG ACC AGA CCA ATT TTG AGG AAA GGA TAC AGA CAG CGC CTG GAA TTG TCA GAC ATA TAC CAA ATC CCT TCT GTT GAT TCT GAA AAA TTG GAA AGA G
  • the full sequence of the Flag-CFTR-N-intN expresses a protein having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 13, wherein SEQ ID NO: 13 is M D Y K D H D G D Y K D H D I D Y K D D D D D K L M Q R S P L E K A S V V S K L FF S W T R P I L R K G Y R Q R L E L S DI Y Q I P S V D S A D N L S E K L E R EW D R E L A S K K K N P K L I N A L R R CF F W R F M F Y G I F L Y L G E V T K AV Q P L L L G R I I A S Y D P D N K E E R
  • the pAAV-SMVP-Flag-CFTR-N-intN nucleic acid construct may be provided in an expression construct, for example, for amplification in bacteria.
  • An exemplary expression construct may comprise an ORI sequence, a linker sequence, an AAV2ITR sequence, and a second linker sequence.
  • the construct may comprise an ORI sequence, the sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to
  • the pAAV-SMVP-Flag-CFTR-N-intN may comprise a first linker sequence.
  • the first linker sequence may be used to link (operatively connect) the ORI sequence above and the AAV2 ITR sequence.
  • the sequence may comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 15, wherein SEQ ID NO: 15 is
  • the pAAV-SMVP-Flag-CFTR-N-intN may comprise an AAV2 ITR sequence.
  • the AAV2 ITR sequence may comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 16, wherein SEQ ID NO: 16 is CTGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC
  • the pAAV-SMVP-Flag-CFTR-N-intN may comprise a second linker sequence.
  • the second linker sequence may comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 17, wherein SEQ ID NO: 17 is
  • the pAAV-SMVP-Flag-CFTR-N-intN may comprise a SMPV promoter sequence.
  • the SMPV promoter sequence may comprise a sequence having at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 9, wherein SEQ ID NO: 9 is
  • the pAAV-SMVP-Flag-CFTR-N-intN may comprise one or more further linker sequences, for example, a third and/or fourth linker sequence.
  • the one or more linker sequences may comprise gggtaccgaagccgctagcgctaccggt (linker, SEQ ID NO: 18) and/or AGAATTAACC (linker, SEQ ID NO: 19).
  • the pAAV-SMVP-Flag-CFTR-N-intN may comprise a tag.
  • the one or more linkers of the preceding paragraph may be operatively bound to a tag such as a Flag tag.
  • the Flag tag may have a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 10, wherein SEQ ID NO: 10 is
  • the Flag sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 11, wherein SEQ ID NO: 11 is
  • the flag sequence may be operatively connected to the N terminal CFTR fragment (CFTR-N).
  • the CFTR-N sequence has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 5, wherein SEQ ID NO: 5 is A TGC A GA GGTCGCCTCTGGAAAA GGCCA GCGTTGTCTCCAAA CTTTTTTTCA GCTGGA CCA
  • the CFTR-N sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 6, wherein SEQ ID NO: 6 is
  • the human CFTR-N region above may be operatively connected to Rhodothermus marinus Intein-N.
  • Intein-N has a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 1, wherein SEQ ID NO: 1 is
  • the Intein N (IntN) sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 2, wherein SEQ ID NO: 2 is
  • the pAAV-SMVP-Flag-CFTR-N-intN may comprise a further linker which operatively connects the intein-N sequence and a poly-A signal.
  • the linker may comprise CTCGAGCTCGATGAGTTTGGACAAACCACAACTAGAAT (linker, SEQ ID NO: 20).
  • the SV50 Poly(A) signal may be, for example, a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to that of SEQ ID NO: 21, wherein SEQ ID NO: 21 is GCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACC
  • ATTATAAGCTGCAATAAACAAGTT (SEQ ID NO: 21), which may be further operatively connected to a linker sequence having a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 22, wherein SEQ ID NO: 22 is:
  • the pAAV-SMVP-Flag-CFTR-N-intN may comprise an AAV2 ITR sequence.
  • the AAV2 ITR sequence may comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 23, wherein SEQ ID NO: 23 is
  • An exemplary ORI-Linker- Promoter- AAV2-Linker-ORI sequence which may be used in conjunction with a CFTR fragment- Intein sequence is as follows: CCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCC TGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCG AATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC AGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTT CCTT CCTTTCTCTCCCTT CCTT CCTTTCTCTCCCTT CCTT CCTTTCTCTCCCTT CCTT CCTTTCTCTCCCTT CCTTTCTCTCCCTT CCTTTCTCTCCCTT CCTTTCTCTCCCTT CCTT
  • a second construct comprising a second CFTR sequence and a second intein is disclosed.
  • An exemplary second construct is the “intC-CFTR-C-HA” construct. It should be noted, however, that the “HA” portion is optional and may be omitted for clinical administration to an individual. Further disclosed is an intC- CFTR-C-HA construct which may be used for carrying out the disclosed methods.
  • the intC- CFTR-C-HA construct for use in the disclosed compositions and methods may be made by employing routine molecular biology techniques as understood in the art.
  • An exemplary AAV vector is provided at www.addgene.org/browse/sequence/152875/.
  • the disclosed constructs employ a human CFTR fragment operatively connected to an Rhodothermus marin s intein.
  • the intC-CFTR construct may comprise an intein C operatively connected to a CFTR-C fragment.
  • the CFTR-C sequence comprises a nucleic acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to
  • the intein-C (intC) sequence is a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 3, wherein SEQ ID NO: 3 is ATGGCGGCGGCGTGCCCGGAACTGCGTCAGCTGGCGCAGAGCGATGTGTATTGGGA
  • the CFTR-C sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 8, wherein SEQ ID NO: 8 is
  • the intC-CFTR-C-HA construct may comprise a tag.
  • the tag is an HA sequence.
  • the HA sequence can serve as a marker for the detection of the CFTR protein following its delivery to a cell or an individual.
  • the HA component is included for experimental applications.
  • the HA component is incorporated for therapeutic applications.
  • the construct may be designed without the inclusion of a flag.
  • a suitable HA sequence is TACCCATACGATGTTCCAGATTACGCTTAG (HA SEQ ID NO: 25).
  • the HA sequence comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to YPYDVPDYA (SEQ ID NO: 26).
  • the intC-CFTR-HA component of the construct may comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 27, wherein bold italic font indicates the Int-C portion of the construct, and bold indicates the optional “HA” portion of the construct as indicated below:
  • the intC-CFTR-HA sequences comprises a nucleic acid sequence that encodes for an amino acid sequence having at least 90%, at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 28, wherein SEQ ID NO: 28 is:
  • the pAAV-SMVP-intC-CFTR-C-HA construct may comprise an Origin of Replication (ORI) sequence.
  • the ORI sequence may have at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to ACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG CGGAGCCTATGGAA (SEQ ID NO: 14).
  • the pAAV-SMVP-intC-CFTR-C-HA construct may comprise a linker sequence.
  • the linker sequence may be
  • the linker sequence may be used to link the ORI sequence to the AAV 2 ITR sequence.
  • the pAAV-SMVP-intC-CFTR-C-HA may comprise an AAV 2 ITR sequence.
  • AAV 2 ITR (inverted terminal repeat) sequences may be provided for AAV vector replication and expression of the CFTR gene fragment.
  • the AAV 2ITR sequence may comprise SEQ ID NO:
  • the pAAV-SMVP-intC-CFTR-C-HA may comprise a second linker sequence.
  • the second linker sequence may be used to link the AAV 2 ITR sequence and the SMVP promoter sequence.
  • the second linker sequence may comprise SEQ ID NO: 17, wherein SEQ ID NO: 17 is
  • the pAAV-SMVP-intC-CFTR-C-HA may comprise an SMVP promoter sequence.
  • the SMVP promoter sequence may comprise SEQ ID NO: 9, wherein SEQ ID NO: 9 is GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGAC CGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACG GTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT
  • the pAAV-SMVP-intC-CFTR-C-HA may comprise a third linker sequence.
  • the third linker sequence may be used to link the SMVP promoter sequence and a fourth linker.
  • the second third sequence may comprise GGGTACCGAAGCCGCTAGCGCTACCGGT (SEQ ID NO: 18).
  • the pAAV-SMVP-intC-CFTR-C-HA may comprise a fourth linker sequence.
  • the fourth linker sequence may be used to link the SMVP promoter sequence and the intein C sequence.
  • the fourth sequence may comprise CGCCACC.
  • the C-intein is a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 3, as set forth above.
  • the intC sequence may be operatively connected to the C terminal CFTR fragment (CFTR-C).
  • the CFTR-C sequence has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to CFTR-C, SEQ ID NO: 7, as set forth above.
  • the CFTR-C sequence may be operatively connected to a tag.
  • the tag may be hemagglutinin (“HA”).
  • HA hemagglutinin
  • the HA sequence has at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to
  • TACCCATACGATGTTCCAGATTACGCTTAG (HA, SEQ ID NO: 25), or the sequence encoding for the protein, YPYDVPDYA (HA, SEQ ID NO: 26).
  • the HA tag may be joined to a SV40 polyA signal via a linker.
  • the linker may comprise
  • the SV40 poly (A) signal may have the sequence gcagtgaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagtt (SV40 poly(A) signal SEQ ID NO: 21). This sequence may be further operatively connected to a further linker having the sequence
  • An exemplary ORI-Linker-Promoter-AAV2-Linker-ORI sequence which may be used in conjunction with a CFTR fragment- Intein sequence is as follows: CCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCC TGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA
  • the present invention also concerns pharmaceutical compositions comprising the vector system or the viral vector system or the host cells of the invention optionally in combination with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • a pharmaceutically acceptable carrier diluent, excipient or adjuvant.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as — or in addition to — the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system).
  • the construct or vector can be administered in vivo or ex vivo.
  • compositions adapted for topical or parenteral administration comprising an amount of a compound, constitute a preferred embodiment of the invention.
  • the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • the pharmaceutical composition of the present invention may be delivered to the retina preferentially via the subretinal injection or it can also be prepared in the form of injectable suspension, eye lotion or ophthalmic ointment that can be delivered to the retina with a non-invasive procedure.
  • the dose administered to a patient, particularly a human should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity.
  • dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.
  • the present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the vector system or viral vector system or host cell of the present invention comprising one or more deliverable therapeutic and/or diagnostic transgenes(s) or a viral particle produced by or obtained from same.
  • the pharmaceutical composition may be for human or animal usage.
  • Dosage regimes and effective amounts to be administered can be determined by ordinarily skilled clinicians. Administration may be in the form of a single dose or multiple doses.
  • General methods for performing gene therapy using polynucleotides, expression constructs, and vectors are known in the art (see, for example, Gene Therapy: Principles and Applications, Springer Verlag 1999; and U.S. Pat. Nos. 6,461,606; 6,204,251 and 6,106,826).
  • the subject invention also concerns methods for expressing a selected polypeptide in a cell.
  • the method comprises incorporating in the cell the vector system of the invention that comprises polynucleotide sequences encoding the selected polypeptide and expressing the polynucleotide sequences in the cell.
  • the selected polypeptide can be one that is heterologous to the cell.
  • the cell is a mammalian cell. In one embodiment, the cell is a human cell.
  • delivery of the compositions may be via intranasal delivery.
  • the delivery may be via pulmonary delivery.
  • the delivery of the composition is directly into the nasal cavity of a subject.
  • the active compound may be formulated in a suitable pharmaceutical composition, such as a nasal spray, which can be easily administered using a standard nasal delivery device.
  • the intranasal administration route is particularly advantageous for the delivery of viral vectors, as it bypasses the potential for systemic side effects and allows for targeted delivery to respiratory tissues.
  • compositions may be inhaled into the lungs of a subject. This route of administration allows for the direct delivery of the active compound to the respiratory tract, facilitating localized treatment and potentially reducing systemic side effects.
  • the active compound may be formulated in a suitable pharmaceutical composition, such as an aerosol or dry powder, which can be administered using a standard inhaler or nebulizer.
  • the pulmonary administration route is particularly advantageous for the delivery of viral vectors, as it allows for targeted delivery to the lung tissues.
  • kits comprising the construct system or viral vector system or the host cells of the invention in one or more containers.
  • Kits of the invention can optionally include pharmaceutically acceptable carriers and/or diluents.
  • a kit of the invention includes one or more other components, adjuncts, or adjuvants as described herein.
  • a kit of the invention includes instructions or packaging materials that describe how to administer a vector system of the kit.
  • Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration.
  • the construct system or viral vector system or the host cells of the invention is provided in the kit as a solid.
  • the construct system or viral vector system or the host cells of the invention is provided in the kit as a liquid or solution.
  • the kit comprises an ampoule or syringe containing the construct system or viral vector system or the host cells of the invention in liquid or solution form.
  • a cell comprising the construct system as disclosed herein.
  • the cell may be a bacterial, yeast, plant, or mammalian cells which comprises at least one of the construct comprising a nucleic acid sequence corresponding to a first portion of a CFTR gene and a nucleic acid sequence corresponding to a an intein gene, as described herein.
  • Cystic Fibrosis is a lethal genetic disease resulting in multiorgan dysfunction that is caused by loss of function of the anion channel CFTR. Delivery of functional CFTR into diseased organs is an ideal approach to treat CF. Success in this approach has been limited by the large size of the CFTR gene, inability to deliver a construct through the thick airway mucus of CF, and lack of durability in construct expression in the airway epithelium.
  • a novel CFTR delivery system was developed using adeno-associated virus (AAV) and intein technology, which allows for the delivery of a split CFTR gene using two distinct AAV constructs.
  • AAV adeno-associated virus
  • CFTR protein a fully functional, recombined CFTR protein
  • Fig. 1 The approach has been validated in patient- derived primary human nasal epithelial cells from six people with CF (PwCF) who are insensitive to CFTR modulators, with CFTR rescue confirmed by ion transport studies.
  • PwCF patient- derived primary human nasal epithelial cells from six people with CF
  • CFTR rescue confirmed by ion transport studies.
  • CFTR-fused inteins are created and packaged into AAVs capable of infecting patient-derived airway epithelial cells, ultimately producing recombined intact CFTR in the cells.
  • the CFTR cDNA was split and an intein coding fragment (IntN or IntC from the eubacterium Rhodothermus marinus') attached to each CFTR fragment.
  • the split fusion genes were then packaged into AAV plasmids which are termed AAV.CFTR-N-intN and AAV.intC-CFTR-C (FIG. 2A; named based on which half of the CFTR cDNA is included).
  • Linker DNA between CFTR and intein was removed to minimize DNA length and avoid potential protein dysfunction due to inserted amino acids from the linker. Additionally, AAV.CFTR-N- intN was tagged with “Flag” and AAV.intC-CFTR-C with “HA” to identify the fraction of cells co-expressing both CFTR.N-intN and intC-CFTR-C, as confirmed by immunofluorescence in 293T cells (FIG. 2B). These plasmids were packaged into two AAV6 (serotype 6) vectors.
  • AAV6.2FF with split CFTRs restores CFTR function in vitro in human nasal epithelial (HNE) cells from people with CF (PwCF) who are insensitive to CFTR modulators
  • Applicant infected HNE cells which are in a proliferating progenitor state, from PwCF homozygous for c.850dup or 1525-1G>A CFTR with AAV6.2FF containing split CFTRs (Dual AAV) and grew them at air-liquid interface to a differentiated state (FIG. 3A).
  • AAV6.2FF which is a new version of AAV6 (F129L, Y445F and Y731F) that can transduce lung epithelial cells, including airway epithelial cells, better than AAV632 was created using QuikChange II XL Site-Mutagenesis Kit (Agilent). In vitro physiological CFTR function was assessed four weeks after infection.
  • HNEs were obtained by non-invasive nasal brushing, processed, and expanded in P100 dishes as previously described (Brewington JJ, Filbrandt ET, EaRosa FJ, 3rd, Moncivaiz JD, Ostmann AJ, Strecker EM, Clancy JP. Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies. JCI insight 2018; 3: e99385.). Once cells reached 80% confluence, they were passaged by adding 0.1% trypsin for 5 minutes to facilitate cell detachment and manual detachment with a cell scraper. The cell mix was centrifuged and the pellet reconstituted in media and counted.
  • HNEs were then seeded onto Transwell®-Clear permeable supports (0.33cm2 filters, 0.4pm pore size) pre-coated with type IV collagen at approximately 260,000 cells/cm2 (80,000 cells/insert).
  • AAV vectors were then added directly to the cell compartment of the Transwell. All cells were maintained in Differentiation Media, changing daily, removing apical media once confluent (approximately 3-4 days). Cells were maintained with basolateral media only, changing daily, for 4-5 weeks (depending on schedule) until testing. [000124] Ion Transport Analysis. Once mature, select inserts were pretreated with VX809 (3pM), VX661 (3pM), and/or VX445 (3pM) for 48h prior to study.
  • Inserts were removed from media and rinsed of any pre-treatment drugs, then mounted in Ussing chambers and studied as previously described (Brewington JJ, Filbrandt ET, LaRosa FJ, 3rd, Ostmann AJ, Strecker LM, Szczesniak RD, Clancy JP. Detection of CFTR function and modulation in primary human nasal cell spheroids. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 2017; 17: 26-33). All studies were performed in an asymmetric chloride ringer buffer, producing a basolateral-to-apical Cl- secretory gradient.
  • FIGS. 6-11 show cellular response data in human nasal epithelial (HNE) cultures from six individuals with cystic fibrosis (CF) without CFTR modulator access.
  • HNE human nasal epithelial
  • CFTR function >10% of wild-type is easily achieved in five of six subjects, with a statistically significant response to dAAV still present in the sixth subject.
  • n 4 inserts. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001 by one-way ANOVA with Dunnet’s multiple comparisons test against control samples.
  • Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol. 2000 Apr;74(8):3852-8. doi: 10.1128/jvi.74.8.3852-3858.2000. PMID: 10729159; PMCID: PMC111893.
  • Zabncr J Binding of adcno-associatcd virus type 5 to 2,3-linkcd sialic acid is required for gene transfer. J Biol Chem. 2001 Jun 8;276(23):20610-6. doi: 10.1074/jbc.M101559200. Epub 2001 Mar 21. PMID: 11262413.
  • a revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature. 2018 Aug;560(7718):319-324. doi: 10.1038/s41586-018-0393-7. Epub 2018 Aug 1. PMID: 30069044; PMCID: PMC6295155.
  • McCray PB Jr. Increased CFTR expression and function from an optimized lentiviral vector for cystic fibrosis gene therapy. Mol Ther Methods Clin Dev. 2021 Feb 27;21:94-106. doi: 10.1016/j.omtm.2021.02.020. PMID: 33768133; PMCID: PMC7973238.

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Abstract

L'invention concerne des systèmes de constructions d'acide nucléique appariées pour l'administration d'une protéine régulatrice de la conductance transmembranaire de la fibrose kystique humaine (CFTR) à un individu en ayant besoin. Le système en deux parties comprend une première construction comprenant une première partie d'un gène CFTR humain fusionné à l'ADNc de Rhodothermus marinus intéine-N (CFTR-N-inteinN) et une seconde construction comprenant une seconde partie d'un gène CFTR humain fusionné à l'ADNc de Rhodothermus marinus inteine-C (inteinC-CFTR-C). L'invention concerne en outre des méthodes de traitement d'un individu ayant une fibrose kystique (CF) comprenant l'administration des constructions d'acide nucléique appariées.
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