EP4211151A1 - Für gjb2 codierendes rekombinantes adeno-assoziiertes virus (raav) und verwendungen davon - Google Patents

Für gjb2 codierendes rekombinantes adeno-assoziiertes virus (raav) und verwendungen davon

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Publication number
EP4211151A1
EP4211151A1 EP21867807.6A EP21867807A EP4211151A1 EP 4211151 A1 EP4211151 A1 EP 4211151A1 EP 21867807 A EP21867807 A EP 21867807A EP 4211151 A1 EP4211151 A1 EP 4211151A1
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EP
European Patent Office
Prior art keywords
gjb2
nucleic acid
isolated nucleic
seq
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP21867807.6A
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English (en)
French (fr)
Inventor
David P. COREY
Kevin T. BOOTH
Cole W. D. PETERS
Maryna V. IVANCHENKO
Michael E. Greenberg
Sinisa HRVATIN
Mark Aurel NAGY
Eric C. Griffith
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Harvard College
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Harvard College
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Publication date
Application filed by Harvard College filed Critical Harvard College
Publication of EP4211151A1 publication Critical patent/EP4211151A1/de
Pending legal-status Critical Current

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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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • GJB2 gap junction beta 2
  • DFNB1 nonsyndromic Hearing Loss and Deafness
  • the present disclosure relates to an isolated nucleic acid comprising an expression cassette, wherein the expression cassette comprises a gap junction beta 2 (GJB2) gene regulatory element (GRE), and a nucleotide sequence encoding a GJB2 protein.
  • the expression cassette further comprises a promoter (e.g., GJB2 promoter).
  • the expression cassette is flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the isolated nucleic acid described herein is capable of expressing the GJB2 protein in inner ear cells that normally express the GJB2 gene (e.g., connective tissue cells of the cochlea and supporting cells of the organ of Corti and nearby regions), but not in the cell that do not normally express GJB2 (e.g., hair cells and spiral ganglion neurons).
  • GJB2 e.g., connective tissue cells of the cochlea and supporting cells of the organ of Corti and nearby regions
  • the present disclosure provides an isolated nucleic acid comprising an expression cassette, wherein the expression cassette comprises a gap junction beta 2 (GJB2) gene regulatory element (GRE), and a nucleotide sequence encoding a GJB2 protein.
  • the GJB2 protein is a human GJB2 protein.
  • the GJB2 protein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1.
  • the nucleotide sequence encoding a human GJB2 protein comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 2.
  • the expression cassette further comprises a promoter operably linked to the nucleotide sequence encoding a GJB2 protein.
  • the promoter is a human GJB2 promoter.
  • the promoter comprises 500 nucleotides of a human GJB2 promoter.
  • the promoter comprises a nucleic acid sequence at least 80% identical to SEQ ID NO: 5.
  • the promoter comprises a nucleic acid sequence at least 80% identical to SEQ ID NO: 102.
  • the promoter comprises a nucleic acid sequence 100% identical to SEQ ID NO: 102.
  • the promoter is a human GJB2 basal promoter.
  • the human GJB2 basal promoter comprises a nucleic acid sequence at least 80% identical to SEQ ID NO: 47.
  • the expression cassette comprises a nucleotide sequence encoding a 5' UTR.
  • the 5' UTR is positioned between the promoter and the nucleotide sequence encoding the GJB2 protein.
  • the 5' UTR comprises about 300 nucleotides of a human GJB2 gene 5' UTR.
  • the promoter and the 5' UTR comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 30.
  • the GJB2 gene regulatory element comprises an enhancer.
  • the enhancer is positioned 5' to the promoter.
  • the enhancer is normally present within approximately 200 kb upstream or downstream of a GJB2 gene.
  • the enhancer is normally present within approximately 95 kb of a GJB2 gene.
  • the GJB2 GRE comprises one or more enhancers.
  • the one or more enhancers are the same enhancers or different enhancers.
  • the enhancer comprises a nucleotide sequence at least 80% identical to nucleotide sequence or a fragment thereof as set forth in any one of SEQ ID NOs: 6 to 29.
  • the enhancer comprises a nucleotide sequence at least 80% identical to a GJB2 enhancer as set forth in any of SEQ ID NOs: 37-46 and 55-60. In some embodiments, the enhancer comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 42.
  • the present disclosure also provides an isolated nucleic acid comprising an expression cassette, wherein the expression cassette comprises a Gap Junction beta 2 (GJB2 ) promoter, and a nucleotide sequence encoding a GJB2 protein.
  • the expression cassette comprises a Gap Junction beta 2 (GJB2 ) promoter, and a nucleotide sequence encoding a GJB2 protein.
  • GJB2 Gap Junction beta 2
  • the GJB2 promoter comprises a nucleic acid sequence at least 80% identical to SEQ ID NO: 102. In some embodiments, the GJB2 promoter comprises a nucleic acid sequence 100% identical to SEQ ID NO: 102.
  • the expression cassette further comprises a 5 ' UTR.
  • the 5' UTR comprises: a first nucleic acid sequence at least 80% identical to SEQ ID NO: 103; and/or a second nucleic acid sequence at least 80% identical to SEQ ID NO: 104.
  • the expression cassette further comprises a 5 ' UTR.
  • the 5' UTR comprises: a first nucleic acid sequence 100% identical to SEQ ID NO: 103; and/or a second nucleic acid sequence 100% identical to SEQ ID NO: 104.
  • the isolated nucleic acid comprises a nucleic acid sequence at least 80% identical to SEQ ID NO: 105. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence 100% identical to SEQ ID NO: 105.
  • the isolated nucleic acid is capable of expressing GJB2 in cells that normally express the GJB2 gene. In some embodiments, the isolated nucleic acid is capable of expressing GJB2 in cochlear connective tissue cells and supporting cells of the organ of Corti. In some embodiments, the supporting cell of the organ of Corti are pillar cells, Deiter cells, Hensen’s cells, Claudius cells, inner phalangeal cells, and border cells.
  • the cochlear connective tissue cells are strial intermediate cells, fibrocytes of the lateral wall and suprastrial zone, basal cells of the stria vascularis, fibrocytes in the spiral ligament, fibrocytes in the spiral limbus, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, and supralimbal dark cells.
  • the expression cassette is flanked by two adeno-associated virus inverted terminal repeats (ITRs).
  • the AAV ITR is from a serotype selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR.
  • the AAV ITR is AAV2 ITR.
  • the expression cassette comprises: a 5' ITR having a nucleotide sequence at least 80% identical to SEQ ID NO: 106; and/or a 3 ' ITR having a nucleotide sequence at least 80% identical to SEQ ID NO: 107.
  • the expression cassette comprises: a 5 ' ITR having a nucleotide sequence 100% identical to SEQ ID NO: 106; and/or a 3 ' ITR having a nucleotide sequence 100% identical to SEQ ID NO: 107.
  • the expression cassette further comprises a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) 3' to the nucleotide sequence encoding the GJB2 protein.
  • WP Woodchuck Hepatitis Virus
  • WPRE Posttranscriptional Regulatory Element
  • the WPRE comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 108. In some embodiments, the WPRE comprises a nucleotide sequence 100% identical to SEQ ID NO: 108.
  • the expression cassette further comprises a nucleotide sequence encoding a 3' UTR located 5' of the WPRE.
  • the 3' UTR is a GJB2 exon 2 3' UTR.
  • the GJB2 exon 2 3' UTR comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 32.
  • the expression cassette further comprises one or more miRNA binding site positioned in the 3' UTR.
  • the miRNA binding site is a neuron-associated miRNA binding site.
  • the neuron- associated miRNA is selected from: miR-124, miR-127, miR-129, miR-129*, miR-136, miR-136*, miR-137, miR-154, miR-3OO-3p, miR-323, miR-329, miR-341, miR-369-5p, miR-376a, miR-376b-3p, miR-376c, miR-379, miR-382, miR-382*, miR-410, miR-411, miR-433, miR-434, miR-495, miR-541, miR-543*, miR-551b, miR-143, miR-449a, miR-219-2-3p, miR-126, miR-126*, miR-141, miR-
  • the miRNA binding site is a cochlear hair cell- associated miRNA binding site.
  • the cochlear hair cell-associated miRNA binding site is selected from: miR-124, miR-96, miR- 182, and miR-183.
  • the expression cassette further comprises a poly A signal.
  • the poly A signal is a bovine growth hormone poly A signal.
  • the poly A signal comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 109. In some embodiments, the poly A signal comprises a nucleotide sequence 100% identical to SEQ ID NO: 109.
  • the present disclosure also provides an isolated nucleic acid comprising a nucleotide sequence 100% identical to SEQ ID NO: 110 or 111. In some aspects, the present disclosure also provides an isolated nucleic acid comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 110 or 111.
  • the present disclosure also provides a vector comprising the isolated nucleic acid as described herein.
  • the vector is a plasmid or a viral vector.
  • the viral vector is an AAV vector.
  • the present disclosure also provides a vector comprising from 5' to 3': (a) an AAV 5' ITR; (b) a GJB2 promoter, or a basal GJB2 promoter sequence thereof; (c) a GJB2 5' UTR (e.g., a GJB2 exon 1 5' UTR); (d) a nucleotide sequence encoding a GJB2 protein; (e) a GJB2 3' UTR (e.g., a GJB2 exon 2 3' UTR), optionally the GJB2 3' UTR comprises one or more miR-124 binding site; (f) a bovine growth hormone poly A signal; and (g) an AAV 3’ ITR.
  • a vector comprising from 5' to 3': (a) an AAV 5' ITR; (b) a GJB2 promoter, or a basal GJB2 promoter sequence thereof; (c) a GJB2 5' UTR (
  • the present disclosure also provides a vector comprising from 5' to 3': (a) an AAV 5' ITR; (b) a GJB2 enhancer; (c) a GJB2 promoter, or a basal GJB2 promoter sequence thereof; (d) a GJB2 5' UTR (e.g., a GJB2 exon 1 5' UTR); (e) a nucleotide sequence encoding a GJB2 protein; (f) a GJB2 3' UTR (e.g., a GJB2 exon 2 3' UTR), optionally the GJB2 3' UTR comprises one or more miR-124 binding site; (g) a bovine growth hormone poly A signal; and (h) an AAV 3' ITR.
  • a GJB2 enhancer e.g., a GJB2 promoter, or a basal GJB2 promoter sequence thereof
  • a GJB2 5' UTR e.
  • the vector comprises a nucleotide sequence at least 80% identical to any one of SEQ ID NOs: 36, 48-62 and 61-83.
  • the vector is an AAV vector.
  • the vector is capable of expressing a GJB2 gene in cells that normally express GJB2.
  • the present disclosure also provides a recombinant adeno-associated virus (rAAV) comprising: (i) a capsid protein; and (ii) the isolated nucleic acid described herein.
  • rAAV recombinant adeno-associated virus
  • the present disclosure also provides a recombinant adeno-associated virus (rAAV) comprising: (i) a capsid protein; and (ii) an isolated nucleic acid comprising: (a) an AAV 5' ITR (e.g., a GJB2 exon 1 5' UTR); (b) a GJB2 promoter, or a basal GJB2 promoter sequence thereof; (c) a GJB2 5' UTR (e.g., a GJB2 exon 2 3' UTR), optionally the GJB2 exon 2 3' UTR comprises one or more miR-124 binding site; (d) a nucleotide sequence encoding a GJB2 protein; (e) a GJB2 3' UTR; (f) a bovine growth hormone poly A signal; and (g) an AAV 3' ITR.
  • rAAV recombinant adeno-associated virus
  • the present disclosure also provides a recombinant adeno-associated virus (rAAV) comprising: (i) a capsid protein; and (ii) an isolated nucleic acid comprising: (a) an AAV 5' ITR; (b) a GJB2 enhancer; (c) a GJB2 promoter, or a basal GJB2 promoter sequence thereof; (d) a GJB2 5' UTR (e.g., a GJB2 exon 1 5' UTR); (e) a nucleotide sequence encoding a GJB2 protein; (f) a GJB2 3' UTR (e.g., a GJB2 exon 2 3' UTR), optionally the GJB2 exon 2 3' UTR comprises one or more miR-124 binding site; (g) a bovine growth hormone poly A signal; and (h) an AAV 3' ITR.
  • rAAV recombinant adeno-
  • the rAAV has tropism for a subset of cochlea cells that normally express the GJB2 gene. In some embodiments, the rAAV has tropism for cells of the inner ear.
  • the capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV5 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV-S capsid protein, or a variant thereof.
  • the AAV capsid is AAV9.PHP.B, AAV9.PHP.eB, or AAV-S.
  • the AAV capsid protein is AAV-S.
  • the present disclosure provides a host cell comprising the isolated nucleic acid, the vector, or the rAAV as described herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated nucleic acid, the vector, the rAAV, or the host cell as described herein.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the present disclosure provides a method for specifically expressing GJB2 in cells that normally expresses the GJB2 gene in a subject, the method comprising administering to the subject an effective amount of the isolated nucleic acid, the vector, the rAAV, the host cell, or the pharmaceutical composition as described herein.
  • the present disclosure provides a method for treating Non-syndromic Hearing Loss and Deafness (DFNB1) in a subject, the method comprising administering to the subject an effective amount of the isolated nucleic acid, the vector, the rAAV, the host cell, or the pharmaceutical composition as described herein.
  • DFNB1 Non-syndromic Hearing Loss and Deafness
  • a method for treating a GJB2-associated disease in a subject in need thereof comprising administering to the subject an effective amount of the isolated nucleic acid, the vector, the rAAV, the host cell, or the pharmaceutical composition as described herein.
  • the subject is a mammal.
  • the mammal is a human.
  • the mammal is a non-human mammal.
  • the non-human mammal is mouse, rat, or non-human primate.
  • the hearing loss is associated with a mutation in the GJB2 gene.
  • the mutation in the GJB2 gene is a point mutation, a missense mutation, a nonsense mutation, a splice-altering mutation, a synonymous mutation, a deletion, an insertion, or a combination thereof.
  • the subject is human; and the mutation is a mutation listed in Table 2 (below) or a combination thereof.
  • the mutation is NM_004004.6 C.101T>C (GRCh37/hgl9 Chrl3:20763620A>G) or c.35delG (GRCh37/hgl9 chrl3:20763685AC>A).
  • the administration results in expression of GJB2 protein in the cochlea connective tissue cells and supporting cells of the organ of Corti and nearby regions.
  • the supporting cell of the organ of Corti are pillar cells, Deiters’ cells, Hensen’s cells, Claudius cells, inner phalangeal cells, and border cells.
  • the connective tissue cells are strial intermediate cells, fibrocytes of the lateral wall and suprastrial zone, basal cells of the stria vascularis, fibrocytes in the spiral ligament, fibrocytes in the spiral limbus, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, and supralimbal dark cells.
  • the administration is via injection.
  • the injection is through round window membrane of the cochlea, into the scala media of the cochlea, into the scala tympani of the cochlea, into the scala vestibuli of the cochlea, into a semicircular canal of the inner ear, or into the saccule or the utricle of the inner ear.
  • FIGs. 1A-1C show the structure and expression distribution of GJB2, and how loss of GJB2 expression affects the patients.
  • FIG. 1A shows the structure of the GJB2 hemichannel. Six subunits of GJB2 protein, each with four trans-membrane helices, assemble in the plane of the membrane to form a large central pore. GJB2 hemichannels from adjacent cells join to create a channel from the cytoplasm of one cell to the cytoplasm of the other. Gap junctions are formed by hundreds or thousands of channels packed in a junctional plaque.
  • FIGs. 1B-1C show the network of fibrocytes and epithelial cells in which GJB2 is expressed (FIG.
  • FIG. ID shows that many patients carrying GJB2 mutation(s) who have some residual hearing at birth show further hearing loss over the next 3-6 years. A window for treatment is present for 1-5 years after birth, with -10,000 affected children in the United State aged 0-5 possibly treatable.
  • FIGs. 2A-2B show the delivery of viral vector to the cochlea by direct injection through the round window membrane (RWM) and the deleterious effect of promiscuous expression of Gjb2 to the hearing of injected mice.
  • FIG. 2A is a cartoon illustrating the round window membrane (RWM) injection.
  • FIG. 2B shows that promiscuous expression of Gjb2 in the inner ear damaged hearing in wild-type mice.
  • FIGs. 3A-3N show the identification of cis-regulatory elements (e.g., enhancers) that are critical for GJB2 expression in the subset of cochlea cells that naturally express the GJB2 gene.
  • FIGs. 3A-3B show that certain patients with GJB2-associated deafness have upstream deletions occurring in trans with GJB2 coding sequence mutations, which suggests that some patients carry mutation(s) in the cis-regulatory element, and the region next to the CRYL1 gene is of particular importance for identification of such cis-regulatory elements.
  • 3C shows the identification of gene regulatory elements (GREs), in UCSC Genome Browser views of ATAC-Seq from mouse cochlea at developmental stages P2, P5 and P8, over -300 kb in the region of the mouse Gjb2 gene. Shaded regions mark regions containing putative GREs.
  • X-axis is the genomic region on chrl4 in the mouse genome.
  • Y-axis is the number of reads from the AT AC- Seq that align to a specific region in the genome.
  • Light shading denotes regions of open chromatin, which are the hallmarks of transcriptionally active regions that are enriched for read pile up, suggesting higher activity in these regions.
  • Regions A and B mark the transcriptionally active sequences within mouse Gjb2 itself.
  • Regions C-M are regions that are transcriptionally active around Gjb2 that might be part of a cis-regulatory network.
  • FIG. 3C (bottom) shows transcriptionally active regions in and around the light-shaded regions that have been tested as specific GREs (dark highlight). Note that the GREs were initially identified in mouse. Human GJB2 GREs were identified in silico by modeling the mouse GREs. Human GJB2 GREs were tested in subsequent experiments.
  • FIGs. 3D-3E show various vector designs with or without incorporation of GJB2 promoter and/or enhancers. These vectors were tested in mouse inner ear.
  • the C15 vector which is the GJB2 enhancer vector, concatenates 500 bp of the human GJB2 promoter, the human GJB2 5’ UTR followed by a coding sequence for GFP and human GJB2 3’ UTR, and three human GJB2 enhancers that match mouse sequences identified by ATAC-seq.
  • Vectors c20-23 were constructed to test the toxicity of promiscuous expression of Gjb2 in mouse.
  • Vector c20 was lethal at doses over 2xl0 9 genomic copies.
  • FIG. 3F shows a segment of the mouse cochlea, from the lateral wall (top) to the interdental cells (bottom).
  • FIG. 3G shows the expression of Gjb2 in inner hair cells driven by construct c20.
  • FIG. 3D reconstruction of the organ of Corti in an uninjected mouse cochlea, with outer hair cells and inner hair cells is shown in the top panel.
  • GJB2-containing gap junctions in supporting cells were labeled with an antibody to GJB2 protein. Hair cells do not make gap junctions.
  • Vector c20 with a promiscuous promoter, drives GJB2 expression in inner hair cells and other cell types (see bottom panel).
  • FIG. 3H shows that promiscuous Gjb2 expression damages hearing in wildtype mice, but targeted expression rescues hearing in Gjb2 knockout mice.
  • FIGs. 3I-3L shows the map of the c70 vector plasmid encoding mouse GJB2 or human GJB2 with or without an HA tag.
  • FIG. 3M shows schematics of vector c.70 encoding mouse GJB2 or human GJB2 with or without the HA tag.
  • FIG. 3N shows additional vectors that were created and tested.
  • FIG. 4 shows that AAV-S encoding eGFP with a CBA promoter efficiently transduces hair cells, supporting cells, and cells of the lateral wall, in both neonatal mouse and juvenile NHP cochlea.
  • FIGs. 5A-5V show vector maps of the AAV vectors including the identified GJB2 GREs 1, 2, 3, 4, 5, 7, 8, and 9, respectively.
  • the vectors include, from 5' to 3 ', a 5' ITR, a human GJB2 GRE, a GJB2 basal promoter, a GJB2 exon 1 5 ' UTR, a nucleotide sequence encoding an eGFR, and GJB2 exon 2 3 ' UTR.
  • FIG. 5A shows vector c.81.1, which includes human GJB2 GRE1, and encodes human GJB2
  • FIG. 5B shows vector c.81.1, which includes human GJB2 GRE1, and encodes mouse GJB2;
  • FIG. 5C shows vector c.81.2, which includes human GJB2 GRE2, and encodes eGFP
  • FIG. 5D shows vector c.81.2, which includes human GJB2 GRE2, and encodes human GJB2
  • FIG. 5E shows vector c.81.2, which includes human GJB2 GRE2, and encodes mouse GJB2
  • FIG. 5F shows vector c.81.3, which includes human GJB2 GRE3, and encodes eGFP
  • FIG. 5G shows vector c.81.3, which includes human GJB2 GRE3, and encodes human GJB2
  • FIG. 5H shows vector c.81.3, which includes human GJB2 GRE3, and encodes mouse GJB2;
  • FIG. 5D shows vector c.81.2, which includes human GJB2 GRE2, and encodes human GJB2
  • FIG. 5E shows vector c.81.2, which includes human GJB2 GRE2, and encodes mouse GJB2
  • FIG. 51 shows vector c.81.4, which includes human GJB2 GRE4, and encodes human GJB2;
  • FIG. 5J shows vector c.81.4, which includes human GJB2 GRE4, and encodes mouse GJB2;
  • FIG. 5K shows vector c.81.5, which includes human GJB2 GRE5, and encodes eGFP;
  • FIG. 5L shows vector c.81.5, which includes human GJB2 GRE5, and encodes human GJB2;
  • FIG. 5M shows vector c.81.5, which includes human GJB2 GRE5, and encodes mouse GJB2;
  • FIG. 5N shows vector c.81.7, which includes human GJB2 GRE7, and encodes eGFP;
  • FIG. 50 shows vector c.81.7, which includes human GJB2 GRE7, and encodes human GJB2;
  • FIG. 5P shows vector c.81.7, which includes human GJB2 GRE7, and encodes mouse GJB2;
  • FIG. 5Q shows vector c.81.8, which includes human GJB2 GRE8, and encodes human GJB2;
  • FIG. 5R shows vector c.81.8, which includes human GJB2 GRE8, and encodes mouse GJB2;
  • FIG. 5S shows vector c.81.9, which includes human GJB2 GRE9, and encodes eGFP;
  • FIG. 5T shows vector c.81.9, which includes human GJB2 GRE9, and encodes human GJB2;
  • FIG. 5P shows vector c.81.7, which includes human GJB2 GRE7, and encodes mouse GJB2;
  • FIG. 5Q shows vector c.81.8, which includes human GJB2 G
  • FIG. 5U shows vector c.81.9, which includes human GJB2 GRE9, and encodes mouse GJB2.
  • FIG. 5V shows schematics of c81.2, c81.3, c81.5, c81.7 and c81.9 encoding eGFP, mouse GJB2 and human GJB2 as described above.
  • FIGs. 6A-6D show GFP expression by vector c81.5 in the cells of the organ of Corti
  • FIG. 6A shows a fluorescent image of GFP expressing cells, including a variety of supporting cells in, and medial to, the organ of Corti.
  • FIG. 6B shows antibody label of endogenous GJB2 in the region of the organ of Corti. Gjb2 expression largely overlapped that of exogenous GFP.
  • FIG. 6C is an overlay of FIGs. 6A and 6B, with a third staining of actin, which revealed stereocilia of hair cells. No GFP was expressed in the hair cells.
  • FIG. 6D shows a frozen section immunofluorescence image of GFP and a protein marker for hair cells, MY07A. GFP was expressed in a variety of supporting cells in the organ of Corti, but did not overlap with MY07A expression, which was expressed in hair cells.
  • FIGs. 7A-7E show GFP expression pattern by vector 81.5 in the lateral wall of the cochlea.
  • FIG. 7A shows GFP expression in cells including fibrocytes of the lateral wall.
  • FIG. 7B shows an antibody labeling of endogenous Gjb2 in the region of the lateral wall. GJB2 expression largely overlaps that of exogenous GFP.
  • FIG. 7C is an overlay image of FIGs. 7A and 7B. Note that GFP was expressed in the cells expressing Gjb2.
  • FIGs. 7D-7E show frozen section immunofluorescences of GFP (FIG. 7D) and GJB2 in supporting cells of the organ of Corti and fibrocytes of the lateral wall (FIG. 7E).
  • the present disclosure at least in part, relates to an isolated nucleic acid comprising an expression cassette, wherein the expression cassette comprises a gap junction beta 2 (GJB2) gene regulatory element (GRE), and a nucleotide sequence encoding a GJB2 protein.
  • the expression cassette further comprises a promoter (e.g., GJB2 promoter).
  • the expression cassette is flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the isolated nucleic acid described herein is capable of expressing the GJB2 protein in inner ear cells that normally express the GJB2 gene (e.g., connective tissue cells of the cochlea and supporting cells of the organ of Corti and nearby regions), but not in the cell that do not normally express GJB2 gene (e.g., hair cells and spiral ganglion neurons).
  • GJB2 gene e.g., connective tissue cells of the cochlea and supporting cells of the organ of Corti and nearby regions
  • GJB2 gene e.g., hair cells and spiral ganglion neurons
  • the present disclosure relates to compositions and methods for treating certain autosomal recessive genetic diseases, for example, non-syndromic hearing loss (DFNB1).
  • DFNB1 is caused by mutations in the GJB2 gene.
  • the GJB2 gene encodes the GJB2 protein, also known as connexin 26.
  • Connexin 26 is a member of the connexin protein family.
  • GJB2 protein forms channels in clusters called gap junctions, which allow communication between neighboring cells, including cells in the inner ear. Mutations in the GJB2 gene eliminate or change the structure of gap junctions and affect the function or survival of cells that are needed for hearing.
  • Gene replacement therapy e.g., gene therapy by recombinant adeno-associated virus (rAAVs)
  • rAAVs recombinant adeno-associated virus
  • the present disclosure is based, in part, on the surprising discovery that successful GJB2 gene therapy requires GJB2 expression in cells that normally express the GJB2 protein (e.g., connective tissue cells of the cochlea and supporting cells of the organ of Corti and nearby regions) and not in other cells (e.g., hair cells and spiral ganglion neurons). Excluding sensory cells, most cells in the cochlea are connected via gap junctions, and these gap junctions appear to play a critical role in cochlear function. GJB2 protein occurs in gap junctions connecting most cell classes in the cochlea. There are two independent systems of cells, which are defined by interconnecting gap junctions.
  • the first system is mainly composed of all organs of Corti supporting cells e.g., epithelial cells of the inner and outer sulcus, and interdental cells), and also includes interdental cells in the spiral limbus and root cells within the spiral ligament.
  • the sensory region of the cochlea termed the organ of Corti, includes one row of inner hair cells (IHC) and three to four rows of outer hair cells (OHC) that are surrounded by various supporting cells.
  • the supporting cells play crucial roles in the development, function, and maintenance of inner ear sensory epithelia.
  • supporting cells span the entire depth of the epithelium, from the basal lamina to the lumen.
  • Supporting cells are linked to each other and to hair cells by tight and adherens junctions; they communicate directly with other supporting cells by gap junctions (e.g., Wan et al., Inner ear supporting cells: Rethinking the silent majority, Semin Cell Dev Biol. 2013 May; 24(5): 448-459).
  • Non-limiting examples of supporting cells for the organ of Corti include pillar cells, Deiters’ cells, Hensen’s cells, Claudius cells, inner phalangeal cells, and border cells.
  • the second system includes strial intermediate cells, fibrocytes of the lateral wall and suprastrial zone, basal cells of the stria vascularis, fibrocytes in the spiral ligament, fibrocytes in the spiral limbus, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, and supralimbal dark cells.
  • GJB2 in the cochlea, is normally expressed in supporting cells of the organ of Corti and nearby regions (e.g., pillar cells, Deiters’ cells, Hensen’s cells, Claudius cells, inner phalangeal cells; and border cells), and the connective tissue system comprising strial intermediate cells, fibrocytes of the lateral wall and suprastrial zone, basal cells of the stria vascularis, fibrocytes in the spiral ligament, fibrocytes in the spiral limbus, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, and supralimbal dark cells (See, e.g., Kikuchi et al.
  • cells of the organ of Corti and nearby regions e.g., pillar cells, Deiters’ cells, Hensen’s cells, Claudius cells, inner phalangeal cells; and border cells
  • the connective tissue system comprising strial intermediate cells, fibrocytes of the lateral wall and suprastrial zone, basal
  • Gap junctions in the rat cochlea immunohistochemical and ultrastructural analysis.
  • GJB2 expression is critical for cochlear function.
  • the K + that enters hair cells through transduction channels and leaves through basal K + channels is shuttled away from the organ of Corti by the epithelial system and conveyed by the cytoplasmic system to the stria, where it is pumped back into endolymph.
  • GJB2 plays a role in the development of the cochlea, as mice lacking GJB2 protein in the inner ear have reduced endocochlear potential and profound apoptotic loss of hair cells and supporting cells by postnatal day 30 (P30), even though hair cells do not express Gjb2 (Cohen-Salmon et al., 2002; Wang et al., 2009; Sun et al., 2009; Crispino et al., 2011; Johnson et al., 2017). If Gjb2 is deleted after P6, the phenotype is much milder (Chang et al., 2015).
  • GJB2 function in shuttling K + may be related to its role in the development of the cochlea: If K + is not carried away from hair cells by a gap junction network, K + accumulation could depolarize hair cells, leading to Ca 2+ influx and eventual cell death.
  • the gap junction network may also be required to transport glucose and nutrients from blood vessels to the sensory epithelium, and its absence could lead to cell death.
  • the present disclosure provides an isolated nucleic acid comprising two adeno-associated virus (AAV) inverted terminal repeats (ITRs) flanking an expression cassette, wherein the expression cassette comprises a promoter (e.g., a human GJB2 promoter) operably linked to a nucleotide sequence encoding a GJB2 gene regulatory element (GRE), and a nucleotide sequence encoding a gap junction beta 2 (GJB2) protein.
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • an expression cassette refers to component of vector DNA comprising a protein coding sequence to be expressed by a cell having the vector and its regulatory sequences. Once delivered to the target cell, the expression cassette directs the cell’s machinery to make RNA and/or protein(s) (e.g., GJB2 protein).
  • a “nucleic acid” sequence refers to a DNA or RNA sequence.
  • proteins and nucleic acids of the disclosure are isolated.
  • the term “isolated” means artificially produced.
  • the term “isolated” means: (i) amplified in vitro by, for example, the polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, for example, by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
  • An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
  • nucleotide sequence contained in a vector in which 5 ' and 3 ' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not.
  • An isolated nucleic acid may be substantially purified, but need not be.
  • a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
  • isolated refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).
  • the GJB2 protein is a human GJB2 protein.
  • the human GJB2 protein comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.
  • MDWGTLQTILGGVNKHSTS IGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGCKN VCYDHYFP I SHIRLWALQLIFVSTPALLVAMHVAYRRHEKRKFIKGEIKSEFKDIEEIKTQK VRIEGSLWWTYTSS IFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTE KTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPV
  • the expression cassette of the isolated nucleic acid encodes a human GJB2 protein having the amino acid sequence as set forth in SEQ ID NO: 1.
  • the nucleotide sequence encoding a human GJB2 protein comprises a nucleotide sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2.
  • An exemplary nucleotide sequence encoding a human GJB2 protein is set forth in SEQ ID NO: 2: ATGGATTGGGGCACGCTGCAGACGATCCTGGGGGGTGTGAACAAACACTCCACCAGCATTGG AAAGATCTGGCTCACCGTCCTCTTCATTTTTCGCATTATGATCCTCGTTGTGGCTGCAAAGG AGGTGTGGGGAGATGAGCAGGCCGACTTTGTCTGCAACACCCTGCAGCCAGGCTGCAAGAAC GTGTGCTACGATCACTACTTCCCCATCTCCCACATCCGGCTATGGGCCCTGCAGCTGATCTT CGTGTCCACGCCAGCTCCTAGTGGCCATGCACGTGGCCTACCGGAGACATGAAGAAGA GGAAGTTCATCAAGGGGGAGATAAAGAGTGAATTTAAGGACATCGAGGAGATCAAAACCCAG AAGGTCCGCATCGAAGGCTCCCTGTGGTGGACCTACACAAGCAGCATCTTCTTCCGGGTCATCTCTCTCTGGGTCAT CTTCGAAGCCG
  • the GJB2 protein is a mouse GJB2 protein.
  • the mouse GJB2 protein comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3.
  • MDWGTLQS ILGGVNKHSTS IGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGCKN VCYDHHFP I SHIRLWALQLIMVSTPALLVAMHVAYRRHEKKRKFMKGEIKNEFKDIEEIKTQ KVRIEGSLWWTYTTS IFFRVIFEAVFMYVFYIMYNGFFMQRLVKCNAWPCPNTVDCFI SRPT EKTVFTVFMI SVSGICILLNITELCYLFVRYCSGKSKRPV
  • the isolated nucleic acid comprises a nucleotide sequence encoding a mouse GJB2 protein having an amino acid sequence as set forth in SEQ ID NO: 3.
  • the nucleotide sequence encoding a mouse GJB2 protein comprises a nucleotide sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4.
  • the nucleotide sequence encoding the GJB2 protein is codon optimized for expression in a host (e.g., a human).
  • Codon optimization refers to the design process of altering codons to codons known to increase maximum protein expression efficiency in a desired cell.
  • codon optimization is described, wherein codon optimization can be performed by using algorithms that are known to those skilled in the art to create synthetic genetic transcripts optimized for high protein yield.
  • Programs containing algorithms for codon optimization are known to those skilled in the art. Programs can include, for example, OptimumGeneTM, GeneGPS® algorithms, etc.
  • synthetic codon optimized sequences can be obtained commercially, for example from Integrated DNA Technologies and other commercially available DNA sequencing services.
  • sequence identity refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence, e.g., GJB2 protein disclosed herein and its coding sequences, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alteration of the amino acid sequence or nucleic acid coding sequences can be obtained by deletion, addition, or substitution of residues of the reference sequence.
  • Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill of one in the art, for instance, using publicly available computer software, such as BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU- B LAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software.
  • publicly available computer software such as BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU- B LAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software.
  • Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence is calculated as follows:
  • a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits from, e.g., 50% to 100% identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence.
  • the length of the candidate sequence aligned for comparison purpose is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the length of the reference sequence.
  • a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence (e.g., GJB2 amino acid sequences, coding sequences, nucleotide sequences for GJB2 gene regulatory elements (GREs), or any other sequences described herein)
  • GJB2 amino acid sequences e.g., GJB2 amino acid sequences, coding sequences, nucleotide sequences for GJB2 gene regulatory elements (GREs), or any other sequences described herein
  • An expression cassette of an isolated nucleic acid sequence described herein may further comprise a promoter operably linked to the coding sequence (e.g., GJB2 protein coding sequence).
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the transcription of a gene.
  • the phrases “operatively linked,” “under control,” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • a promoter may be a constitutive promoter, inducible promoter, or a tissue- specific promoter.
  • the promoter is a tissue/cell-specific promoter.
  • a tissue/cell specific promoter refers to a promoter that has activity in only certain cell types.
  • the promoter used in the isolated nucleic acid described herein has activity in cochlear cells that normally express the GJB2 gene. Use of a tissue/cell- specific promoter in the isolated nucleic acid described herein can restrict unwanted transgene (e.g., GJB2 gene) expression as well as facilitate persistent transgene expression.
  • the expression cassette of the isolated nucleic acid comprises a tissue/cell specific promoter.
  • the expression cassette of the isolated nucleic acid comprises a GJB2 promoter (e.g., a GJB2 promoter for any species where cell specific GJB2 expression is desired). In some embodiments, the expression cassette of the isolated nucleic acid comprises a human GJB2 promoter. In some embodiments, the expression cassette of the isolated nucleic acid comprises at least 300 bp (e.g., 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, or more) of any consecutive nucleotides of a human GJB2 promoter.
  • the expression cassette of the isolated nucleic acid comprises a promoter having 500 bp consecutive nucleotides of a human GJB2 promoter. In some embodiments, the expression cassette of the isolated nucleic acid comprises a promoter having a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5.
  • An exemplary nucleotide sequence of 500 bp of a human GJB2 promoter is set forth in SEQ ID NO: 5: ACCTGTCTCCCGCCGTGGCGCCTTTTAACCGCACCCCACACCCCGCCTCTTCCCTCGGAGAC TGGGAAAGTTACGGAGGGGGCGGCGCCGCGGGCGGAGCGCCCGGCCTCTGGGTCCTCAGA GCTTCCCGGGTCCGCGAACCCCCGACCGCCCCCGAAAGCCCCGAACCCCCCCCAAGTCCCCTTC GAGGTCCCGATCCTAGTTCCTTTGAGCCCCCATGAGTTCCCCAAGTGCCCCCAGCGCCCT GAGTCTCCCCCGGTTACCCCGAGCGCCGCCTCCCAGCCTTGGCGGCCCGGGTGAAGCG GGGGCGGCTGAGAGTCGGGACCCCCCAGGAAGCGGCGCCCCCCAGACCCCGGCTGT GCCGTGGGCGGGGTTCAGGGATGGCTGTGGTCGTTGTCCTCTGTACTCCGCATAGTGCGAGA GGACT
  • the expression cassette of the isolated nucleic acid comprises a GJB2 basal promoter (e.g., a human GJB2 basal promoter).
  • a GJB2 basal promoter is a promoter region of a GJB2 gene highly conserved across different species (e.g., human and mouse).
  • the GJB2 basal promoter has been previous described, for example, in Tu, Z. J., and Kiang, D. T. (1998). Mapping and characterization of the basal promoter of the human connexin26 gene. Biochim. Biophys. Acta 1443, 169-181; Kiang, D. T., Jin, N., Tu, Z. J., and Lin, H. H. (1997).
  • the expression cassette of the isolated nucleic acid comprises a GJB2 basal promoter having a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 47.
  • An exemplary nucleotide sequence of a human GJB2 basal promoter is set forth in SEQ ID NO: 47: GGGTTCGGGGCTTTCGGGGGCGGTCGGGGGTTCGCGGACCCGGGAAGCTCTGAGGACCCAGA GGCCGGGCGCGCTCCGCCCGCGGCGCCGCCCCCTCCGTAACTTTCCCAGTCTCCGAGGGAAG AGGCGGGGTGT
  • constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (See, e.g., Boshart et al., Cell, 41:521-530 (1985)) the simian vacuolating virus 40 (SV40) promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the elongation factor 1 -alpha 1 (EFla) promoter.
  • RSV Rous sarcoma virus
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • the promoter is a chicken beta-actin (CBA) promoter. In some embodiments, the promoter is an enhanced chicken P-actin promoter. In some embodiments, the promoter is a U6 promoter. Since the CBA promoter is constitutively active in all cell types, using a CBA promoter in the isolated nucleic acid described herein leads to promiscuous expression of GJB2 protein in all cell types, including cells that do notnormally express GJB2 protein (e.g., hair cells of the cochlea). Accordingly, in some embodiments, a CBA promoter is not used in the isolated nucleic acid described herein.
  • CBA chicken beta-actin
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Many other promoters have been described and can be readily selected by one of skill in the art.
  • inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex) -inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346- 3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci.
  • MT zinc-inducible sheep metallothionine
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system WO 98/10088
  • ecdysone insect promoter No et al., Proc. Natl. Acad. Sci. USA, 93:3346- 3351
  • the isolated nucleic acid comprises a gene regulatory element (GRE) (e.g., GJB2 GRE).
  • GRE gene regulatory element
  • Gene regulatory elements refer to a variety of DNA sequences that are involved in the regulation of gene expression.
  • a GRE may rely on the interactions involving DNA, cellular proteins (e.g., histones), and transcription factors to regulate gene expression.
  • the isolated nucleic acid comprises gene regulatory elements which are cis-regulatory elements (e.g., cis-regulatory elements for the GJB2 gene).
  • Cis- regulatory elements are regions of non-coding DNA which regulate the transcription of neighboring genes. Cis-regulatory elements are found in the vicinity of the genes that they regulate. Cis-regulatory elements typically regulate gene transcription by binding to transcription factors.
  • the gene regulatory elements impart cell-specific gene expression capabilities (e.g., cell specific GJB2 gene expression).
  • the gene regulatory elements are cis-regulatory elements associated with the GJB2 gene.
  • the cis-regulatory elements of the GJB2 gene are enhancers.
  • An enhancer refers to DNA sequences, which are located more distal to the transcription start site as compared to a promoter, capable of interacting with site- specific transcription factors to regulate gene expression in a cell-type specific manner. Enhancers confer cell-specific gene expression regulation by binding to the collection of transcription factors in a cell, which leads to transcriptional activation or inhibition through various mechanisms, e.g., recruitment of epigenetic enzymes that catalyze post-translational histone modifications, and recruitment of cofactors that promote DNA looping.
  • Enhancers can be identified in the vicinity of the gene they regulate, or at a distance of hundreds of kilobases from their target genes. Multiple enhancers can act additively and redundantly to regulate gene expression e.g., Doane el al, Regulatory elements in molecular networks, Wiley Interdiscip Rev Syst Biol Med. 2017 May; 9(3)).
  • the enhancers described herein are enhancers capable of regulating genomic GJB2 gene expression.
  • the GJB2 enhancers are identified in the transcriptionally active sequences of the GJB2 gene.
  • a transcriptionally active sequence refers to a region of DNA in a chromosome in which the DNA is in open chromatin confirmation such that the sequence is exposed, thereby allowing binding of transcription factors and transcription to take place.
  • the GJB2 enhancers are identified within approximately 1000 kb of a genomic GJB2 gene (e.g., within 1000 kb, within 900 kb, within 800 kb, within 700 kb, within 600 kb, within 500 kb, within 450 kb, within 400 kb, within 350 kb, within 300 kb, within 250 kb, within 200 kb, within 150 kb, within 100 kb, within 95 kb, within 90 kb, within 85 kb, within 85 kb, within 80 kb, within 75 kb, within 70 kb, within 65 kb, within 60 kb, within 55 kb, within 50 kb, within 45 kb, within 40
  • the GJB2 enhancers are identified within approximately 200 kb of the GJB2 gene. In some embodiments, the GJB2 enhancers are identified within approximately 95 kb of the GJB2 gene (e.g., regions C-M listed in FIG. 3C) In some embodiments, the GJB2 enhancers are within the regions of DNA sequences near the GJB2 gene (FIG. 3C) listed in Table 1. Table 1. Human and mouse DNA regions that include GJB2 enhancers.
  • a GJB2 GRE (e.g., a GJB2 enhancer) sequences can be identified from the regional sequence listed in Table 2.
  • a GJB2 GRE (e.g., a GJB2 enhancer) comprises at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900 at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800, at least 2800, at least 2900, at least 3000, at least 3100, at least 3200, at least
  • a GJB2 GRE (e.g., a GJB2 enhancer) is identified with the transcriptionally active regions of the GJB2 gene (e.g., regions A and/or B).
  • the GJB2 GRE (e.g., a GJB2 enhancer) comprises at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900 at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800, at least 2800, at least 2900, at least 3000, at least
  • the GJB2 GRE (e.g., a GJB2 enhancer) comprises at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900 at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800, at least 2800, at least 2900, at least 3000, at least 3100, at least 3200, at least 3300, at least 3400, at least 3500, at least 3600, at least 3700, at least 3800, at least 3900, at least 4000, at least 4100, at least 4000, at least
  • a GJB2 GRE e.g., a GJB2 enhancer
  • GJB2 GRE is located on the reverse complement strand of the GJB2 coding sequence in the genome. It is within the skill of one in the art to select the appropriate sequence (e.g., GRE sequence on the sense strand, or GRE sequences on the reverse complement strand) when designing a vector using the enhancer sequences as described herein.
  • a GJB2 GRE (e.g., a GJB2 enhancer) comprises at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900 at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800, at least 2800, at least 2900, at least 3000, at least 3100, at least 3200, at least 3300, at least 3400, at least 3500, at least 3600, at least 3700, at least 3800, at least 3900, at least 4000, at least 4
  • a GJB2 GRE (e.g., a GJB2 enhancer) comprises 200-500 nucleotides or any number of nucleotides in between, 300-600 nucleotides or any number of nucleotides in between, 400-700 nucleotides or any number of nucleotides in between, 500-800 nucleotides or any number of nucleotides in between, 600-900 nucleotides or any number of nucleotides in between, 700-1000 nucleotides or any number of nucleotides in between, 1000-1500 nucleotides or any number of nucleotides in between, 1500-2000 nucleotides or any number of nucleotides in between.
  • a GJB2 GRE (e.g., a GJB2 enhancer) comprises 700 nucleotides.
  • the GJB2 GRE is a human GJB2 enhancer.
  • the GJB2 GRE (e.g., a human GJB2 enhancer) comprises nucleotide sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the GRE sequences as listed in Table 3.
  • the GJB2 GRE is a non-human primate (e.g., Cynomolgus macaque) GJB2 enhancer.
  • the GJB2 GRE e.g., a Cynomolgus macaque GJB2 enhancer
  • the human GJB2 GREs share homology with the mfGJB2
  • the human GJB2 GREs correspond to mfGJB2 GREs as set forth in Table 5:
  • the isolated nucleic acid comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 9, or more) enhancers e.g., GJB2 enhancers).
  • the isolated nucleic acid comprises more than one enhancer, and the more than one enhancer are the same enhancers or different enhancers.
  • the GJB2 GRE is positioned 5’ to the promoter. In other embodiments, the GJB2 GRE is positioned 3’ to the promoter.
  • the presence of the GJB2 enhancer(s) in the isolated nucleic acid facilitates cell-type specific expression of the GJB2 protein encoded by the isolated nucleic acid.
  • cells that normally express the GJB2 gene e.g., fibrocytes and supporting cells of the organ of Corti and nearby regions
  • the expression cassette of the isolated nucleic acid further comprises a 5 ' UTR.
  • the 5' UTR is a native 5' UTR of the genomic GJB2 gene.
  • the 5' untranslated region (5' UTR) (also known as a leader sequence or leader RNA) is the region of an mRNA that is directly upstream of the initiation codon.
  • the 5' UTR plays important roles in both transcriptional and translational regulation of the downstream gene (e.g., the GJB2 gene).
  • the isolated nucleic acid comprising a nucleotide sequence encoding a GJB2 5 ' UTR is also capable of expression GJB2 in a cell- specific manner (e.g., expressing GJB2 in cells that normally express it).
  • the nucleotide sequence encoding the GJB2 5 ' UTR comprises a portion of a nucleotide sequence encoding a full-length human GJB2 gene 5 ' UTR.
  • the 5 ' UTR is a human GJB2 gene exon 1 5 ' UTR.
  • the nucleotide sequence encoding a 5' UTR comprises at least 100 consecutive nucleotides, at least 200 consecutive nucleotides, at least 300 consecutive nucleotides, at least 400 consecutive nucleotides, at least 500 consecutive nucleotides, at least 600 consecutive nucleotides, at least 700 consecutive nucleotides, at least 800 consecutive nucleotides, at least 900 consecutive nucleotides, at least 1000 consecutive nucleotides, or more of a native full-length 5' UTR e.g., the human GJB2 gene exon 1 5' UTR).
  • the expression cassette comprises a nucleotide sequence encoding the 5' UTR having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleotide sequence encoding a human GJB2 gene 5' UTR (e.g., human GJB2 exon 1 5' UTR).
  • a human GJB2 gene 5' UTR e.g., human GJB2 exon 1 5' UTR.
  • the expression cassette comprises a nucleotide sequence encoding the 5' UTR having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleotide sequence encoding a consecutive 300 bp of a human GJB2 gene 5' UTR (e.g., the human GJB2 gene exon 1 5' UTR) as set forth in SEQ ID NO: 53.
  • a human GJB2 gene 5' UTR e.g., the human GJB2 gene exon 1 5' UTR
  • an exemplary nucleotide sequence encoding the 300 bp of the human GJB2 gene exon 1 5' UTR has a nucleotide sequence as set forth in SEQ ID NO: 53: GGGGTGCGGTTAAAAGGCGCCACGGCGGGAGACAGGTGTTGCGGCCCCGCAGCGCCCGCGCGCGCTCCTCTCCCCGACTCGGAGCCCCTCGGCGGCGCCCGGCCCAGGACCCGCCTAGGAGCGCAG GAGCCCCAGCGCAGAGACCCCAACGCCGAGACCCCCGCCCCGGCCCCGCCGCGCTTCCTCCC GACGCAGAGCAAACCGCCCAGAGTAG
  • the cell specific GJB2 expression is achieved by incorporation of a nucleotide sequence encoding a basal promoter and a GJB2 5 ' UTR or a portion thereof (basal promoter/5 ' UTR).
  • an expression cassette e.g., GJB2 expression cassette
  • the isolated nucleic acid can further comprise additional nucleotide sequence encoding one or more GJB2 GREs (e.g., GJB2 enhancers).
  • the nucleotide sequence encoding the GJB2 GREs and the nucleotide sequence encoding the basal promoter/5 ' UTR can be placed in any order.
  • the nucleotide sequence encoding the GJB2 GREs is placed 5 ' to the nucleotide sequence encoding the basal promoter/5 ' UTR.
  • the isolated nucleic acid comprising a nucleotide sequence encoding a GJB2 basal promoter/5 ' UTR is also capable of expressing GJB2 in a cell-specific manner (e.g., expressing GJB2 in cells that normally express it).
  • the nucleotide sequence encoding the basal promoter/5 ' UTR comprises a portion of a nucleotide sequence encoding a full-length human GJB2 gene 5 ' UTR.
  • the 5' UTR comprises at least 100 consecutive nucleotides, at least 200 consecutive nucleotides, at least 300 consecutive nucleotides, at least 400 consecutive nucleotides, at least 500 consecutive nucleotides, at least 600 consecutive nucleotides, at least 700 consecutive nucleotides, at least 800 consecutive nucleotides, at least 900 consecutive nucleotides, at least 1000 consecutive nucleotides, or more of a native full-length 5' UTR e.g., the GJB2 5' UTR).
  • the 5 ' UTR is a human GJB2 gene exon 1 5 ' UTR.
  • the expression cassette comprises a nucleotide sequence encoding a basal promoter/5' UTR having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleotide sequence encoding the basal promoter and about 300 bp of a human GJB2 gene 5' UTR (e.g., the human GJB2 gene exon 1 5' UTR) (SEQ ID NO: 30).
  • an exemplary nucleotide sequence encoding the 300 bp of the human GJB2 gene basal promoter/exon 1 5' UTR has a nucleotide sequence as set forth in SEQ ID NO
  • a nucleotide sequence encoding a basal promoter/5 ' UTR (e.g., a human GJB2 basal promoter/exon 1 5 ' UTR) within the expression cassette (e.g., GJB2 expression cassette) further comprises an intron or a portion thereof.
  • the expression cassette of the isolated nucleic acid e.g., GJB2 expression cassette
  • the nucleotide sequence encoding an intron has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 54.
  • An exemplary nucleotide sequence encoding the conserved sequence of GJB2 intron 1 is set forth in SEQ ID NO: 54:
  • UTR/intron has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31.
  • An exemplary nucleotide sequence encoding human GJB2 basal promoter/5 'UTR/conserved sequence of intron 1 is set forth in SEQ ID NO: 31:
  • the expression cassette (e.g., GJB2 expression cassette) comprises a nucleotide sequence encoding a proximal promoter of the human GJB2 gene.
  • the proximal promoter of the human GJB2 gene has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 102.
  • an exemplary nucleotide sequence encoding the human GJB2 gene proximal promoter has a nucleotide sequence as set forth in SEQ ID NO: 102.
  • the expression cassette (e.g., GJB2 expression cassette) comprises SEQ ID NO: 102: GACCTCGAAGGGGACTTGGGGGGTTCGGGGCTTTCGGGGGCGGTCGGGGGTTCGCGGACCCG GGAAGCTCTGAGGACCCAGAGGCCGGGCGCGCTCCGCCCGCGGCGCCGCCCCCTCCGT
  • the expression cassette (e.g., GJB2 expression cassette) comprises a nucleotide sequence encoding a 5' UTR of the human GJB2 gene.
  • the 5' UTR of the human GJB2 gene has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 103 or CC.
  • an exemplary nucleotide sequence encoding the human GJB2 gene 5' UTR has a nucleotide sequence as set forth in SEQ ID NO: 103 or CC. In some embodiments, an exemplary nucleotide sequence encoding the human GJB2 gene 5' UTR has a nucleotide sequence comprising SEQ ID NO: 103 and SEQ ID NO: 104. In some embodiments, the expression cassette (e.g., GJB2 expression cassette) comprises SEQ ID NO: 103:
  • the expression cassette (e.g., GJB2 expression cassette) comprises SEQ ID NO: 104:
  • the expression cassette (e.g., GJB2 expression cassette) comprises a nucleotide sequence encoding a proximal promoter and a 5' UTR of the human GJB2 gene.
  • the proximal promoter and the 5' UTR of the human GJB2 gene has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 105.
  • an exemplary nucleotide sequence encoding the human GJB2 gene proximal promoter and 5' UTR has a nucleotide sequence as set forth in SEQ ID NO: 105.
  • the expression cassette (e.g., GJB2 expression cassette) comprises SEQ ID NO: 105: GACCTCGAAGGGGACTTGGGGGGTTCGGGGCTTTCGGGGGCGGTCGGGGGTTCGCGGACCCG GGAAGCTCTGAGGACCCAGAGGCCGGGCGCGCTCCGCCCGCGGCGCCGCCCCCTCCGTAACT TTCCCAGTCTCCGAGGGAAGAGGCGGGGTGTGGGGTGCGGTTAAAAAAGGCGCCACGGCGGGAG ACAGGTGTTGCGGCCCCGCAGCGCCCGCGCGCTCCTCTCCCCGACTCGGAGCCCCTCGGCGG CGCCCGGCCCAGGACCCGCCTAGGAGCGCAGGAGCCCCAGCGCAGAGACCCCAACGCCGAGA CCCCCCGCCCGCCCGCCCGCCCGCCCGCTCCCGACTCG
  • An isolated nucleic acid described herein may also contain an artificial intron, desirably located between the promoter/enhancer sequence and the protein coding sequence (e.g., nucleotide sequence encoding GJB2 protein).
  • an intron is a synthetic or artificial (e.g., heterologous) intron. Examples of synthetic introns include an intron sequence derived from SV-40 (referred to as the SV-40 T intron sequence) and intron sequences derived from chicken beta-actin gene.
  • a transgene described by the disclosure comprises one or more (1, 2, 3, 4, 5, or more) artificial introns.
  • the one or more artificial introns are positioned between a promoter and a nucleotide sequence encoding the GJB2 protein.
  • the expression cassette (e.g., the GJB2) further comprises a nucleotide sequence encoding a 3 ' UTR located 3 ' of the nucleotide sequence encoding the GJB2 protein.
  • the 3' UTR is a GJB2 gene 3 ' UTR.
  • the 3 ’UTR is a GJB2 gene exon 2 3 ' UTR.
  • the nucleotide sequence encoding the 3 ' UTR has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32.
  • An exemplary nucleotide sequence encoding GJB2 gene exon 2 3' UTR is set forth in SEQ ID NO: 32:
  • the expression cassette of the isolated nucleic acid comprises a de-targeting agent that restricts or reduces the transgene expression (e.g., GJB2 expression) in a cell type (e.g., hair cell or spiral ganglion neurons).
  • a de-targeting agent that restricts or reduces the transgene expression (e.g., GJB2 expression) in a cell type (e.g., hair cell or spiral ganglion neurons).
  • incorporation of one or more miRNA binding sites into an expression allows for de-targeting of transgene expression in a cell-type specific manner (e.g., in hair cell or spiral ganglion neurons).
  • one or more miRNA binding sites are positioned in the 3 ' UTR (e.g., GJB2 exon 2 3 ' UTR of the expression cassette of the isolated nucleic acid).
  • an expression cassette comprises one or more (e.g., 1, 2, 3, 4, 5, or more) miRNA binding sites that de-target expression of GJB2 from cells that do not normally express GJB2 (e.g., hair cell or spiral ganglion neurons).
  • the expression cassette of the isolated nucleic acid comprises one or more miR binding sites for detargeting neuron cells (e.g., spiral ganglion neurons), e.g., binding sites for neuron enriched miRs as described in Jovicic et al., Comprehensive Expression Analyses of Neural Cell- Type-Specific miRNAs Identify New Determinants of the Specification and Maintenance of Neuronal Phenotypes, J Neurosci.
  • Non-limiting examples of neuron enriched miRs include miR- 124, miR- 127, miR-129, miR-129*, miR-136, miR-136*, miR-137, miR-154, miR-3OO-3p, miR-323, miR-329, miR-341, miR-369-5p, miR-376a, miR-376b-3p, miR-376c, miR-379, miR-382, miR-382*, miR-410, miR-411, miR-433, miR-434, miR-495, miR-541, miR-543*, miR- 551b, miR-143, miR-449a, miR-219-2-3p, miR-126, miR-126*, miR-141, miR-142-3p, miR-142-5p, miR-146a, miR-150, miR-200c, or
  • the expression cassette of the isolated nucleic acid comprises one or more miR binding sites for detargeting hair cells (e.g., inner or outer hair cell), e.g., binding sites for hair cell enriched miRs as described in Li et al., MicroRNAs in hair cell development and deafness, Curr Opin Otolaryngol Head Neck Surg. 2010 Oct; 18(5): 459-465, which is incorporated herein by reference.
  • Non-limiting examples of neuron enriched miRs include miR-96, miR-182, miR- 183, miR-18a, or miR-99a.
  • the GJB2 exon 2 3 ' UTR of the expression cassette comprises one or more miR binding sites for detargeting neuron cells and hair cells. In some embodiments, the GJB2 exon 2 3 ' UTR of the expression cassette comprises one or more miR binding sites for miR- 124.
  • a gene therapy vector may be a viral vector (e.g., a lentiviral vector, an adeno-associated virus vector, an adenoviral (Ad) vector, etc.), a plasmid, a closed- ended DNA (e.g., ceDNA), a lipid/DNA nanoparticle, etc.
  • a gene therapy vector is a viral vector.
  • an expression cassette encoding a protein is flanked by one or more viral replication sequences, for example, lentiviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • viral replication sequences for example, lentiviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • the isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors).
  • AAV adeno-associated virus
  • an isolated nucleic acid as described by the disclosure comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences, or variants thereof.
  • the isolated nucleic acid e.g., the recombinant AAV vector
  • “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, an expression cassette (e.g., expression cassette for GJB2), and 5 ' and 3 ' AAV inverted terminal repeats (ITRs).
  • the isolated nucleic acids may also comprise a region encoding, for example, 5 ' and 3 ' untranslated regions (UTRs), and/or an expression control sequence (e.g., a poly- A tail).
  • ITR sequences are about 145 bp in length. Preferably, substantially the entire sequence encoding the ITR is used in the isolated nucleic acid, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of one in the art. (See, e.g., texts such as Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)).
  • An example of such a molecule employed in the present invention is an isolated nucleic acid comprising an expression cassette encoding a GJB2 protein, in which the expression cassette comprising the nucleotide sequences GJB2 protein and GJB2 gene regulatory elements (GREs) are flanked by the 5 ' and 3 ' AAV ITR sequences.
  • the AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types.
  • the isolated nucleic acid (e.g., the rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof.
  • the isolated nucleic acid comprises a region e.g., a first region) encoding an AAV2 ITR.
  • the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR.
  • the second AAV ITR has a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof.
  • the second AAV ITR is an AAV2 ITR.
  • the second ITR is a mutant ITR that lacks a functional terminal resolution site (TRS).
  • the term “lacking a terminal resolution site” can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non-synonymous mutation, or missense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ATRS ITR, or AfTR).
  • TRS terminal resolution site
  • an rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example, as described by McCarthy (2008) Molecular Therapy 16( 10): 1648- 1656.
  • the isolated nucleic acid comprises a 5 ' AAV2 ITR and a 3 ' AAV2 ITR.
  • AAV2 ITR nucleotide sequence is set forth in SEQ ID NO: 34:
  • exemplary 3 ' AAV2 ITR nucleotide sequence is set forth in SEQ ID NO: 35:
  • the isolated nucleic acid (e.g., rAAV vector) described herein comprises a 5' ITR sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34 or 106.
  • the isolated nucleic acid e.g., rAAV vector) described herein comprises a 3' ITR sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35 or 107.
  • the isolated nucleic acid (e.g., rAAV vector) described herein comprises a posttranscriptional response element.
  • posttranscriptional response element refers to a nucleic acid sequence that, when transcribed, adopts a tertiary structure that enhances expression of a gene.
  • posttranscriptional regulatory elements include, but are not limited to, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), mouse RNA transport element (RTE), constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), the CTE from the Mason-Pfizer monkey virus (MPMV), and the 5' untranslated region of the human heat shock protein 70 (Hsp70 5' UTR).
  • the isolated nucleic acid e.g., rAAV vector
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • the isolated nucleic acid e.g., rAAV vector
  • the isolated nucleic acid comprises a posttranscriptional response element having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 108.
  • An exemplary posttranscriptional response element is set forth in SEQ ID NO: 108: GATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTA TGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGG CCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTG GGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCTCCCTATTGCCA CGGCGGAACTCATCGCCGCCTGCCTGCCTTGCCCGCTGCTGGACAGGGGGGCTCGGCTGTTGGGCACT GACAATTCCGTGGTGTTGTCGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGC CACCTGGATTCTGCTCGCCTGTGTTGC CACCTGGATT
  • Expression control sequences include appropriate transcription initiation, termination; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability.
  • a polyadenylation sequence generally is inserted following the coding sequences and optionally before a 3 ' AAV ITR sequence.
  • a rAAV construct useful in the disclosure may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene.
  • the isolated nucleic acid e.g., rAAV vector) described herein comprises a polyadenylation signal sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 109.
  • An exemplary polyadenylation signal sequence is set forth in SEQ ID NO: 109: GTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTT GCCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAA AATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA
  • an AAV vector described herein comprises a GJB2 proximal promoter (e.g., SEQ ID NO: 102), a GJB2 5' UTR (e.g., SEQ ID NO: 103 and CC), a nucleotide sequence encoding a GJB2 gene product (e.g., SEQ ID NO: 2), a GJB2 3 ' UTR (e.g., SEQ ID NO: 32), a WPRE (e.g., SEQ ID NO: 108), and a bovine growth hormone poly A signal (e.g., SEQ ID NO: 109).
  • GJB2 proximal promoter e.g., SEQ ID NO: 102
  • GJB2 5' UTR e.g., SEQ ID NO: 103 and CC
  • a nucleotide sequence encoding a GJB2 gene product e.g., SEQ ID NO: 2
  • an AAV vector described herein comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 110.
  • An exemplary AAV vector sequence is set forth in SEQ ID NO: 110: GACCTCGAAGGGGACTTGGGGGGTTCGGGGCTTTCGGGGGCGGTCGGGGGTTCGCGGACCCG GGAAGCTCTGAGGACCCAGAGGCCGGGCGCGCTCCGCCCGCGGCGCCGCCCCCTCCGTAACT TTCCCAGTCTCCGAGGGAAGAGGCGGGGTGTGGGGTGCGGTTAAAAGGCGCCACGGCGGGAG ACAGGTGTTGCGGCCCCGCAGCGCCCGCGCGCTCCTCCCCGACTCGGAGCCCCTCGGCGG CGCCCGGCCCAGGACCCGCCTAGGAGCGCAGGAGCCCCAGCGCAGAGACCCCAACGCCGAGA CCCCCCCCGGCCCCGCCGCGCTTCCTCCCGACGCAGAGCAAACCGCCCAGAGTAGAAGCG GATCCGCCACCATGGATTGGGGCACGCTGCAGACGATCCTGGGGGGTGTGAACAAACACTCC ACCAGCATTGGAAAGATCTGGCTCACCGTCCTCTTCATTTT
  • an AAV vector described herein comprises a 5 ' ITR (e.g.,
  • SEQ ID NO: 106 a GJB2 proximal promoter e.g., SEQ ID NO: 102
  • GJB2 5' UTR e.g.,
  • SEQ ID NO: 103 and CC a nucleotide sequence encoding a GJB2 gene product (e.g., SEQ ID NO: 2), a GJB2 3 ' UTR (e.g., SEQ ID NO: 32), a WPRE (e.g., SEQ ID NO: 108), a bovine growth hormong poly A signal (e.g., SEQ ID NO: 109), and a 3 ' ITR (e.g., SEQ ID NO: 107).
  • GJB2 gene product e.g., SEQ ID NO: 2
  • GJB2 3 ' UTR e.g., SEQ ID NO: 32
  • WPRE e.g., SEQ ID NO: 108
  • a bovine growth hormong poly A signal e.g., SEQ ID NO: 109
  • 3 ' ITR e.g., SEQ ID NO: 107
  • an AAV vector described herein comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 111.
  • An exemplary AAV vector sequence is set forth in SEQ ID NO: 111:
  • an AAV vector described herein comprises 5 ' ITR, a GJB2 basal promoter, a 5 ' UTR (e.g., GJB2 exon 1 5 ' UTR), Kozak sequence, nucleotide sequence encoding a gene product (e.g., GJB2 or GFP), an optional HA tag, a 3 ' UTR (e.g., GJB2 exon 2 3 ' UTR), a WPRE, a bovine growth hormone poly A signal, and a 3 ' ITR (e.g., vector c70).
  • 5 ' ITR e.g., GJB2 exon 1 5 ' UTR
  • Kozak sequence e.g., nucleotide sequence encoding a gene product (e.g., GJB2 or GFP)
  • an optional HA tag e.g., GJB2 or GFP
  • a 3 ' UTR e.g., GJB2 ex
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36.
  • An exemplary nucleotide sequence for vector c70 encoding a mouse GJB2 protein with an HA tag is set forth in SEQ ID NO: 36 (mouse GJB2 coding sequence in boldface; HA tag underlined):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 61.
  • An exemplary nucleotide sequence for vector c70 encoding a human GJB2 protein with an HA tag is set forth in SEQ ID NO: 61 (human GJB2 coding sequence in boldface; HA tag underlined):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 62.
  • An exemplary nucleotide sequence for vector c70 encoding a mouse GJB2 protein with an HA tag is set forth in SEQ ID NO: 62 (mouse GJB2 coding sequence in boldface; no HA tag):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63.
  • An exemplary nucleotide sequence for vector c70 encoding a mouse GJB2 protein with a HA tag is set forth in SEQ ID NO: 63 (human GJB2 coding sequence in boldface; no HA tag): CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAG GCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTG GTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAAT TTAAAAGGATCTAGGTGAAGATCCTTTGATAATCTCATGACCAAAATCCCTTAACGTGAG TTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGTTTGCCGGATCAAGCTACCA
  • an AAV vector described herein comprises an AAV 5 ' ITR, a GJB2 GRE enhancer (hGJB2 GRE1 ), a GJB2 basal promoter, a GJB2 exon 1 5 ' UTR, Kozak sequence, nucleotide sequence encoding a gene product (e.g., GJB2 or GFP), a GJB2 exon 2 3 ' UTR, a WPRE, a bovine growth hormone poly A signal, and an AAV 3 ' ITR (e.g., vector c81.1).
  • a gene product e.g., GJB2 or GFP
  • GJB2 exon 2 3 ' UTR e.g., a WPRE
  • a bovine growth hormone poly A signal e.g., vector c81.
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64.
  • An exemplary nucleotide sequence for vector c81.1 encoding eGFP is set forth in SEQ ID NO: 64 (hGJB2 GRE1 underlined; eGFP coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65.
  • An exemplary nucleotide sequence for vector c81.1 encoding human GJB2 is set forth in SEQ ID NO: 65 hGJB2 GRE1 underlined; human GJB2 coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66.
  • An exemplary nucleotide sequence for vector c81.1 encoding mouse GJB2 is set forth in SEQ ID NO: 66 hGJB2 GRE1 underlined; mouse GJB2 coding sequence in bold face):
  • an AAV vector described herein comprises an AAV 5 ' ITR, a GJB2 GRE enhancer (hGJB2 GRE2), a GJB2 basal promoter, a GJB2 exon 1 5 ' UTR, Kozak sequence, nucleotide sequence encoding a gene product (e.g., GJB2 or GFP), a GJB2 exon 2 3 ' UTR, a WPRE, a bovine growth hormone poly A signal, and an AAV 3 ' ITR (e.g., vector c81.2).
  • a gene product e.g., GJB2 or GFP
  • GJB2 exon 2 3 ' UTR e.g., a WPRE
  • a bovine growth hormone poly A signal e.g., vector c81.2
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48.
  • An exemplary nucleotide sequence for vector c81.2 encoding eGFP is set forth in SEQ ID NO: 48 hGJB2 GRE2 underlined; eGFP coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67.
  • An exemplary nucleotide sequence for vector c81.2 encoding human GJB2 is set forth in SEQ ID NO: 67 hGJB2 GRE2 underlined; human GJB2 coding sequence in bold face): CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACG ACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACT GATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC TTCATTTTTAATTTAAAAGGATCTAGGTGA
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68.
  • An exemplary nucleotide sequence for vector c81.2 encoding mouse GJB2 is set forth in SEQ ID NO: 68 (hGJB2 GRE2 underlined; mouse GJB2 coding sequence in bold face):
  • an AAV vector described herein comprises an AAV 5 ' ITR, a GJB2 GRE enhancer (hGJB2 GRE3), a GJB2 basal promoter, a GJB2 exon 1 5 ' UTR, Kozak sequence, nucleotide sequence encoding a gene product (e.g., GJB2 or GFP), a GJB2 exon 2 3 ' UTR, a WPRE, a bovine growth hormone poly A signal, and an AAV 3 ' ITR (e.g., vector c.81.3).
  • a gene product e.g., GJB2 or GFP
  • GJB2 exon 2 3 ' UTR e.g., a WPRE
  • a bovine growth hormone poly A signal e.g., vector c.81.3
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49.
  • An exemplary nucleotide sequence for vector c.81.3 is set forth in SEQ ID NO: 49 (hGJB2 GRE3 underlined; eGFP coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70.
  • nucleotide sequence for vector c.81.3 is set forth in SEQ ID NO: 70 hGJB2 GRE3 underlined; human GJB2 coding sequence in bold face): CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACG ACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACT GATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTGATAATCT
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71.
  • An exemplary nucleotide sequence for vector c.81.3 is set forth in SEQ ID NO: 71 (hGJB2 GRE3 underlined; mouse GJB2 coding sequence in bold face):
  • an AAV vector described herein comprises an AAV 5 ' ITR, a GJB2 GRE enhancer (hGJB2 GRE4), a GJB2 basal promoter, a GJB2 exon 1 5 ' UTR, Kozak sequence, nucleotide sequence encoding a gene product (e.g., GJB2 or GFP), a GJB2 exon 2 3 ' UTR, a WPRE, a bovine growth hormone poly A signal, and an AAV 3 ' ITR (e.g., vector c.81.4).
  • a gene product e.g., GJB2 or GFP
  • GJB2 exon 2 3 ' UTR e.g., a WPRE
  • a bovine growth hormone poly A signal e.g., vector c.81.4
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72.
  • An exemplary nucleotide sequence for vector c.81.4 is set forth in SEQ ID NO: 72 hGJB2 GRE4 underlined; eGFP coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73.
  • nucleotide sequence for vector c.81.4 is set forth in SEQ ID NO: 73 hGJB2 GRE4 underlined; human GJB2 coding sequence in bold face): CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACG ACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACT GATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTGATAATTGATAAT
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74.
  • nucleotide sequence for vector c.81.4 is set forth in SEQ ID NO: 74 hGJB2 GRE4 underlined; mouse GJB2 coding sequence in bold face): CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACG ACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACT GATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTGATAATTGATAAT
  • an AAV vector described herein comprises an AAV 5 ' ITR, a GJB2 GRE enhancer (hGJB2 GRE5), a GJB2 basal promoter, a GJB2 exon 1 5 ' UTR, Kozak sequence, nucleotide sequence encoding a gene product (e.g., GJB2 or GFP), a GJB2 exon 2 3 ' UTR, a WPRE, a bovine growth hormone poly A signal, and an AAV 3 ' ITR (e.g., vector c.81.5).
  • a gene product e.g., GJB2 or GFP
  • GJB2 exon 2 3 ' UTR e.g., a WPRE
  • a bovine growth hormone poly A signal e.g., vector c.81.5
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50.
  • An exemplary nucleotide sequence for vector c.81.5 is set forth in SEQ ID NO: 50 hGJB2 GRE5 underlined; eGFP coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75.
  • An exemplary nucleotide sequence for vector c.81.5 is set forth in SEQ ID NO: 75 hGJB2 GRE5 underlined; human GJB2 coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.
  • An exemplary nucleotide sequence for vector c.81.5 is set forth in SEQ ID NO: 76 hGJB2 GRE5 underlined; mouse GJB2 coding sequence in bold face):
  • an AAV vector described herein comprises an AAV 5 ' ITR, a GJB2 GRE enhancer (hGJB2 GRE7), a GJB2 basal promoter, a GJB2 exon 1 5 ' UTR, Kozak sequence, nucleotide sequence encoding a gene product (e.g., GJB2 or GFP), a GJB2 exon 2 3 ' UTR, a WPRE, a bovine growth hormone poly A signal, and an AAV 3 ' ITR (e.g., vector c.81.7).
  • a gene product e.g., GJB2 or GFP
  • GJB2 exon 2 3 ' UTR e.g., a WPRE
  • a bovine growth hormone poly A signal e.g., vector c.81.7
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 51.
  • An exemplary nucleotide sequence for vector c.81.7 is set forth in SEQ ID NO: 51 (hGJB2 GRE7 underlined; eGFP coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77.
  • An exemplary nucleotide sequence for vector c.81.7 is set forth in SEQ ID NO: 77 hGJB2 GRE7 underlined; human GJB2 coding sequence in bold face):
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.
  • An exemplary nucleotide sequence for vector c.81.7 is set forth in SEQ ID NO: 78 (hGJB2 GRE7 underlined; mouse GJB2 coding sequence in bold face):
  • an AAV vector described herein comprises an AAV 5 ' ITR, a GJB2 GRE enhancer (hGJB2 GRE8), a GJB2 basal promoter, a GJB2 exon 1 5 ' UTR, Kozak sequence, nucleotide sequence encoding a gene product (e.g., GJB2 or GFP), a GJB2 exon 2
  • AAV 3 ' UTR a WPRE, a bovine growth hormone poly A signal, and an AAV 3 ' ITR (e.g., vector c.81.8).
  • an AVV vector described herein comprises a nucleotide sequence at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79.
  • An exemplary nucleotide sequence for vector c.81.8 is set forth in SEQ ID NO: 79 (hGJB2 GRE8 underlined; eGFP coding sequence in bold face):

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EP21867807.6A 2020-09-14 2021-09-14 Für gjb2 codierendes rekombinantes adeno-assoziiertes virus (raav) und verwendungen davon Pending EP4211151A1 (de)

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