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COCHLEAR OUTER HAIR CELL PROMOTERS AND USES THEREOF

20220288236 · 2022-09-15

    Inventors

    Cpc classification

    International classification

    Abstract

    The disclosure provides polynucleotides containing outer hair cell-specific promoters, as well as vectors containing the same, that can be used to promote expression of a transgene specifically in outer hair cells. The polynucleotides described herein may be operably linked to a transgene, such as a transgene encoding a therapeutic protein, so as to promote outer hair cell-specific expression of the transgene. The polynucleotides described herein may be operably linked to a therapeutic transgene and used for the treatment of subjects having or at risk of developing hearing loss.

    Claims

    1. A nucleic acid vector comprising a polynucleotide having at least 85% sequence identity to any one of SEQ ID NOs: 1-3.

    2. The nucleic acid vector of claim 1, wherein the polynucleotide has at least 85% sequence identity to SEQ ID NO: 1.

    3. The nucleic acid vector of claim 1, wherein the polynucleotide has at least 85% sequence identity to SEQ ID NO: 2.

    4. The nucleic acid vector of claim 1, wherein the polynucleotide has at least 85% sequence identity to SEQ ID NO: 3.

    5. The nucleic acid vector of any one of claims 1-4, wherein the polynucleotide is operably linked to a transgene.

    6. The nucleic acid vector of claim 5, wherein the transgene is a heterologous transgene.

    7. The nucleic acid vector of claim 5 or 6, wherein the transgene encodes a therapeutic protein, a short interfering RNA (siRNA), an antisense oligonucleotide (ASO), a nuclease, or is a microRNA.

    8. The nucleic acid vector of any one of claims 5-7, wherein the polynucleotide is capable of directing cochlear outer hair cell (OHC)-specific expression of the transgene in a mammalian OHC.

    9. The nucleic acid vector of claim 8, wherein the mammalian OHC is a human OHC.

    10. The nucleic acid vector of any one of claims 7-9, wherein the therapeutic protein is selected from the group consisting of Actin Gamma 1 (ACTG1), Fascin Actin-Bundling Protein 2, Retinal (FSCN2), Radixin (RDX), POU Class 4 Homeobox 3 (POU4F3), TRIO and F-Actin Binding Protein (TRIOBP), Taperin (TPRN), Xin Actin Binding Repeat Containing 2 (XIRP2), Atonal BHLH Transcription Factor 1 (ATOH1), Growth Factor Independent 1 Transcriptional Repressor (GFI1), Cholinergic Receptor Nicotinic Alpha 9 Subunit (CHRNA9), Cholinergic Receptor Nicotinic Alpha 10 Subunit (CHRNA10), Calcium and Integrin Binding Family Member 3 (CIB3), Cadherin 23 (CDH23), Protocadherin 15 (PCDH15), Kinocilin (KNCN), Pejvakin (DFNB59), Otoferlin (OTOF), MKRN2 Opposite Strand (MKRN2OS), LIM Homeobox Protein 3 (LHX3), Transmembrane Channel Like 1 (TMC1), Myosin 15 (MYO15), Myosin 7A (MYO7A), Myosin 6 (MYO6), Myosin IIIA (MYO3A), Myosin IIIB (MYO3B), Glutaredoxin Domain Containing Cysteine-Rich Protein 1 (GRXCR1), Protein Tyrosine Phosphatase, Receptor Type Q (PTPRQ), Late Cornified Envelope 6A (LCE6A), Lipoxygenase Homology Domain-containing Protein 1 (LOXHD1), ADP-Ribosyltransferase 1 (ART1), ATPase Plasma Membrane Ca2+ Transporting 2 (ATP2B2), Calcium and Integrin Binding Family Member 2 (CIB2), Calcium Voltage-Gated Channel Auxiliary Subunit Alpha2delta 4(CACNA2D4), Calcium Binding Protein 2 (CABP2), Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8), EPS8 Like 2 (EPS8L2), Espin (ESPN), Espin Like (ESPNL), Peripherin 2 (PRPH2), Stereocilin (STRC), Solute Carrier Family 8 Member A2 (SLC8A2), Zinc Finger CCHC-Type Containing Protein 12 (ZCCHC12), Leucine Rich Transmembrane and 0-methyltransferase Domain Containing (LRTOMT2, LRTOMT1), USH1 Protein Network Component Harmonin (USH1C), Solute Carrier Family 26 Member 5 (SLC26A5), Piezo Type Mechanosensitive Ion Channel Component 2 (PIEZO2), Extracellular Leucine Rich Repeat and Fibronectin Type III Domain Containing 1 (ELFN1), Tetratricopeptide Repeat Protein 24 (TTC24), Dystrotelin (DYTN), Kielin/Chordin-Like Protein (KCP), Coiled-coil Glutamate Rich Protein 2 (CCER2), Leucine-rich Repeat and Transmembrane Domain-containing protein 2 (LRTM2), Potassium Voltage-Gated Channel Subfamily A Member 10 (KCNA10), Clarin 1 (CLRN1), Clarin 2 (CLRN2), SKI Family Transcriptional Corepressor 1 (SKOR1), Tctex1 Domain Containing Protein 1 (TCTEX1 D1), Fc Receptor Like B (FCRLB), Solute Carrier Family 17 Member 8 (SLC17A8), Glutaredoxin Domain Containing Cysteine-Rich Protein 2 (GRXCR2), Brain-derived Neurotrophic Factor (BDNF), Serpin Family E Member 3 (SERPINE3), Nescient Helix-loop Helix 1 (NHLH1), Heat Shock Protein 70 (HSP70), Heat Shock Protein 90 (HSP90), Activating Transcription Factor 6 (ATF6), Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3 (PERK), Serine/Threonine-Protein Kinase/Endoribonuclease IRE1 (IRE1), Whirlin (WHRN), Oncomodulin (OCM), LIM Homeobox 1 (Isl1), Neurotrophin 3 (NTF3), Transmembrane and Tetratricopeptide Repeat Containing 4 (TMTC4), and Binding Immunoglobulin Protein (BIP).

    11. The nucleic acid vector of any one of claims 1-10, wherein the nucleic acid vector is selected from the group consisting of a viral vector, a plasmid, a cosmid, or an artificial chromosome.

    12. The nucleic acid vector of claim 11, wherein the nucleic acid vector is a viral vector selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, and a lentivirus.

    13. The nucleic acid vector of claim 12, wherein the viral vector is an AAV vector.

    14. The nucleic acid vector of claim 13, wherein the AAV vector has an AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ/8, DJ/9, 7m8, PHP.B, PHP.eb, or PHP.S capsid.

    15. A composition comprising the nucleic acid vector of any one of claims 1-14.

    16. The composition of claim 15, further comprising a pharmaceutically acceptable excipient.

    17. A polynucleotide having at least 85% sequence identity to any one of SEQ ID NOs: 1-3 operably linked to a transgene.

    18. The polynucleotide of claim 17, wherein the polynucleotide has at least 85% sequence identity to SEQ ID NO: 1.

    19. The polynucleotide of claim 17, wherein the polynucleotide has at least 85% sequence identity to SEQ ID NO: 2.

    20. The polynucleotide of claim 17, wherein the polynucleotide has at least 85% sequence identity to SEQ ID NO: 3.

    21. The polynucleotide of any one of claims 17-20, wherein the transgene is a heterologous transgene.

    22. The polynucleotide of claim 21, wherein the transgene encodes a therapeutic protein, an siRNA, an ASO, a nuclease, or is a microRNA.

    23. The polynucleotide of claim 22, wherein the therapeutic protein is selected from the group consisting of ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CHRNA10, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, SLC26A5, PIEZO2, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, WHRN, OCM, ISL1, NTF3, TMTC4, and BIP.

    24. A cell comprising the polynucleotide of any one of claims 17-23 or the nucleic acid vector of any one of claims 1-14.

    25. The cell of claim 24, wherein the cell is a mammalian OHC.

    26. The cell of claim 25, wherein the mammalian OHC is a human OHC.

    27. A method of expressing a transgene in a mammalian OHC, comprising contacting the mammalian OHC with the nucleic acid vector of any one of claims 1-14 or the composition of claim 15 or 16.

    28. The method of claim 27, wherein the transgene is not substantially expressed in inner ear cells that are not OHCs.

    29. A method of treating a subject having or at risk of developing hearing loss, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 1-14 or the composition of claim 15 or 16.

    30. The method of claim 29, wherein the hearing loss is genetic hearing loss.

    31. The method of claim 30, wherein the genetic hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss.

    32. The method of claim 29, wherein the hearing loss is acquired hearing loss.

    33. The method of claim 32, wherein the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss.

    34. A method of promoting OHC regeneration in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 1-14 or the composition of claim 15 or 16.

    35. A method of preventing or reducing ototoxic drug-induced OHC damage or death in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 1-14 or the composition of claim 15 or 16.

    36. The method of claim 33 or 35, wherein the ototoxic drug is selected from the group consisting of aminoglycosides, antineoplastic drugs, ethacrynic acid, furosemide, salicylates, and quinine.

    37. A method of treating a subject having or at risk of developing tinnitus, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 1-14 or the composition of claim 15 or 16.

    38. A method of preventing or reducing OHC damage or death in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 1-14 or the composition of claim 15 or 16.

    39. A method of increasing OHC survival in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 1-14 or the composition of claim 15 or 16.

    40. The method of any one of claims 29-39, wherein the method further comprises evaluating the hearing of the subject prior to administering the nucleic acid vector or composition.

    41. The method of any one of claims 29-40, wherein the method further comprises evaluating the hearing of the subject after administering the nucleic acid vector or composition.

    42. The method of any one of claims 29-41, wherein the nucleic acid vector or composition is locally administered.

    43. The method of any one of claims 29-42, wherein the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, prevent or reduce tinnitus, delay the development of hearing loss, slow the progression of hearing loss, improve hearing, improve hair cell function, prevent or reduce hair cell damage, prevent or reduce hair cell death, promote or increase hair cell survival, or increase hair cell numbers.

    44. The method of any one of claims 29-43, wherein the subject is a human.

    45. A kit comprising the nucleic acid vector of any one of claims 1-14 or the composition of claim 15 or 16.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0062] FIGS. 1A-1B are a series of fluorescent images of mouse cochlea transduced with either an adeno-associated virus (AAV) vector expressing green fluorescent protein (GFP) under the control of the ubiquitous cytomegalovirus (CMV) promoter (FIG. 1A), or an AAV vector expressing GFP under control of an Oncomodulin (OCM) promoter (SEQ ID NO: 1; FIG. 1B). Native GFP fluorescence is shown. Using a ubiquitous promoter, AAV-CMV-GFP induced GFP expression in many cell types within the cochlea including inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, mesenchymal cells, and glia (FIG. 1A). Using an OHC-specific promoter, AAV-OCM (SEQ ID NO: 1)-GFP induced GFP expression exclusively in OHCs (FIG. 1B).

    DETAILED DESCRIPTION

    [0063] Described herein are compositions and methods for inducing transgene expression specifically in cochlear outer hair cells (OHCs). The invention features OHC-specific promoters that are capable of expressing a transgene specifically in OHCs the inner ear. The invention also features nucleic acid vectors containing said promoters operably linked to polynucleotides encoding polypeptides. The compositions and methods described herein can be used to express polynucleotides encoding proteins (e.g., therapeutic proteins, reporter proteins, or other proteins of interest) specifically in OHCs, and, therefore, the compositions described herein can be administered to a subject (such as a mammalian subject, for instance, a human) to treat disorders caused by dysfunction of OHCs, such as hearing loss.

    Hair cells

    [0064] Hair cells are sensory cells of the auditory and vestibular systems that reside in the inner ear. Cochlear hair cells are the sensory cells of the auditory system, and are made up of two main cell types: inner hair cells, which are responsible for sensing sound, and OHCs, which are thought to amplify low-level sound. Hair cells are named for the stereocilia that protrude from the apical surface of the cell, forming a hair cell bundle. Deflection of the stereocilia (e.g., by sound waves in cochlear hair cells) leads to the opening of mechanically gated ion channels, which allows hair cells to release neurotransmitters to activate nerves, thereby converting mechanical sound signals into electrical signals that can be transmitted to the brain. Cochlear hair cells are essential for normal hearing, and damage to cochlear hair cells and genetic mutations that disrupt cochlear hair cell function are implicated in hearing loss and deafness. Gene therapy has recently emerged as an attractive therapeutic approach for treating hearing loss; however, the field lacks methods for specifically targeting the nucleic acid vectors used in gene therapy to hair cells.

    [0065] The present invention is based, in part, on the discovery of genes that are specifically expressed in cochlear OHCs as compared to other cochlear cell types. The promoters of these genes, therefore, can induce gene expression specifically in OHCs of the inner ear. The compositions and methods described herein can, thus, be used to express a gene of interest in OHCs such as, for example, a gene implicated in OHC development, OHC function, OHC fate specification, OHC regeneration, OHC survival, or OHC maintenance, or a gene known to be disrupted, e.g., mutated, in subjects with hearing loss, to treat subjects having or at risk of developing hearing loss (e.g., sensorineural hearing loss).

    Oncomodulin

    [0066] The present invention is based, in part, on the discovery of a region of 1,140 base pairs (bp) located upstream of the OCM translation start site that is sufficient for driving gene expression in outer hair cells. The compositions and methods described herein can, thus, be used to express a gene of interest in OHCs (e.g., a gene implicated in OHC cell development, function, cell fate specification, regeneration, survival, or maintenance, or a gene known to be disrupted, e.g., mutated, in subjects with hearing loss) to treat subjects having or at risk of developing hearing loss (e.g., sensorineural hearing loss).

    [0067] The compositions and methods described herein include OCM promoters listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3) that are capable of expressing a transgene specifically in OHCs, such as polynucleotide sequences that have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of SEQ ID NOs: 1-3). The polynucleotides described herein can include regions located both upstream and downstream of the TSS of the OCM gene or may include only regions upstream of the TSS of the OCM gene.

    [0068] Exemplary promoter sequences for OCM are listed in Table 2.

    TABLE-US-00002 TABLE 2 OCM promoter sequences SEQ Description of promoter ID NO: sequence Promoter sequence 1 Murine OCM promoter AGCAGGTTTGTTACAGAAACCTTAGTTAAGGTTTGTTGAGG sequence (1140 bp) GTTTTTTTTCTCTCTCTCTCTCTTAATTGGCTGTCCCAATCC ATCCTTCTATAAATAGAAAAGAGAGACAGGGAGTGTGTGTG GTTTCATTACTAAGGTAAAGACACTTGAGCTACACACACTT GATCCCTGAACATGAAATCTAAGAGGTTGAACGATCACAGT TTCAGGACTATATAAGGTGGTGAAAGACCATCTGCTTCGTT TTTCTGTTTGTTCCTACAACTCTTTCCCTCCGCTTGATTTTA ACTCTAAATTGGTGAGTAGCTGGTGGGCTCACCAGACTCC GAGATCCTCTTCTCTGCACGCACTGTATTAGACTTGGCACC CGGGAGGATTTTCACCTCTGCTGCATGGGCTAATCTTCCA CAAGGGATCTGTGGTATTGCAATCTCGGGTTGATGCATGA CGGTGATGTTGTGTTTATAGCATGGCTAAGGTTTAGCTGCC TATGATGATTGGTTAGGGAAGGATAATTTTTGCTAGAAGAT TGGACTTTAGGGAAAAAAAACCCCACTTTTATTTGCTTTTAG AATTTTAAAAGACTGGGCCATGTAGCTCAGGCTGGTTTGGA GTTCATTATGTAGTCAAGGATGCTCTTGGACTCTTTAGCAT CCTCCTCCTCCTCTTTTTCCTCCTCCTCCTTCTTGTTCTTCT TCTTGTTCTTCCTCTTCCCCTTCTCTTCCCCCTTCTCTCCCT CTTCCTCCTCTTCCTCCTCCTTCTTGTTCTTCTTCCTCTTCC CCTTCTCCTCTCCCCCTTGTCCTCCTCTTCCTTCTCCTCCT CCTCCTCTTCTTCTTTCTGAGTACCAAGATTGCAAGTGTGC ACACGATGACCAGCTTGGTCTTTCTTTGTCTTTTTTTTTTAA CTTCAATTTTGGAGTGAATTCAAGAGCAACCATGTAGTCAA GAGGTGGCTGGAGTCTTTTCTGTATCTGGGTTTGGTTTAGT ACTCTGCCCCATCACTTAACAGGTCCTTATGGCCACATCTT AAAAAAATTCTAGAGATACACGGTGTCGGTGAGTGGCTGA GAATGTGTGGTCTTCCCATTTCTCTGTCACCGTGGCTCACA TCTTGTTTCCTCTGTTCGGCCAGGTAGAAA 2 Human OCM promoter TTTTACCACAATAATTAAAAAGAACAGTCTAGCACAGTGCT sequence containing a GGCCATATAAAGGCTCAATAAATGTTTGCTGAAAGTTAAAA polynucleotide located -2 kb AAAAAAAAAAAAAAAAAAAAGCCAGGCGCAGTGGTTCATTC to +0.5 kb to the TSS of the CTGTAATCCCAGCACTTTAGGAGGATGAGGTGGGAGAATT human OCM gene ACTTGAGCCCAGGAGTTCGAGACCAGCCTAGGCAACATGG CAAAACCCTGTCAAAACCCTGTCTCTCCAAAAAATATGCAT ATTTAAAAAATTAGCCAGGCATGGTGGTGTGTGCCTGTAGT ACCAGCTACTCGGGAGACTGAAGTGGGAGGATCGCTTGAG CCTGGGAGGTCAAGGCTGCAATGAGCTGAGATCGTGCCAC TGCACTCCAGCCGGGGCAACAGAGCAAGACCCTGTCACAA CAGAAACAAAATCTTGAGGTGTCTAGTCCTGGCCTCAGCCT CAGAATATTTGTTTCTGAACATGTTAGTTTTGGGGGTTGGG GATGCTGGTTTGATTTCCTCCTTTTTGCCTTTTGAGTGTGTG CAATTTATGGTATAGCTGGGAAACGTCAAAGTCAAGAGTTT TGTAGGAAAGTCACGTCACTTAGCCCTGTCTCCTGTGCCG GGTGAGACCTGTGTGTGCACTTGGTGACAATGGCTTTGAG TCTGTCAACTCCAGACTGAGGTCAGCCTTACACACCCATAG TTCCCAAAGCTGAAAACAGGCCTGCCTCCAACGGTACCTG CTAATATCAGGGGAGCCTTTTCAGCTTACAGAGCACCCTGT ATGTGTTTGTCTTAGTTCAGGCCACCATCTCCACCTTACCA GGCATCTAGAACCTTCTCCACACTTTGCCAACAGGGTTCGT TTGCAGAATTGAAATCTTAGTTAAGGTTTGTTGAAGTTTGTT GTTGTTTTTTTTTTTTTTTTACAATTGGCTGTTCCCACCCACA TTCCCTTGAGACATAAATAGAAAAAAAAAAAAAAAGAGGTTT CATGAGTAAGACAAGACATTTGAGCTGCATCCACTTGATCC TTGAAAAGGAAATCTAAGAGGTTGTAACTATCACTTTTTCTA GCCTATATAAGGTAGGTCAGTAAGGTAGCAAAAACACATCT GTTGTTTTGCTCCTTCAACTCTTTTTCCTGATTCTTCCTGGG GGGAAACCGAAAACGGTGAGTAACTGGTGGACACATCAGA CCCCAGACTCTTTTCTTCACTGCATGCATTCATATTAGGCT CAGGTGCTTAGACTCCTGTTTTCCGGTGGCTCTGACACCT GGAAGGATTTTAATCTCTGGGAGATGGGCTTTTCATCCATC TGCTTCCCACCTTTCAGGACAGGTGCATGCCTTCTTCCACA GAATGTCTGCAAGCAGCCCAAACTGTATCCTTTCCCACGTG GAATTTGCAACATTGCATCTCTCGGGCTGCTGTAGGAAAAT GCCAGTGCATGTGTAACATGGTTTACGGCTGCCTATGCAA ATGACTGATTATGTCAGTATAATTTTTATAAGAAAACAATTG AATCCTTCTTTGGGTCATTTTTTTTTTCCATTTTTGGCATGTA TTCAAAAGAAGGCTCTGAGACAAAAAAGGCTGGGGTGTTTT CCGTATCTGGTTTTAATTTGGATATTCTGTCCCGTCACTTAA TACAAAACCATGCTTATCACATTTTAAAAATTCTAGACAGGC CTGGCTCGGTGGCTTGCATCTGTCATCCCAGCACTTTGTG AGGCCAAGGCAGGCAGATCACCTGAGGTCAGGAGCTCAA GACCAGCCTGGCCAACATGGCAAAACCCCGTCTCTACTAA AAACACAAAAATTAGCCAGGCATGGTAGTGCGCACCTGTA ATCCCAGCTACTGGGAAGGCTTAGGCAGGAGAATCACTTG AGCCCAGGAGGCGGAGGTTGCGGTGAGCCGAGATCACGC TCTTGCACTCCAGCCTGGGTGACAGAGTGAGACTCCGTCT TAATTTAAAAAAAAAAATAATCTAGACACACATACAGTTTCA GTGGGCCTGGGAAGATGTGTTTCCCCTGGATGTGCACATT CCTGTTTGTGGCTTATCGCCTCTCATTTATTCTGTGTGAGT AGGTAGAAAATGAGCATCACGGACGTGCTCAGTGCTGACG ACATTGCAGCAGCGCTCCAGGAATGCCGAGGTAGAGGGG ACGTGAGGCGGGGGTGGGATTTCCTCACAGCTTTGCACCT CCAGCGAGTCAACACAAAATCAAAATGTAGGCCAGGCGGC CAGACGCAGTGGCTCACACCTGTAATCCCAGCACTTTGGG AGGCCGAGGCGGGTGGATCACGAGGTCAGGAGTTCGAGA CCAGCCTGGCCAAGATGGTGAAACCCCATCTCTACTAAAA ATACAAAAAAATTAACCGGGCGTGGTGGTGGGTGCCTGTA ATCCCAGCTACTCGGGAGGCTGAGGCAGAGAATTGCTTGA ACCCGGGAGGCAGAAGTTGCAGTGAGCTGAGATCATGCCA CTGCACTCCAGCCTGGGCA 3 Human OCM promoter GTTCCCAAAGCTGAAAACAGGCCTGCCTCCAACGGTACCT sequence containing GCTAATATCAGGGGAGCCTTTTCAGCTTACAGAGCACCCT regions from SEQ ID NO: 2 GTATGTGTTTGTCTTAGTTCAGGCACCTTACCAGGCATCTA that are conserved across GAACCTTCTCCACACTTTGCCAACAGGGTTCGTTTGCAGAA mammalian species TTGAAATCTTAGTTAAGGTTTGTTGAAGTTTGTTGTTGTTTT TTTTTTTTTTTTACAATTGGCTGTTCCCACCCACATTCCCTT GAGACATAAATAGAAAAAAAAAAAAAAAGAGGTTTCATGAG TAAGACAAGACATTTGAGCTGCATCCACTTGATCCTTGAAA AGGAAATCTAAGAGGTTGTAACTATCACTTTTTCTAGCCTAT ATAAGGTAGGTCAGTAAGGTAGCAAAAACACATCTGTTGTT TTGCTCCTTCAACTCTTTTTCCTGATTCTTCCTGGGGGGAA ACCGAAAACGGTGAGTAACTGGTGGACACATCAGACCCCA GACTCTTTTCTTCACTGCATGCATTCATATTAGGCTCAGGT GCTTAGACTCCTGTTTTCCGGTTTACGGCTGCCTATGCAAA TGACTGATTATGTCAGTATAATTTTTATAAGAAAACAATTGA ATCCTTCTTTGGGTCATTTTTTTTTTCCATTTTTGGCATGTAT GTGCACATTCCTGTTTGTGGCTTATCGCCTCTCATTTATTCT GTGTGAGTAGGTAGAAAATGAGCATCACGGACGTGCTCAG TGCTGACGACATTGCAGCAGCGCTCCAGGAATGCCGAGGT AGAGGGGACGTGAGGCGGGGGTGGGATTTCCTCACAGCT TTGCACCTCCAGC

    [0069] The foregoing polynucleotides can be included in a nucleic acid vector and operably linked to a transgene to express the transgene specifically in OHCs. In some embodiments, the transgene encodes a protein that is implicated in OHC function, OHC development, OHC fate specification, OHC regeneration, OHC survival, or OHC maintenance, or the transgene is the wild-type version of a gene that has been found to be mutated in subjects having hearing loss, deafness, auditory neuropathy, or tinnitus. According to the methods described herein, a subject can be administered a composition containing one or more of the foregoing polynucleotides (e.g., an OHC-specific promoter, e.g., any one the polynucleotide sequences listed in Table 2 (e.g., SEQ ID NOs: 1-3)) operably linked to a transgene encoding a therapeutic protein for the treatment of hearing loss, deafness, auditory neuropathy, or tinnitus. In some embodiments, the transgene encodes a protein selected from the group including Actin Gamma 1 (ACTG1), Fascin Actin-Bundling Protein 2, Retinal (FSCN2), Radixin (RDX), POU Class 4 Homeobox 3 (POU4F3), TRIO and F-Actin Binding Protein (TRIOBP), Taperin (TPRN), Xin Actin Binding Repeat Containing 2 (XIRP2), Atonal BHLH Transcription Factor 1 (ATOH1), Growth Factor Independent 1 Transcriptional Repressor (GFI1), Cholinergic Receptor Nicotinic Alpha 9 Subunit (CHRNA9), Cholinergic Receptor Nicotinic Alpha 10 Subunit (CHRNA10), Calcium and Integrin Binding Family Member 3 (CIB3), Cadherin 23 (CDH23), Protocadherin 15 (PCDH15), Kinocilin (KNCN), Pejvakin (DFNB59), Otoferlin (OTOF), MKRN2 Opposite Strand (MKRN2OS), LIM Homeobox Protein 3 (LHX3), Transmembrane Channel Like 1 (TMC1), Myosin 15 (MYO15), Myosin 7A (MYO7A), Myosin 6 (MYO6), Myosin IIIA (MYO3A), Myosin IIIB (MYO3B), Glutaredoxin Domain Containing Cysteine-Rich Protein 1 (GRXCR1), Protein Tyrosine Phosphatase, Receptor Type Q (PTPRQ), Late Cornified Envelope 6A (LCE6A), Lipoxygenase Homology Domain-containing Protein 1 (LOXHD1), ADP-Ribosyltransferase 1 (ART1), ATPase Plasma Membrane Ca2+Transporting 2 (ATP2B2), Calcium and Integrin Binding Family Member 2 (CIB2), Calcium Voltage-Gated Channel Auxiliary Subunit Alpha2delta 4(CACNA2D4), Calcium Binding Protein 2 (CABP2), Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8), EPS8 Like 2 (EPS8L2), Espin (ESPN), Espin Like (ESPNL), Peripherin 2 (PRPH2), Stereocilin (STRC), Solute Carrier Family 8 Member A2 (SLC8A2), Zinc Finger CCHC-Type Containing Protein 12 (ZCCHC12), Leucine Rich Transmembrane and O-methyltransferase Domain Containing (LRTOMT2, LRTOMT1), USH1 Protein Network Component Harmonin (USH1C), Solute Carrier Family 26 Member 5 (SLC26A5), Piezo Type Mechanosensitive Ion Channel Component 2 (PIEZO2), Extracellular Leucine Rich Repeat and Fibronectin Type III Domain Containing 1 (ELFN1), Tetratricopeptide Repeat Protein 24 (TTC24), Dystrotelin (DYTN), Kielin/Chordin-Like Protein (KCP), Coiled-coil Glutamate Rich Protein 2 (CCER2), Leucine-rich Repeat and Transmembrane Domain-containing protein 2 (LRTM2), Potassium Voltage-Gated Channel Subfamily A Member 10 (KCNA10), Clarin 1 (CLRN1), Clarin 2 (CLRN2), SKI Family Transcriptional Corepressor 1 (SKOR1), Tctex1 Domain Containing Protein 1 (TCTEX1 D1), Fc Receptor Like B (FCRLB), Solute Carrier Family 17 Member 8 (SLC17A8), Glutaredoxin Domain Containing Cysteine-Rich Protein 2 (GRXCR2), Brain-derived Neurotrophic Factor (BDNF), Serpin Family E Member 3 (SERPINE3), Nescient Helix-loop Helix 1 (NHLH1), Heat Shock Protein 70 (HSP70), Heat Shock Protein 90 (HSP90), Activating Transcription Factor 6 (ATF6), Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3 (PERK), Serine/Threonine-Protein Kinase/Endoribonuclease IRE1 (IRE1), Whirlin (WHRN), Oncomodulin (OCM), LIM Homeobox 1 (1511), Neurotrophin 3 (NTF3), Transmembrane and Tetratricopeptide Repeat Containing 4 (TMTC4), and Binding Immunoglobulin Protein (BIP).

    Expression of Exogenous Polynucleotides in Mammalian Cells

    [0070] Mutations in a variety of genes, such as MYO7A, POU4F3, SLC17A8, and TMC1, have been linked to sensorineural hearing loss. The compositions and methods described herein can be used to induce or increase the expression of proteins encoded by genes of interest (e.g., the wild-type form of genes implicated in hearing loss, or genes involved in OHC development, OHC function, OHC fate specification, OHC regeneration, OHC survival, or OHC maintenance) specifically in, e.g., OHCs by administering a nucleic acid vector that contains an OHC-specific promoter sequence (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3)) operably linked to a polynucleotide sequence that encodes a protein of interest. A wide array of methods has been established for the delivery of proteins to mammalian cells and for the stable expression of genes encoding proteins in mammalian cells.

    [0071] Proteins that can be expressed in connection with the compositions described herein (e.g., when the transgene encoding the protein is operably linked to an OHC-specific promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3))) are proteins that are expressed in healthy OHCs (e.g., proteins that play a role in OHC development, OHC function, OHC regeneration, OHC fate specification, OHC survival, or OHC maintenance, or proteins that are deficient in subjects with sensorineural hearing loss) or other proteins of interest. Proteins that can be expressed in hair cells using the compositions and methods described herein include ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CHRNA10, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, SLC26A5, PIEZO2, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, WHRN, OCM, ISL1, NTF3, TMTC4, and BIP. The polynucleotides (e.g., OHC-specific promoters) described herein can also be used to express a short interfering RNA (siRNA), an antisense oligonucleotide (ASO), a nuclease (e.g., CRISPR Associated Protein 9 (Cas9), Transcription Activator-Like Effector Nuclease (TALEN), Zinc Finger Nuclease (ZFN), or guide RNA (gRNA)), or a microRNA in OHCs.

    Polynucleotides Encoding Proteins of Interest

    [0072] One platform that can be used to achieve therapeutically effective intracellular concentrations of proteins of interest in mammalian cells is via the stable expression of the gene encoding the protein of interest (e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell, or by episomal concatemer formation in the nucleus of a mammalian cell). The gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein. In order to introduce exogenous genes into a mammalian cell, genes can be incorporated into a vector. Vectors can be introduced into a cell by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and by encapsulation of the vector in a liposomes. Examples of suitable methods of transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. Such methods are described in more detail, for example, in Green, et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014); and Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

    [0073] Proteins of interest can also be introduced into a mammalian cell by targeting a vector containing a gene encoding a protein of interest to cell membrane phospholipids. For example, vectors can be targeted to the phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to a VSV-G protein, a viral protein with affinity for all cell membrane phospholipids. Such a construct can be produced using methods well known to those of skill in the field.

    [0074] Recognition and binding of the polynucleotide encoding a protein of interest by mammalian RNA polymerase is important for gene expression. As such, one may include sequence elements within the polynucleotide that exhibit a high affinity for transcription factors that recruit RNA polymerase and promote the assembly of the transcription complex at the transcription initiation site. Such sequence elements include, e.g., a mammalian promoter, the sequence of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters have been described in Smith, et al., Mol. Sys. Biol., 3:73, online publication, the disclosure of which is incorporated herein by reference. The promoter used in the methods and compositions described herein is an OHC-specific promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3)).

    [0075] Once a polynucleotide encoding a protein of interest has been incorporated into the nuclear DNA of a mammalian cell, the transcription of this polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the mammalian cell to an external chemical reagent, such as an agent that modulates the binding of a transcription factor and/or RNA polymerase to the mammalian promoter and thus regulates gene expression. The chemical reagent can serve to facilitate the binding of RNA polymerase and/or transcription factors to the mammalian promoter, e.g., by removing a repressor protein that has bound the promoter. Alternatively, the chemical reagent can serve to enhance the affinity of the mammalian promoter for RNA polymerase and/or transcription factors such that the rate of transcription of the gene located downstream of the promoter is increased in the presence of the chemical reagent. Examples of chemical reagents that potentiate polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, Calif.) and can be administered to a mammalian cell in order to promote gene expression according to established protocols.

    [0076] Other DNA sequence elements that may be included in polynucleotides for use in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce a conformational change in the polynucleotide containing the gene of interest such that the DNA adopts a three-dimensional orientation that is favorable for binding of transcription factors and RNA polymerase at the transcription initiation site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode a protein of interest and additionally include a mammalian enhancer sequence. Many enhancer sequences are now known from mammalian genes, and examples include enhancers from the genes that encode mammalian globin, elastase, albumin, a-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those that are derived from the genetic material of a virus capable of infecting a eukaryotic cell. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription include the CMV enhancer and RSV enhancer. An enhancer may be spliced into a vector containing a polynucleotide encoding a protein of interest, for example, at a position 5′ or 3′ to this gene. In a preferred orientation, the enhancer is positioned at the 5′ side of the promoter, which in turn is located 5′ relative to the polynucleotide encoding a protein of interest.

    [0077] The nucleic acid vectors containing an OHC-specific promoter described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the mRNA level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cell. The addition of the WPRE to a vector can result in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo.

    [0078] In some embodiments, the nucleic acid vectors containing an OHC-specific promoter described herein include a reporter sequence, which can be useful in verifying the expression of a gene operably linked to an OHC-specific promoter, for example, in cells and tissues (e.g., in OHCs). Reporter sequences that may be provided in a transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. When associated with regulatory elements that drive their expression, such as an OHC-specific promoter, the reporter sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for β-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.

    Methods for the Delivery of Exogenous Polynucleotides to Target Cells

    [0079] Techniques that can be used to introduce a transgene, such as a transgene operably linked to an OHC-specific promoter described herein, into a target cell (e.g., a mammalian cell) are well known in the art. For instance, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous polynucleotides. Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection™ and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.

    [0080] Additional techniques useful for the transfection of target cells include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of polynucleotides into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

    [0081] Lipofection represents another technique useful for transfection of target cells. This method involves the loading of polynucleotides into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous polynucleotides, for instance, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for instance, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign polynucleotides include contacting a cell with a cationic polymer-polynucleotide complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane include activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) polyethylenimine, and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for instance, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of polynucleotides. This technology is described in detail, for instance, in US 2010/0227406, the disclosure of which is incorporated herein by reference.

    [0082] Another useful tool for inducing the uptake of exogenous polynucleotides by target cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.

    [0083] Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107: 1870 (2010), the disclosure of which is incorporated herein by reference.

    [0084] Magnetofection can also be used to deliver polynucleotides to target cells. The magnetofection principle is to associate polynucleotides with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications. Their association with the gene vectors (DNA, siRNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

    [0085] Another useful tool for inducing the uptake of exogenous polynucleotides by target cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane to permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

    [0086] Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein. For instance, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

    Vectors for Delivery of Exogenous Polynucleotides to Target Cells

    [0087] In addition to achieving high rates of transcription and translation, stable expression of an exogenous gene in a mammalian cell can be achieved by integration of the polynucleotide containing the gene into the nuclear genome of the mammalian cell. A variety of vectors for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of a mammalian cell have been developed. Examples of expression vectors are described in, e.g., Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, Mass., 2006). Expression vectors for use in the compositions and methods described herein contain an OHC-specific promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3)) operably linked to a polynucleotide sequence that encodes a protein of interest, as well as, e.g., additional sequence elements used for the expression of these agents and/or the integration of these polynucleotide sequences into the genome of a mammalian cell. Vectors that can contain a hair cell-specific promoter operably linked to a transgene encoding a protein of interest include plasmids (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmids (e.g., pWE or sCos vectors), artificial chromosomes (e.g., a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC)), and viral vectors. Certain vectors that can be used for the expression of a protein of interest include plasmids that contain regulatory sequences, such as enhancer regions, which direct gene transcription. Other useful vectors for expression of a protein of interest contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5′ and 3′ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

    Viral Vectors for Polynucleotide Delivery

    [0088] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of a gene of interest into the genome of a target cell (e.g., a mammalian cell, such as a human cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors include a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, U.S. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.

    AAV Vectors for Polynucleotide Delivery

    [0089] In some embodiments, polynucleotides of the compositions and methods described herein are incorporated into rAAV vectors and/or virions in order to facilitate their introduction into a cell. rAAV vectors useful in the compositions and methods described herein are recombinant polynucleotide constructs that include (1) an OHC-specific promoter described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3)), (2) a heterologous sequence to be expressed, and (3) viral sequences that facilitate integration and expression of the heterologous genes. The viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion. In typical applications, the transgene encodes a protein that can promote hair cell development, hair cell function, hair cell regeneration, hair cell fate specification, hair cell survival, or hair cell maintenance, or a wild-type form of a hair cell protein that is mutated in subjects with forms of hereditary hearing loss that may be useful for improving hearing in subjects carrying mutations that have been associated with hearing loss or deafness,. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs can be AAV2 ITRs. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

    [0090] The polynucleotides and vectors described herein (e.g., an OHC-specific promoter operably linked to a transgene encoding a protein of interest) can be incorporated into a rAAV virion in order to facilitate introduction of the polynucleotide or vector into a cell. The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for instance, in U.S. Pat. Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

    [0091] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ/8, DJ/9, 7m8, PHP.B, PHP.eb, and PHP.S. For targeting hair cells, AAV1, AAV2, AAV2quad(Y-F), AAV6, AAV8, AAV9, Anc80, Anc80L65, DJ/9, 7m8, and PHP.B may be particularly useful. Serotypes evolved for transduction of the retina may also be used in the methods and compositions described herein. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for instance, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

    [0092] Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for instance, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

    [0093] AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

    Pharmaceutical Compositions

    [0094] The polynucleotides described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3)) may be operably linked to a transgene (e.g., a transgene encoding a protein of interest) and incorporated into a vehicle for administration into a patient, such as a human patient suffering from sensorineural hearing loss. Pharmaceutical compositions containing vectors, such as viral vectors, that contain a polynucleotide described herein operably linked to a therapeutic transgene can be prepared using methods known in the art. For example, such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.

    [0095] Mixtures of nucleic acid vectors (e.g., viral vectors) containing a polynucleotide described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3)) operably linked to a transgene) may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

    [0096] For example, a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition may be formulated to contain a synthetic perilymph solution. An exemplary synthetic perilymph solution includes 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl.sub.2, 1-10 mM glucose, and 2-50 mM HEPEs, with a pH between about 6 and 9 and an osmolality of about 300 mOsm/kg. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

    Methods of Treatment

    [0097] The compositions described herein may be administered to a subject having or at risk of developing sensorineural hearing loss by a variety of routes, such as local administration to the inner ear (e.g., administration into the perilymph or endolymph, such as through the oval window, round window, or semicircular canal (e.g., horizontal canal), or by transtympanic or intratympanic injection, e.g., administration to an OHC), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most suitable route for administration in any given case will depend on the particular composition administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patients age, body weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. Compositions may be administered once, or more than once (e.g., once annually, twice annually, three times annually, bi-monthly, or monthly).

    [0098] Subjects that may be treated as described herein are subjects having or at risk of developing sensorineural hearing loss. The compositions and methods described herein can be used to treat subjects having or at risk of developing damage to OHCs (e.g., damage related to acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging), subjects having or at risk of developing sensorineural hearing loss, deafness, or auditory neuropathy, subjects having tinnitus (e.g., tinnitus alone, or tinnitus that is associated with sensorineural hearing loss), subjects having a genetic mutation associated with hearing loss, or subjects with a family history of hereditary hearing loss, deafness, auditory neuropathy, or tinnitus. In some embodiments, the disease associated with damage to or loss of hair cells (e.g., OHCs) is an autoimmune disease or condition in which an autoimmune response contributes to hair cell damage or death. Autoimmune diseases linked to sensorineural hearing loss include autoimmune inner ear disease (AIED), polyarteritis nodosa (PAN), Cogan's syndrome, relapsing polychondritis, systemic lupus erythematosus (SLE), Wegener's granulomatosis, Sjögren's syndrome, and Behcet's disease. Some infectious conditions, such as Lyme disease and syphilis can also cause sensorineural hearing loss (e.g., by triggering autoantibody production). Viral infections, such as rubella, cytomegalovirus (CMV), lymphocytic choriomeningitis virus (LCMV), HSV types 1&2, West Nile virus (WNV), human immunodeficiency virus (HIV) varicella zoster virus (VZV), measles, and mumps, can also cause sensorineural hearing loss. In some embodiments, the subject has hearing loss that is associated with or results from loss of OHCs. The methods described herein may include a step of screening a subject for one or more mutations in genes known to be associated with hearing loss prior to treatment with or administration of the compositions described herein. A subject can be screened for a genetic mutation using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein may also include a step of assessing hearing function in a subject prior to treatment with or administration of the compositions described herein. Hearing can be assessed using standard tests, such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions. These tests can also be used to assess hearing function in a subject after treatment with or administration of the compositions described herein. The compositions and methods described herein may also be administered as a preventative treatment to patients at risk of developing hearing loss, e.g., patients who have a family history of hearing loss (e.g., inherited hearing loss), patients carrying a genetic mutation associated with hearing loss who do not yet exhibit hearing impairment, or patients exposed to risk factors for acquired hearing loss (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging).

    [0099] The compositions and methods described herein can be used to promote or induce hair cell regeneration in a subject (e.g., OHC regeneration). Subjects that may benefit from compositions that promote or induce OHC regeneration include subjects suffering from hearing loss as a result of loss of OHCs (e.g., loss of OHCs related to trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), and subjects with abnormal OHCs (e.g., OHCs that do not function properly when compared to normal OHCs), damaged OHCs (e.g., OHC damage related to trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or reduced OHC numbers due to genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase OHC survival (e.g., increase survival of damaged OHCs, promote repair of damaged OHCs, or preserve OHCs in a subject at risk of loss of OHCs (e.g., loss of OHCs due to age, exposure to loud noise, disease or infection, head trauma, or ototoxic drugs)). The compositions and methods described herein can also be used to promote or increase OHC maturation, which can lead to improved auditory function.

    [0100] The compositions and methods described herein can also be used to prevent or reduce ototoxic drug-induced hair cell damage or death (e.g., OHC damage or death) in subjects who have been treated with ototoxic drugs, or who are currently undergoing or soon to begin treatment with ototoxic drugs. Ototoxic drugs are toxic to the cells of the inner ear, and can cause sensorineural hearing loss, tinnitus, or a combination of these symptoms. Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamycin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, antineoplastic drugs (e.g., platinum-containing chemotherapeutic agents, such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, particularly at high doses), and quinine. In some embodiments, the methods described herein prevent or reduce hair cell damage or death (e.g., OHC damage or death) related to acoustic trauma, disease or infection, head trauma, or aging.

    [0101] The transgene operably linked to an OHC-specific promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3)) for treatment of a subject as described herein can be a transgene that encodes a protein expressed in healthy OHCs (e.g., a protein that plays a role in OHC development, OHC function, OHC fate specification, OHC regeneration, OHC survival, or OHC maintenance, or a protein that is deficient in a subject with sensorineural hearing loss), another protein of interest (e.g., a therapeutic protein or a reporter protein, such as a fluorescent protein, lacZ, or luciferase), an siRNA, an ASO, a nuclease, or a microRNA. The transgene may be selected based on the cause of the subject's hearing loss (e.g., if the subject's hearing loss is associated with a particular genetic mutation, the transgene can be a wild-type form of the gene that is mutated in the subject, or if the subject has hearing loss associated with loss of hair cells, the transgene can encode a protein that promotes hair cell regeneration), the severity of the subject's hearing loss, the health of the subject's hair cells, the subject's age, the subject's family history of hearing loss, or other factors. The proteins that may be expressed by a transgene operably linked a hair cell-specific promoter for treatment of a subject as described herein include ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CHRNA10, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, SLC26A5, PIEZO2, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, WHRN, OCM, ISL1, NTF3, TMTC4, and BIP.

    [0102] Treatment may include administration of a composition containing a nucleic acid vector (e.g., an AAV viral vector) containing an OHC-specific promoter described herein in various unit doses. Each unit dose will ordinarily contain a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route of administration and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Dosing may be performed using a syringe pump to control infusion rate in order to minimize damage to the inner ear (e.g., the cochlea). In cases in which the nucleic acid vectors are AAV vectors (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ/8, DJ/9, 7m8, PHP.B, PHP.eb, or PHP.S vectors), the viral vectors may be administered to the patient at a dose of, for example, from about 1×10.sup.9 vector genomes (VG)/mL to about 1×10.sup.16 VG/mL (e.g., 1×10.sup.9 VG/mL, 2×10.sup.9 VG/mL, 3×10.sup.9 VG/mL, 4×10.sup.9 VG/mL, 5×10.sup.9 VG/mL, 6×10.sup.9 VG/mL, 7×10.sup.9 VG/mL, 8×10.sup.9 VG/mL, 9×10.sup.9 VG/mL, 1×10.sup.10 VG/mL, 2×10.sup.10 VG/mL, 3×10.sup.10 VG/mL, 4×10.sup.10 VG/mL, 5×10.sup.10 VG/mL, 6×10.sup.10 VG/mL, 7×10.sup.10 VG/mL, 8×10.sup.10 VG/mL, 9×10.sup.10 VG/mL, 1×10.sup.11 VG/mL, 2×10.sup.11 VG/mL, 3×10.sup.11 VG/mL, 4×10.sup.11 VG/mL, 5×10.sup.11 VG/mL, 6×10.sup.11 VG/mL, 7×10.sup.11 VG/mL, 8×10.sup.11 VG/mL, 9×10.sup.11 VG/mL, 1×10.sup.12 VG/mL, 2×10.sup.12 VG/mL, 3×10.sup.12 VG/mL, 4×10.sup.12 VG/mL, 5×10.sup.12 VG/mL, 6×10.sup.12 VG/mL, 7×10.sup.12 VG/mL, 8×10.sup.12 VG/mL, 9×10.sup.12 VG/mL, 1×10.sup.13 VG/mL, 2×10.sup.13 VG/mL, 3×10.sup.13 VG/mL, 4×10.sup.13 VG/mL, 5×10.sup.13 VG/mL, 6×10.sup.13 VG/mL, 7×10.sup.13 VG/mL, 8×10.sup.13 VG/mL, 9×10.sup.13 VG/mL, 1×10.sup.14 VG/mL, 2×10.sup.14 VG/mL, 3×10.sup.14 VG/mL, 4×10.sup.14 VG/mL, 5×10.sup.14 VG/mL, 6×10.sup.14 VG/mL, 7×10.sup.14 VG/mL, 8×10.sup.14 VG/mL, 9×10.sup.14 VG/mL, 1×10.sup.15 VG/mL, 2×10.sup.15 VG/mL, 3×10.sup.15 VG/mL, 4×10.sup.15 VG/mL, 5×10.sup.15 VG/mL, 6×10.sup.15VG/mL, 7×10.sup.15VG/mL, 8×10.sup.15 VG/mL, 9×10.sup.15 VG/mL, or 1×10.sup.16 VG/mL) in a volume of 1 μL to 200 μL (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μL). The AAV vectos may be administered to the subject at a dose of about 1×10.sup.7 VG/ear to about 2×10.sup.15 VG/ear (e.g., 1×10.sup.7 VG/ear, 2×10.sup.7 VG/ear, 3×10.sup.7 VG/ear, 4×10.sup.7 VG/ear, 5×10.sup.7 VG/ear, 6×10.sup.7 VG/ear, 7×10.sup.7 VG/ear, 8×10.sup.7 VG/ear, 9×10.sup.7 VG/ear, 1×10.sup.8 VG/ear, 2×10.sup.8 VG/ear, 3×10.sup.8 VG/ear, 4×10.sup.8 VG/ear, 5×10.sup.8 VG/ear, 6×10.sup.8 VG/ear, 7×10.sup.8 VG/ear, 8×10.sup.8 VG/ear, 9×10.sup.8 VG/ear, 1×10.sup.9 VG/ear, 2×10.sup.9 VG/ear, 3×10.sup.9 VG/ear, 4×10.sup.9 VG/ear, 5×10.sup.9 VG/ear, 6×10.sup.9 VG/ear, 7×10.sup.9 VG/ear, 8×10.sup.9 VG/ear, 9×10.sup.9 VG/ear, 1×10.sup.10 VG/ear, 2×10.sup.10 VG/ear, 3×10.sup.10 VG/ear, 4×10.sup.10 VG/ear, 5×10.sup.10 VG/ear, 6×10.sup.10 VG/ear, 7×10.sup.10 VG/ear, 8×10.sup.10 VG/ear, 9×10.sup.10 VG/ear, 1×10.sup.11 VG/ear, 2×10.sup.11 VG/ear, 3×10.sup.11 VG/ear, 4×10.sup.11 VG/ear, 5×10.sup.11 VG/ear, 6×10.sup.11 VG/ear, 7×10.sup.11 VG/ear, 8×10.sup.11 VG/ear, 9×10.sup.11 VG/ear, 1×10.sup.12 VG/ear, 2×10.sup.12 VG/ear, 3×10.sup.12 VG/ear, 4×10.sup.12 VG/ear, 5×10.sup.12 VG/ear, 6×10.sup.12 VG/ear, 7×10.sup.12 VG/ear, 8×10.sup.12 VG/ear, 9×10.sup.12 VG/ear, 1×10.sup.13 VG/ear, 2×10.sup.13 VG/ear, 3×10.sup.13 VG/ear, 4×10.sup.13 VG/ear, 5×10.sup.13 VG/ear, 6×10.sup.13 VG/ear, 7×10.sup.13 VG/ear, 8×10.sup.13 VG/ear, 9×10.sup.13 VG/ear, 1×10.sup.14 VG/ear, 2×10.sup.14 VG/ear, 3×10.sup.14 VG/ear, 4×10.sup.14 VG/ear, 5×10.sup.14 VG/ear, 6×10.sup.14 VG/ear, 7×10.sup.14 VG/ear, 8×10.sup.14 VG/ear, 9×10.sup.14 VG/ear, 1×10.sup.15 VG/ear, or 2×10.sup.15 VG/ear).

    [0103] The compositions described herein are administered in an amount sufficient to improve hearing, reduce tinnitus, increase expression of a protein encoded by a transgene operably linked to an OHC-specific promoter, increase function of a protein encoded by a transgene operably linked to an OHC-specific promoter, prevent or reduce OHC damage (e.g., OHC damage related to acoustic trauma, head trauma, ototoxic drugs, disease or infection, or aging), prevent or reduce OHC death (e.g., ototoxic drug-induced OHC death, noise-related OHC death, age-related OHC death, disease or infection-related OHC death, or head trauma-related OHC death), promote or increase OHC development, increase OHC numbers (e.g., promote or induce OHC regeneration), promote or increase OHC survival, promote or increase OHC maturation, or improve OHC function. Hearing may be evaluated using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions) and may be improved by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to hearing measurements obtained prior to treatment. In some embodiments, the compositions are administered in an amount sufficient to improve the subject's ability to understand speech. The compositions described herein may also be administered in an amount sufficient to slow or prevent the development or progression of sensorineural hearing loss (e.g., in subjects who carry a genetic mutation associated with hearing loss, who have a family history of hearing loss (e.g., hereditary hearing loss), or who have been exposed to risk factors associated with hearing loss (e.g., ototoxic drugs, head trauma, disease or infection, or acoustic trauma) but do not exhibit hearing impairment, or in subjects exhibiting mild to moderate hearing loss). Expression of the protein encoded by the transgene operably linked to an OHC-specific promoter in the nucleic acid vector administered to the subject may be evaluated using immunohistochemistry, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detection protein or mRNA, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to expression prior to administration of the compositions described herein. OHC numbers, OHC function, or function of the protein encoded by the nucleic acid vector administered to the subject may be evaluated indirectly based on hearing tests, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to OHC numbers, OHC function, or function of the protein prior to administration of the compositions described herein. OHC damage or death may be reduced by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to OHC damage and death typically observed in untreated subjects. These effects may occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more, following administration of the compositions described herein. The patient may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more following administration of the composition depending on the dose and route of administration used for treatment. Depending on the outcome of the evaluation, the patient may receive additional treatments.

    Kits

    [0104] The compositions described herein can be provided in a kit for use in treating sensorineural hearing loss. Compositions may include a polynucleotide described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to any one of the promoter sequences listed in Table 2 (e.g., any one of SEQ ID NOs: 1-3)), nucleic acid vectors containing such polynucleotides, and nucleic acid vectors containing a polynucleotide described herein operably linked to a transgene encoding a protein of interest (e.g., a protein that can be expressed in hair cells to treat hearing loss). The nucleic acid vectors may be packaged in an AAV virus capsid (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV6, AAV8, AAV9, Anc80, Anc80L65, DJ/9, 7m8, or PHP.B). The kit can further include a package insert that instructs a user of the kit, such as a physician, to perform the methods described herein. The kit may optionally include a syringe or other device for administering the composition.

    EXAMPLES

    [0105] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

    Example 1

    OCM Promoter Sequence Induces Transgene Expression in OHCs in Murine Cochlea In Vivo

    [0106] To determine the efficacy of the constructed OCM promoter (SEQ ID NO: 1) in inducing transgene expression in OHCs in vivo, mouse cochlea was transduced with either an AAV vector expressing GFP under the control of the cytomegalovirus (CMV) promoter, or an AAV vector expressing GFP under control of the OCM promoter. Specifically, AAV-OCM-GFP virus was infused via the posterior semicircular canal to two day-old CBA/CaJ mice at a dose of 7.7E+9 vector genomes per ear. Mice recovered from surgery and were euthanized and perfused with 10% normal buffered formalin 19 days later. The inner ear temporal bone was harvested and decalcified in 8% EDTA for three days. The cochlea was dissected from the de-calcified temporal bone, immunostained with Myosin 7a (Myo7a) antibody to label all hair cells, and mounted on a slide for confocal imaging. Native GFP fluorescence is shown. Using a ubiquitous promoter, AAV-CMV-GFP induced GFP expression in many cell types within the cochlea including inner hair cells, outer hair cells, spiral ganglion neurons, mesenchymal cells, and glia (FIG. 1A). Using the outer hair cell-specific promoter, AAV-OCM (SEQ ID NO: 1)-GFP induced GFP expression exclusively in outer hair cells (FIG. 1B).

    Example 2

    Administration of a Composition Containing a Nucleic Acid Vector Containing a Hair Cell-Specific Promoter to a Subject with Sensorineural Hearing Loss

    [0107] According to the methods disclosed herein, a physician of skill in the art can treat a patient, such as a human patient, with sensorineural hearing loss so as to improve or restore hearing. To this end, a physician of skill in the art can administer to the human patient a composition containing an AAV vector (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV6, AAV9, Anc80, Anc80L65, DJ/9, 7m8, or PHP.B) containing an outer hair cell-specific promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the sequence of any one of SEQ ID NOs: 1-3) operably linked to a transgene that encodes a therapeutic protein. The composition containing the AAV vector may be administered to the patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or through the round window membrane), to treat sensorineural hearing loss.

    [0108] Following administration of the composition to a patient, a practitioner of skill in the art can monitor the expression of the therapeutic protein encoded by the transgene, and the patient's improvement in response to the therapy, by a variety of methods. For example, a physician can monitor the patient's hearing by performing standard tests, such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions following administration of the composition. A finding that the patient exhibits improved hearing in one or more of the tests following administration of the composition compared to hearing test results prior to administration of the composition indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

    Other Embodiments

    [0109] Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. Other embodiments are in the claims.