AAV1 vectors and their use for treatment of ear indications

Abstract

The present invention provides AAV1 vectors and their use for the treatment of ear indications. In particular, the present invention provides AAV1 vectors that can be used to transduce a variety of inner ear cell types and their use for the treatment of hearing loss, deafness, tinnitus and vestibular dysfunction.

Description

[0001] This application is a divisional application of a patent application with an original filing date of October 11, 2019, application number 201980082153.6 (international application number PCT / US2019 / 055979), and invention title "AAV1 Vectors and Their Use in Treating Ear Indications".

[0002] Sequence Listing

[0003] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on October 11, 2019, is named 51124 - 054WO3_Sequence_Listing_10.11.2019_ST25 and is 38,358 bytes in size. Technical Field

[0004] The present invention provides AAV1 vectors that can be used to transduce multiple inner ear cell types and their use in treating hearing loss, tinnitus, and vestibular dysfunction. Background of the Invention

[0006] Gene therapy has recently emerged as a promising approach for treating inner ear disorders such as hearing loss and vestibular dysfunction, as it can be used to treat the genetic causes of these disorders, induce the expression of genes encoding therapeutic proteins, and can preserve or restore hearing, with a more natural sound perception compared to cochlear implants. However, mutations causing hearing loss and / or vestibular dysfunction have been identified in multiple different cell types of the inner ear, and preclinical studies in rodent models are pending to identify a single viral vector that can be used to transduce most or all inner ear cell types. In addition, few studies have been conducted in larger animal models that are more predictive of human therapy. Therefore, there is a need for viral vectors that exhibit pan - tropic transduction of inner ear cells in clinically relevant animal models. Summary of the Invention

[0007] This disclosure is based on the inventors' discovery that adeno-associated virus (AAV) vectors having serotype AAV1 (e.g., AAV1 capsids) unexpectedly have pan-tropism in the inner ear of non-human primates. Compared to the tropism of AAV1 in the inner ear of mice and also compared to the tropism of other AAV serotypes (e.g., AAV2 and AAV7m8) in non-human primates, the AAV1 vector exhibits strong expression and unexpectedly excellent tropism in multiple cell types of the inner ear of non-human primates. Accordingly, the present invention provides compositions and methods for transducing cell types of the inner ear of primates (e.g., humans) using AAV1 vectors. The AAV1 vectors described herein can be administered to a primate (e.g., human) subject to promote the expression of a polynucleotide, e.g., a polynucleotide corresponding to a gene that promotes or improves the inner ear cell function, regeneration, maintenance, development, proliferation, or survival of one or more inner ear cells of a primate (e.g., human). The compositions and methods described herein can be administered to a primate (e.g., human) patient to treat or prevent hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance).

[0008] Exemplary embodiments of the invention are described in the paragraphs listed below.

[0009] E1. A method of transducing developing inner ear cells in a primate subject, the method comprising administering to the subject an effective amount of a serotype 1 adeno-associated virus (AAV1) vector that transduces an amount of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) inner ear cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocytes, Scarpa's ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiter cells, second row of Deiter cells, third row of Deiter cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocytes, cells of Reissner's membrane, and glial cells.

[0010] E2. The method according to E1, wherein the AAV1 vector transduces three or more inner ear cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, the first row of Deiter cells, the second row of Deiter cells, the third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane, and glial cells.

[0011] E3. The method according to E2, wherein the AAV1 vector transduces five or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, the first row of Deiter cells, the second row of Deiter cells, the third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane, and glial cells.

[0012] E4. The method according to E3, wherein the AAV1 vector transduces ten or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, the first row of Deiter cells, the second row of Deiter cells, the third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane, and glial cells.

[0013] E5. The method according to E4, wherein the AAV1 vector transduces fifteen or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibroblast cells, Scarpa's ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, the first row of Deiter cells, the second row of Deiter cells, the third row of Deiter cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibroblast cells, cells of Reissner's membrane, and glial cells.

[0014] E6. The method according to E5, wherein the AAV1 vector transduces twenty or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibroblast cells, Scarpa's ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, the first row of Deiter cells, the second row of Deiter cells, the third row of Deiter cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibroblast cells, cells of Reissner's membrane, and glial cells.

[0015] E7. The method according to any one of E1 - E6, wherein the AAV1 vector transduces at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cells of a given cell type or all inner ear cell types.

[0016] E8. The method according to any one of E1 - E7, wherein the AAV1 vector transduces all the cells of a given cell type or all inner ear cell types.

[0017] E9. The method according to any one of E1 - E8, wherein transduction occurs throughout the length of the cochlea (e.g., the basal - to - apical axis of the cochlea).

[0018] E10. The method according to any one of E1 - E9, wherein the AAV1 vector transduces at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%) of the cells of a given cell type or all inner ear cell types across the length of the basal - to - apical axis of the cochlea.

[0019] E11. The method according to any one of E1 - E10, wherein the AAV1 vector transduces at least 20% (such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%) of a given cell type or all inner ear cell types of cells in the base of the cochlea.

[0020] E12. The method according to any one of E1 - E11, wherein the AAV1 vector transduces at least 20% (such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%) of a given cell type or all inner ear cell types of cells in the middle of the cochlea.

[0021] E13. The method according to any one of E1 - E12, wherein the AAV1 vector transduces at least 20% (such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%) of a given cell type or all inner ear cell types of cells in the apex of the cochlea.

[0022] E14. The method according to any one of E1 - E13, wherein the AAV1 vector comprises a promoter selected from the list of promoters in Table 5.

[0023] E15. The method according to E14, wherein the promoter is a glial fibrillary acidic protein (GFAP) promoter.

[0024] E16. The method according to E14, wherein the promoter is a synapsin (SYN) promoter.

[0025] E17. The method according to E14, wherein the promoter is selected from the group consisting of Jagged 1 (JAG1) promoter, Notch 1 (NOTCH1 promoter), myosin 7A (MYO7A) promoter, solute carrier family 1 member 3 (GLAST) promoter, and POU class 4 homeobox 3 (POU4F3) promoter.

[0026] E18. The method according to E14, wherein the AAV1 vector comprises a promoter selected from the group consisting of Prestin (SLC26A5) promoter, Oncomodulin (OCM) promoter, Stereocilin (STRC) promoter, Myosin 15 (MYO15) promoter, Growth Factor Independent 1 Transcriptional Repressor (GFI1) promoter, Potassium Voltage-Gated Channel Subfamily Q Member 1 (KCNQ1) promoter, Potassium Voltage-Gated Channel Subfamily J Member 10 (KCNJ10) promoter, Tyrosinase (TYR) promoter, SRY-Box 2 (SOX2) promoter, Calbindin 2 (CALB2) promoter, Basic Helix-Loop-Helix Family Member E22 (BHLHE22) promoter, Leucine-Rich Repeat-Containing G-Protein-Coupled Receptor 5 (LGR5) promoter, Hes Family BHLH Transcription Factor 1 (HES1) promoter, Hes Family BHLH Transcription Factor 5 (HES5) promoter, SRY-Box 9 (SOX9) promoter, ATPase Plasma Membrane Ca2+ Transporter 2 (ATP2B2) promoter, CD44 Molecule (CD44) promoter, Fibroblast Growth Factor Receptor 3 (FGFR3) promoter, Frizzled Related Protein (FRZB) promoter, Solute Carrier Family 26 Member 4 (SLC26A4) promoter, Carcinoembryonic Antigen-Related Cell Adhesion Molecule 16 (CEACAM16) promoter, Claudin 11 (CLDN11) promoter, Peripheral Myelin Protein 22 (PMP22) promoter, Potassium Voltage-Gated Channel Subfamily E Regulatory Subunit 1 (KCNE1) promoter, POU Class 3 Homeobox 4 (POU3F4) promoter, Gap Junction Protein Beta 2 (GJB2) promoter, and Tubulin Beta 3 Class III (TUBB3) promoter.

[0027] E19. The method according to E18, wherein the promoter is the SLC26A5 promoter.

[0028] E20. The method according to E18, wherein the promoter is the OCM promoter.

[0029] E21. The method according to E18, wherein the promoter is the MYO15 promoter.

[0030] E22. The method according to E18, wherein the promoter is the ATP2B2 promoter.

[0031] E23. The method according to E18, wherein the promoter is the GJB2 promoter.

[0032] E24. The method according to E18, wherein the promoter is the TYR promoter.

[0033] E25. The method according to any one of E1 - E13, wherein the promoter is a ubiquitous promoter.

[0034] E26. The method according to E25, wherein the promoter is a cytomegalovirus (CMV) promoter.

[0035] E27. The method according to E25, wherein the promoter is a hybrid promoter (CAG promoter) derived from a CMV early enhancer element, a promoter of the chicken β - actin gene, the first exon and the first intron, and a splice acceptor of the rabbit β - globin gene.

[0036] E28. The method according to E25, wherein the promoter is a truncated CMV - chicken β - actin (smCBA) promoter.

[0037] E29. The method according to any one of E1 - E28, wherein the AAV1 vector comprises a polynucleotide selected from the list in Table 2.

[0038] E30. The method according to E29, wherein the polynucleotide encodes Strc.

[0039] E31. The method according to E29, wherein the polynucleotide encodes transmembrane channel - like 1 (Tmc1).

[0040] E32. The method according to E29, wherein the polynucleotide encodes Harmonin (Ush1c).

[0041] E33. The method according to E29, wherein the polynucleotide encodes Atonal BHLH transcription factor 1 (Atoh1).

[0042] E34. The method according to E29, wherein the polynucleotide encodes Clarin 1 (Clrn1).

[0043] E35. The method according to E29, wherein the polynucleotide encodes SRY - box 4 (Sox4).

[0044] E36. The method according to E29, wherein the polynucleotide encodes brain - derived neurotrophic factor (Bdnf).

[0045] E37. The method according to E29, wherein the polynucleotide encodes neurotrophin 3 (Ntf3).

[0046] E38. The method according to E29, wherein the polynucleotide encodes SRY - box 11 (Sox11).

[0047] E39. The method as described in E29, wherein the polynucleotide encodes myosin 7A (Myo7a).

[0048] E40. The method as described in E29, wherein the polynucleotide encodes Gjb2.

[0049] E41. The method as described in E29, wherein the polynucleotide encodes cholinergic receptor nicotinic alpha 9 subunit (Chrna9).

[0050] E42. The method as described in E29, wherein the polynucleotide encodes cholinergic receptor nicotinic alpha 10 subunit (Chrna10).

[0051] E43. The method as described in E29, wherein the polynucleotide encodes Ocm.

[0052] E44. The method as described in E29, wherein the polynucleotide encodes Tyr.

[0053] E45. The method as described in E29, wherein the polynucleotide encodes transmembrane and tetratricopeptide repeat-containing 4 (Tmtc4).

[0054] E46. The method as described in E29, wherein the polynucleotide encodes TEA domain transcription factor 2 (Tead2).

[0055] E47. The method as in E29, wherein the polynucleotide encodes Yes-associated protein 1 (Yap1).

[0056] E48. A method of expressing a polynucleotide in supporting cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: GFAP promoter, GLAST promoter, HES1 promoter, JAG1 promoter, NOTCH1 promoter, LGR5 promoter, SOX2 promoter, HES5 promoter, and SOX9 promoter.

[0057] E49. The method as described in E48, wherein the promoter is the GFAP promoter.

[0058] E50. The method as described in E48, wherein the promoter is selected from the group consisting of: HES1 promoter, LGR5 promoter, SOX2 promoter, HES5 promoter, and SOX9 promoter.

[0059] E51. The method according to any one of E48 - E50, wherein the polynucleotide encodes Sox9, Spalt-like transcription factor 2 (Sall2), calmodulin-binding transcription activator 1 (Camta1), Hes-related family BHLH transcription factor with YRPW motif 2 (Hey2), Gata-binding protein 2 (Gata2), Hes-related family BHLH transcription factor with YRPW motif 1 (Hey1), ceramide synthase 2 (Lass2), SRY-box 10 (Sox10), GATA-binding protein 3 (Gata3), cut-like homeobox 1 (Cux1), nuclear receptor subfamily 2F member (Nr2f1), Hes family BHLH transcription factor 1 (Hes1), RAR-related orphan receptor B (Rorb), Jun proto-oncogene AP-1 transcription factor subunit (Jun), zinc finger protein 667 (Zfp667), LIM homeobox 3 (Lhx3), Nescient helix-loop-helix 1 (Nhlh1), MAX dimerization protein 4 (Mxd4), zinc finger MIZ type 1-containing (Zmiz1), myelin transcription factor 1 (Myt1), signal transducer and activator of transcription 3 (Stat3), BarH-like homeobox 1 (Barhl1), thymocyte selection-associated high mobility group box (Tox), Prospero homeobox 1 (Prox1), nuclear factor I A (Nfia), thyroid hormone receptor beta (Thrb), MYCL proto-oncogene BHLH transcription factor (Mycl1), lysine demethylase 5A (Kdm5a), CAMP-responsive element-binding protein 3-like 4 (Creb3I4), ETS variant 1 (Etv1), paternally expressed 3 (Peg3), BTB domain and CNC homolog 2 (Bach2), ISL LIM homeobox (Isl1), zinc finger and BTB domain-containing 38 (Zbtb38), limb bud and heart development (Lbh), Tubby bipartite transcription factor (Tub), ubiquitin C (Hmg20), RE1-silencing transcription factor (Rest), zinc finger protein 827 (Zfp827), AF4 / FMR2 family member 3 (Aff3), PBX / knotted 1 homeobox 2 (Pknox2), AT-rich interaction domain 3B (Arid3b), MLX-interacting protein (Mlxip), zinc finger protein (Zfp532), IKAROS family zinc finger 2 (Ikzf2), Spalt-like transcription factor 1 (Sall1), SIX homeobox 2 (Six2), Spalt-like transcription factor 3 (Sall3), Lin-28 homolog B (Lin28b), Pou4f3, regulatory factor X7 (Rfx7), Atoh1, an Atoh1 variant containing mutations at amino acids 328, 331 and / or 334 (e.g.,S328A, S331A, S334A, S328A / S331A, S328A / S334A, S331A / S334A, and S328A / S331A / S334, such as variants having a sequence of any one of SEQ ID NO: 4-10), Gfi1, Sox4, Bdnf, Ntf3, Sox11, Tead2, Yap1, or a nuclease (e.g., CRISPR-associated protein 9 (Cas9), transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN), or guide RNA (gRNA)), or is a microRNA (miRNA, such as miR-183, miR-96, or miR-182).

[0060] E52. The method according to any one of E48-E51, wherein the supporting cell is a vestibular supporting cell.

[0061] E53. The method according to E52, wherein the vestibular supporting cell is located in the utricle.

[0062] E54. The method according to any one of E48-E51, wherein the supporting cell is a cochlear supporting cell, and the promoter is SOX2.

[0063] E55. A method of expressing a polynucleotide in hair cells (e.g., vestibular hair cells and / or cochlear hair cells) of the inner ear of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, and MYO7A promoter.

[0064] E56. The method according to E55, wherein the promoter is the MYO15 promoter.

[0065] E57. The method according to E55 or E56, wherein the polynucleotide encodes Bdnf, Ntf3, Tmtc4, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or is a microRNA (miRNA, such as miR-183, miR-96, or miR-182).

[0066] E58. The method according to any one of E55-E57, wherein the hair cell is a cochlear hair cell.

[0067] E59. The method according to E58, wherein the cochlear hair cell is an inner hair cell.

[0068] E60. The method according to E58, wherein the cochlear hair cell is an outer hair cell.

[0069] E61. The method according to any one of E55 - E57, wherein the hair cells are vestibular hair cells.

[0070] E62. A method for expressing a polynucleotide in outer hair cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: SLC26A5 promoter, OCM promoter, STRC promoter, and ATP2B2 promoter.

[0071] E63. The method according to E62, wherein the promoter is the SLC26A5 promoter.

[0072] E64. The method according to E62, wherein the promoter is the OCM promoter.

[0073] E65. The method according to E62, wherein the promoter is the ATP2B2 promoter.

[0074] E66. The method according to any one of E62 - E65, wherein the polynucleotide encodes Strc, Tmc1, Myo7a, Ush1c, Atoh1, Pou4f3, Gfi1, ISL LIM homeobox 1 (Isl1), Clrn1, protocadherin-related 15 (Pcdh15), protocadherin-related 23 (Cdh23), Chrna9, Chrna10, Ocm, Bdnf, Ntf3, Tmtc4, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or is a microRNA (e.g., miR-183, miR-96, or miR-182).

[0075] E67. A method for expressing a polynucleotide in cochlear hair cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, and MYO7A promoter.

[0076] E68. The method according to E67, wherein the promoter is the GFI1 promoter.

[0077] E69. The method according to E67, wherein the promoter is the MYO15 promoter.

[0078] E70. The method according to any one of E67 - E69, wherein the polynucleotide encodes Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, otoferlin (Otof), solute carrier family 17 member 8 (Vglut3), Strc, Chrna9, Chrna10, Ocm, Tmc1, Myo7a, Ush1c, Whirlin (Whrn), Bdnf, Ntf3, Tmtc4, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or is a microRNA (e.g., miR - 183, miR - 96, or miR - 182).

[0079] E71. A method for expressing a polynucleotide in inner hair cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: OTOF promoter, fibroblast growth factor 8 (FGF8) promoter, and solute carrier family 17 member 8 (SLC17A8) promoter.

[0080] E72. The method according to E71, wherein the promoter is the OTOF promoter.

[0081] E73. The method according to E72 or E72, wherein the polynucleotide encodes Otof, Vglut3, Whirlin, Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, Myo7a, Tmc1, Ush1c, Bdnf, Ntf3, Tmtc4, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or is a microRNA (e.g., miR - 183, miR - 96, or miR - 182).

[0082] E74. A method for expressing a polynucleotide in vestibular hair cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, MYO7A promoter, ATP2B2 promoter, and CALB2 promoter.

[0083] E75. The method according to E74, wherein the promoter is the MYO15 promoter.

[0084] E76. The method according to E74, wherein the promoter is the ATP2B2 promoter.

[0085] E77. The method according to E74, wherein the vestibular hair cells are type I vestibular hair cells, and the promoter is the ATP2B2 promoter.

[0086] E78. The method according to E74, wherein the vestibular hair cells are type II vestibular hair cells, and the promoter is the CALB2 promoter.

[0087] E79. The method according to any one of E74 - E78, wherein the polynucleotide encodes Whirlin, Bdnf, Ntf3, Tmtc4, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or is a microRNA (e.g., miR - 183, miR - 96, or miR - 182).

[0088] E80. A method of expressing a polynucleotide in pillar cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a CD44 promoter or a GJB2 promoter.

[0089] E81. The method according to E80, wherein the promoter is the GJB2 promoter.

[0090] E82. The method according to E80 or E81, wherein the polynucleotide encodes a nerve growth factor receptor (Ngfr), Bdnf, Ntf3, Tectorinβ (Tectb), Tectorinα (Tecta), Gjb2, or connexin β6 (Gjb6).

[0091] E83. A method of expressing a polynucleotide in spiral ganglion neurons of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: BHLHE22 promoter, SYN promoter, and CALB2 promoter.

[0092] E84. The method according to E83, wherein the promoter is the SYN promoter.

[0093] E85. The method according to E83, wherein the spiral ganglion neurons have a high spontaneous firing rate, and the promoter is the CALB2 promoter.

[0094] E86. The method according to E83, wherein the spiral ganglion neurons are afferent spiral ganglion neurons, and the promoter is the BHLHE22 promoter.

[0095] E87. The method according to any one of E83 - E86, wherein the polynucleotide encodes Bdnf, Ntf3, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or is a microRNA (miRNA, e.g., miR-183, miR-96, or miR-182), or an shRNA against RGMA.

[0096] E88. A method of expressing a polynucleotide in the marginal cells of the stria vascularis of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of the KCNQ1 promoter, the KCNE1 promoter, and the GJB2 promoter.

[0097] E89. The method according to E88, wherein the promoter is the GJB2 promoter.

[0098] E90. A method of expressing a polynucleotide in the basal cells of the stria vascularis of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to the CLDN11 promoter or the GJB2 promoter.

[0099] E91. The method according to E90, wherein the promoter is the GJB2 promoter.

[0100] E92. A method of expressing a polynucleotide in the intermediate cells of the stria vascularis of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of the KCNJ10 promoter, the GJB2 promoter, and the TYR promoter.

[0101] E93. The method according to E92, wherein the promoter is the GJB2 promoter.

[0102] E94. The method according to E92, wherein the promoter is the TYR promoter.

[0103] E95. The method according to any one of E90 - E94, wherein the polynucleotide encodes Kcnq1, Kcne1, Tyr, Gjb2, Gjb6, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or is a microRNA (e.g., miR-183, miR-96, or miR-182).

[0104] E96. A method of expressing a polynucleotide in marginal cells and / or inner phalangeal cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a GLAST promoter or a GJB2 promoter.

[0105] E97. The method according to E96, wherein the promoter is a GJB2 promoter.

[0106] E98. The method according to E96 or E97, wherein the polynucleotide encodes Bdnf, Ntf3, Tectb, Tecta, transmembrane protein 16A (Tmem16a), Gjb2 or Gjb6.

[0107] E99. A method of expressing a polynucleotide in Dieter's cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to an FGFR3 promoter or a GJB2 promoter.

[0108] E100. The method according to E99, wherein the promoter is a GJB2 promoter.

[0109] E101. The method according to E99 or E100, wherein the polynucleotide encodes Bdnf, Ntf3, Tectb, Tecta, IKAROS family zinc finger 2 (Ikzf2), Gjb2 or Gjb6.

[0110] E102. A method of expressing a polynucleotide in Hensen's cells and / or Claudius cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to an FRZB promoter or a GJB2 promoter.

[0111] E103. The method according to E102, wherein the promoter is a GJB2 promoter

[0112] E104. The method according to E102 or E103, wherein the polynucleotide encodes Gjb2 or Gjb6.

[0113] E105. A method of expressing a polynucleotide in spiral prominence cells and / or root cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to an SLC26A4 promoter.

[0114] E106. The method according to E105, wherein the polynucleotide is Slc26a4.

[0115] E107. A method for expressing a polynucleotide in the interdental cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a CEACAM16 promoter or a GJB2 promoter.

[0116] E108. The method according to E107, wherein the promoter is a GJB2 promoter

[0117] E109. The method according to E107 or E108, wherein the polynucleotide encodes Ceacam16, otoancorin (Otoa), Gjb2 or Gjb6.

[0118] E110. A method for expressing a polynucleotide in the glial cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a PMP22 promoter.

[0119] E111. The method according to E110, wherein the polynucleotide encodes Pmp22, Bdnf, Ntf3 or myelin protein zero (Mpz).

[0120] E112. A method for expressing a polynucleotide in the vestibular dark cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a KCNE1 promoter.

[0121] E113. The method according to E112, wherein the polynucleotide encodes Kcnq1, Kcne1 or Slc26a4.

[0122] E114. A method for expressing a polynucleotide in the fibroblast cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a POU3F4 promoter or a GJB2 promoter.

[0123] E115. The method according to E114, wherein the promoter is a GJB2 promoter

[0124] E116. The method according to E114 or E115, wherein the polynucleotide encodes Gjb2, Gjb6 or collagen.

[0125] E117. A method of expressing a polynucleotide in Scarpa's ganglion neurons (vestibular ganglion neurons) of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: TUBB3 promoter and SYN promoter.

[0126] E118. The method according to E117, wherein the promoter is the SYN promoter.

[0127] E119. The method according to E117 or E118, wherein the polynucleotide encodes Bdnf or Ntf3, or is an shRNA against repulsive guidance molecule BMP coreceptor A (RGMA).

[0128] E120. A method of expressing a polynucleotide in supporting cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox9, Sall2, Camta1, Hey2, Gata2, Hey1, Lass2, Sox10, Gata3, Cux1, Nr2f1, Hes1, Rorb, Jun, Zfp667, Lhx3, Nhlh1, Mxd4, Zmiz1, Myt1, Stat3, Barhl1, Tox, Prox1, Nfia, Thrb, Mycl1, Kdm5a, Creb3I4, Etv1, Peg3, Bach2, Isl1, Zbtb38, Lbh, Tub, Hmg20, Rest, Zfp827, Aff3, Pknox2, Arid3b, Mlxip, Zfp532, Ikzf2, Sall1, Six2, Sall3, Lin28b, Pou4f3, Rfx7, Atoh1, an Atoh1 variant comprising mutations at amino acids 328, 331 and / or 334 (e.g., S328A, S331A, S334A, S328A / S331A, S328A / S334A, S331A / S334A and S328A / S331A / S334, e.g., a variant having a sequence of any one of SEQ ID NO: 4-10), Gfi1, Sox4, Bdnf, Ntf3, Sox11, Tead2, Yap1 or a nuclease (e.g., Cas9, TALEN, ZFN or gRNA), or is a microRNA (e.g., miR-183, miR-96 or miR-182).

[0129] E121. The method according to E120, wherein the polynucleotide encodes Atoh1.

[0130] E122. The method according to E120, wherein the polynucleotide encodes Sox4.

[0131] E123. The method according to E120, wherein the polynucleotide encodes Sox11.

[0132] E124. The method according to E120, wherein the polynucleotide encodes Bdnf.

[0133] E125. The method according to E120, wherein the polynucleotide encodes Ntf3.

[0134] E126. The method according to E120, wherein the polynucleotide encodes Tead2.

[0135] E127. The method according to E120, wherein the polynucleotide encodes Yap1.

[0136] E128. The method according to any one of E120 - E127, wherein the promoter is selected from the group consisting of: GFAP promoter, GLAST promoter, HES1 promoter, JAG1 promoter, NOTCH1 promoter, LGR5 promoter, SOX2 promoter, HES5 promoter, and SOX9 promoter.

[0137] E129. The method according to E128, wherein the promoter is the GFAP promoter.

[0138] E130. The method according to E128, wherein the promoter is selected from the group consisting of: HES1 promoter, LGR5 promoter, SOX2 promoter, HES5 promoter, and SOX9 promoter.

[0139] E131. The method according to any one of E120 - E130, wherein the supporting cells are vestibular supporting cells.

[0140] E132. The method according to E131, wherein the vestibular supporting cells are located in the utricle.

[0141] E133. The method according to any one of E120 - E130, wherein the supporting cells are cochlear supporting cells, and the promoter is SOX2.

[0142] E134. A method of expressing a polynucleotide in hair cells (e.g., vestibular hair cells and / or cochlear hair cells) of the inner ear of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Bdnf, Ntf3, Tmtc4, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or a microRNA (e.g., miR-183, miR-96, or miR-182).

[0143] E135. The method according to E134, wherein the polynucleotide encodes Bdnf.

[0144] E136. The method according to E134, wherein the polynucleotide encodes Ntf3.

[0145] E137. The method according to E134, wherein the polynucleotide encodes Tmtc4.

[0146] E138. The method according to any one of E134-E137, wherein the promoter is selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, and MYO7A promoter.

[0147] E139. The method according to any one of E134-E138, wherein the hair cells are cochlear hair cells.

[0148] E140. The method according to E139, wherein the cochlear hair cells are inner hair cells.

[0149] E141. The method according to E139, wherein the cochlear hair cells are outer hair cells.

[0150] E142. The method according to any one of E134-E138, wherein the hair cells are vestibular hair cells.

[0151] E143. A method of expressing a polynucleotide in outer hair cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Strc, Tmc1, Myo7a, Ush1c, Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, Chrna9, Chrna10, Ocm, Bdnf, Ntf3, Tmtc4, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or a microRNA (e.g., miR-183, miR-96, or miR-182).

[0152] E144. The method according to E143, wherein the polynucleotide encodes Strc.

[0153] E145. The method according to E143, wherein the polynucleotide encodes Chrna9.

[0154] E146. The method according to E143, wherein the polynucleotide encodes Chrna10.

[0155] E147. The method according to E143, wherein the polynucleotide encodes Ocm.

[0156] E148. The method according to E143, wherein the polynucleotide encodes Tmc1.

[0157] E149. The method according to E143, wherein the polynucleotide encodes Myo7a.

[0158] E150. The method according to E143, wherein the polynucleotide encodes Ush1c.

[0159] E151. The method according to E143, wherein the polynucleotide encodes Tmtc4.

[0160] E152. The method according to any one of E143 - E151, wherein the promoter is selected from the group consisting of: SLC26A5 promoter, OCM promoter, STRC promoter, and ATP2B2 promoter.

[0161] E153. A method for expressing a polynucleotide in cochlear hair cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, Otof, Vglut3, Strc, Chrna9, Chrna10, Ocm, Tmc1, Myo7a, Ush1c, Whirlin, Bdnf, Ntf3, Tmtc4, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or a microRNA (e.g., miR - 183, miR - 96, or miR - 182).

[0162] E154. The method according to E153, wherein the polynucleotide encodes Atoh1.

[0163] E155. The method according to E153, wherein the polynucleotide encodes Clrn1.

[0164] E156. The method as described in E153, wherein the polynucleotide encodes Otof.

[0165] E157. The method as described in E153, wherein the polynucleotide encodes Tmc1.

[0166] E158. The method as described in E153, wherein the polynucleotide encodes Ush1c.

[0167] E159. The method as described in E153, wherein the polynucleotide encodes Myo7a.

[0168] E160. The method as described in E153, wherein the polynucleotide encodes Vglut3.

[0169] E161. The method as described in E153, wherein the polynucleotide encodes Strc.

[0170] E162. The method as described in E153, wherein the polynucleotide encodes Chrna9.

[0171] E163. The method as described in E153, wherein the polynucleotide encodes Chrna10.

[0172] E164. The method as described in E153, wherein the polynucleotide encodes Tmtc4.

[0173] E165. The method as described in E153, wherein the polynucleotide encodes Ocm.

[0174] E166. The method as described in any one of E153 - E165, wherein the promoter is selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, and MYO7a promoter.

[0175] E167. The method as described in E166, wherein the promoter is the GFI1 promoter.

[0176] E168. The method as described in E166, wherein the promoter is the MYO15 promoter.

[0177] E169. A method of expressing a polynucleotide in inner hair cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Otof, Vglut3, Whirlin, Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh14, Cdh23, Myo7a, Ush1c, Tmc1, Bdnf, Ntf3, Tmtc4 or a nuclease (e.g., Cas9, TALEN, ZFN or gRNA) or being a microRNA (e.g., miR-183, miR-96 or miR-182).

[0178] E170. The method according to E169, wherein the polynucleotide encodes Otof.

[0179] E171. The method according to E169, wherein the polynucleotide encodes Atoh1.

[0180] E172. The method according to E169, wherein the polynucleotide encodes Vglut3.

[0181] E173. The method according to E169, wherein the polynucleotide encodes Clrn1.

[0182] E174. The method according to any one of E169 - E173, wherein the promoter is selected from the group consisting of: OTOF promoter, FGF8 promoter and SLC17A8 promoter.

[0183] E175. A method of expressing a polynucleotide in vestibular hair cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Whirlin, Bdnf, Ntf3, Tmtc4 or a nuclease (e.g., Cas9, TALEN, ZFN or gRNA) or being a microRNA (e.g., miR-183, miR-96 or miR-182).

[0184] E176. The method according to E175, wherein the polynucleotide encodes Bdnf.

[0185] E177. The method according to E175, wherein the polynucleotide encodes Ntf3.

[0186] E178. The method according to E175, wherein the polynucleotide encodes Tmtc4.

[0187] E179. The method according to any one of E175 - E178, wherein the promoter is selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, MYO7A promoter, ATP2B2 promoter, and CALB2 promoter.

[0188] E180. The method according to any one of E175 - E179, wherein the vestibular hair cell is a type I vestibular hair cell, and the promoter is ATP2B2.

[0189] E181. The method according to any one of E175 - E179, wherein the vestibular hair cell is a type II vestibular hair cell, and the promoter is CALB2 promoter.

[0190] E182. A method for expressing a polynucleotide in pillar cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Ngfr, Bdnf, Ntf3, Tectb, Tecta, Gjb2, or Gjb6.

[0191] E183. The method according to E182, wherein the polynucleotide encodes Gjb2.

[0192] E184. The method according to E182 or E183, wherein the promoter is CD44 promoter or GJB2 promoter.

[0193] E185. A method for expressing a polynucleotide in spiral ganglion neurons of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Bdnf, Ntf3, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or being a microRNA (e.g., miR - 183, miR - 96, or miR - 182), or an shRNA against RGMA.

[0194] E186. The method according to E185, wherein the polynucleotide encodes Bdnf.

[0195] E187. The method according to E185, wherein the polynucleotide encodes Ntf3.

[0196] E188. The method according to any one of E185 - E187, wherein the promoter is selected from the group consisting of: BHLHE22 promoter, SYN promoter, and CALB2 promoter.

[0197] E189. The method according to any one of E185 - E188, wherein the spiral ganglion neurons have a high spontaneous firing rate, and the promoter is the CALB2 promoter.

[0198] E190. The method according to any one of E185 - E188, wherein the vestibular hair cells are afferent spiral ganglion neurons, and the promoter is the BLHLE22 promoter.

[0199] E191. A method for expressing a polynucleotide in cells in the stria vascularis of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Kcnq1, Kcne1, Gjb2, Gjb6, Tyr or a nuclease (e.g., Cas9, TALEN, ZFN or gRNA) or being a microRNA (e.g., miR - 183, miR - 96 or miR - 182).

[0200] E192. The method according to E191, wherein the polynucleotide encodes Gjb2.

[0201] E193. The method according to E191, wherein the polynucleotide encodes Tyr.

[0202] E194. The method according to any one of E191 - E193, wherein the cells are marginal cells of the stria vascularis, and the promoter is selected from the group consisting of the KCNQ1 promoter, the GJB2 promoter, and the KCNE1 promoter.

[0203] E195. The method according to any one of E191 - E193, wherein the cells are basal cells of the stria vascularis, and the promoter is the CLDN11 promoter or the GJB2 promoter.

[0204] E196. The method according to any one of E191 - E193, wherein the cells are intermediate cells of the stria vascularis, and the promoter is selected from the group consisting of the KCNJ10 promoter, the GJB2 promoter, and the TYR promoter.

[0205] E197. A method for expressing a polynucleotide in marginal cells and / or inner phalangeal cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Bdnf, Ntf3, Tectb, Tecta, Tmem16a, Gjb2 or Gjb6.

[0206] E198. The method as described in E197, wherein the polynucleotide encodes Gjb2.

[0207] E199. The method as described in E197 or E196, wherein the promoter is a GLAST promoter or a GJB2 promoter.

[0208] E200. A method for expressing a polynucleotide in Dieter's cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Bdnf, Ntf3, Tectb, Tecta, Ikzf2, Gjb2 or Gjb6.

[0209] E201. The method as described in E200, wherein the polynucleotide encodes Gjb2.

[0210] E202. The method as described in E200 or E201, wherein the promoter is an FGFR3 promoter or a GJB2 promoter.

[0211] E203. A method for expressing a polynucleotide in Hensen's cells and / or Claudius cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Gjb2 or Gjb6.

[0212] E204. The method as described in E203, wherein the polynucleotide encodes Gjb2.

[0213] E205. The method as described in E203 or E204, wherein the promoter is an FRZB promoter or a GJB2 promoter.

[0214] E206. A method for expressing a polynucleotide in spiral prominence cells and / or root cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Slc26a4.

[0215] E207. The method as described in E206, wherein the promoter is an SLC26A4 promoter.

[0216] E208. A method for expressing a polynucleotide in interdental cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Ceacam16, Otoa, Gjb2 or Gjb6.

[0217] E209. The method as described in E208, wherein the polynucleotide encodes Gjb2.

[0218] E210. The method as described in E208 or E209, wherein the promoter is the CEACAM16 promoter or the GJB2 promoter.

[0219] E211. A method for expressing a polynucleotide in glial cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Pmp22, Bdnf, Ntf3 or Mpz.

[0220] E212. The method as described in E211, wherein the polynucleotide encodes Bdnf.

[0221] E213. The method as described in E211, wherein the polynucleotide encodes Ntf3.

[0222] E214. The method as described in any one of E211 - E213, wherein the promoter is the PMP22 promoter.

[0223] E215. A method for expressing a polynucleotide in vestibular dark cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Kcnq1, Kcne1 or Slc26a4.

[0224] E216. The method as described in E215, wherein the promoter is the KCNE1 promoter.

[0225] E217. A method for expressing a polynucleotide in fibroblast cells of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Gjb2, Gjb6 or collagen.

[0226] E218. The method as described in E217, wherein the polynucleotide encodes Gjb2.

[0227] E219. The method as described in E217 or E218, wherein the promoter is the POU3F4 promoter or the GJB2 promoter.

[0228] E220. A method of expressing a polynucleotide in Scarpa ganglion neurons (vestibular ganglion neurons) of a primate subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf or Ntf3 or an shRNA against RGMA.

[0229] E221. The method of E220, wherein the polynucleotide encodes Bdnf.

[0230] E222. The method of E220, wherein the polynucleotide encodes Ntf3.

[0231] E223. The method according to any one of E220 - E222, wherein the promoter is selected from the group consisting of the TUBB3 promoter and the SYN promoter.

[0232] E224. A method of treating a primate subject suffering from or at risk of developing monogenic hereditary deafness associated with a mutation in OTOF, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Otof.

[0233] E225. The method of E224, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, smCBA promoter, MYO15 promoter, and OTOF promoter.

[0234] E226. The method of E225, wherein the promoter is the MYO15 promoter or the OTOF promoter

[0235] E227. A method of treating a primate subject suffering from or at risk of developing monogenic hereditary deafness associated with a mutation in STRC, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Strc.

[0236] E228. The method of E227, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, smCBA promoter, MYO15 promoter, SLC26A5 promoter, OCM promoter, and ATP2B2 promoter.

[0237] E229. The method of E228, wherein the promoter is selected from the group consisting of: MYO15 promoter, SLC26A5 promoter, OCM promoter, and ATP2B2 promoter.

[0238] E230. A method of treating a primate subject suffering from aminoglycoside-induced bilateral vestibular hypofunction or at risk of developing aminoglycoside-induced bilateral vestibular hypofunction, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Atoh1.

[0239] E231. A method of treating a primate subject suffering from bilateral vestibular hypofunction or at risk of developing bilateral vestibular hypofunction, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Atoh1.

[0240] E232. A method of treating a primate subject suffering from age-related vestibular disorder or at risk of developing age-related vestibular disorder, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Atoh1.

[0241] E233. A method of treating a primate subject suffering from vestibular disorder or at risk of developing vestibular disorder, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Atoh1.

[0242] E234. A method of treating a primate subject suffering from hearing loss or at risk of developing hearing loss, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Atoh1.

[0243] E235. The method according to any one of E230-E234, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, smCBA promoter, and GFAP promoter.

[0244] E236. The method according to E235, wherein the promoter is the GFAP promoter.

[0245] E237. A method of treating a primate subject suffering from monogenic hereditary deafness associated with a mutation in GJB2 or at risk of developing monogenic hereditary deafness, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Gjb2.

[0246] E238. A method of treating a primate subject having age - related hearing loss associated with a mutation in GJB2 or at risk of developing age - related hearing loss, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Gjb2.

[0247] E239. The method according to E237 or E238, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, smCBA promoter, and GJB2 promoter.

[0248] E240. The method according to E239, wherein the promoter is the GJB2 promoter.

[0249] E241. A method of treating a primate subject having monogenic hereditary deafness associated with a mutation in SLC17A8 or at risk of developing monogenic hereditary deafness, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Slc17a8.

[0250] E242. The method according to E241, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, smCBA promoter, MYO15 promoter, and OTOF promoter.

[0251] E243. The method according to E242, wherein the promoter is the MYO15 promoter or the OTOF promoter.

[0252] E244. A method of treating a primate subject having monogenic hereditary deafness associated with a mutation in TMC1 or at risk of developing monogenic hereditary deafness, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Tmc1.

[0253] E245. The method according to E244, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, and smCBA promoter.

[0254] E246. The method according to E244, wherein the promoter is the MYO15 promoter.

[0255] E247. A method of treating a primate subject having hearing loss or at risk of developing hearing loss, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0256] E248. A method for treating a primate subject suffering from tinnitus or at risk of developing tinnitus, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0257] E249. A method for treating a primate subject suffering from difficulty in speech understanding in noise or at risk of developing difficulty in speech understanding in noise, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0258] E250. A method for treating a primate subject suffering from Charcot-Marie-Tooth disease or at risk of developing Charcot-Marie-Tooth disease, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0259] E251. A method for treating a primate subject suffering from Friedreich's ataxia or at risk of developing Friedreich's ataxia, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0260] E252. The method according to any one of E247-E251, wherein the promoter is the SYN promoter.

[0261] E253. The method according to any one of E247-E251, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, and smCBA promoter.

[0262] E254. A method for treating a primate subject suffering from hearing loss or at risk of developing hearing loss, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0263] E255. A method for treating a primate subject suffering from tinnitus or at risk of developing tinnitus, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0264] E256. A method for treating a primate subject suffering from or at risk of developing difficulty in speech understanding in noise, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0265] E257. A method for treating a primate subject suffering from or at risk of developing Charcot-Marie-Tooth disease, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0266] E258. A method for treating a primate subject suffering from or at risk of developing Friedreich's ataxia, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0267] E259. The method according to any one of E254-E258, wherein the promoter is the SYN promoter.

[0268] E260. The method according to any one of E254-E258, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, and smCBA promoter.

[0269] E261. A method for treating a primate subject suffering from or at risk of developing bilateral vestibular hypofunction, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox4.

[0270] E262. A method for treating a primate subject suffering from or at risk of developing age-related vestibular disorder, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox4.

[0271] E263. A method for treating a primate subject suffering from or at risk of developing vestibular disorder, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox4.

[0272] E264. A method of treating a primate subject having hearing loss or at risk of developing hearing loss, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox4.

[0273] E265. The method according to any one of E261-E264, wherein the promoter is a GFAP promoter.

[0274] E266. The method according to any one of E261-E264, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, and smCBA promoter.

[0275] E267. A method of treating a primate subject having bilateral vestibular hypofunction or at risk of developing bilateral vestibular hypofunction, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox11.

[0276] E268. A method of treating a primate subject having age-related vestibular disorder or at risk of developing age-related vestibular disorder, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox11.

[0277] E269. A method of treating a primate subject having vestibular disorder or at risk of developing vestibular disorder, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox11.

[0278] E270. A method of treating a primate subject having hearing loss or at risk of developing hearing loss, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox11.

[0279] E271. The method according to any one of E267-E270, wherein the promoter is a GFAP promoter.

[0280] E272. The method according to any one of E267-E270, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, and smCBA promoter.

[0281] E273. A method of treating a primate subject having or at risk of developing monogenic hereditary deafness associated with a mutation in USH1C, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ush1c.

[0282] E274. The method of E273, wherein the promoter is the MYO15 promoter.

[0283] E275. The method of E273, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, and smCBA promoter.

[0284] E276. A method of treating a primate subject having or at risk of developing monogenic hereditary deafness associated with a mutation in MYO7A, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Myo7a.

[0285] E277. The method of E276, wherein the promoter is the MYO15 promoter.

[0286] E278. The method of E276, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, and smCBA promoter.

[0287] E279. A method of treating a primate subject having or at risk of developing monogenic hereditary deafness associated with a mutation in CLRN1, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Clrn1.

[0288] E280. The method of E279, wherein the promoter is the MYO15 promoter.

[0289] E281. The method of E279, wherein the promoter is selected from the group consisting of: CMV promoter, CAG promoter, and smCBA promoter.

[0290] E282. A method of treating a primate subject having or at risk of developing vestibular dysfunction, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide.

[0291] E283. The method according to E282, wherein the promoter is a supporting cell promoter, Scarpa ganglion promoter, vestibular hair cell promoter or vestibular supporting cell promoter listed in Table 5.

[0292] E284. The method according to E283, wherein the promoter is MYO15.

[0293] E285. The method according to any one of E282-E284, wherein the AAV1 vector comprises a polynucleotide for expression in vestibular hair cells and cochlear hair cells, cochlear supporting cells and vestibular supporting cells or Scarpa ganglion selected from the list in Table 2.

[0294] E286. The method according to any one of E282-E285, wherein the polynucleotide encodes Whirlin, Sox9, Sall2, Camta1, Hey2, Gata2, Hey1, Lass2, Sox10, Gata3, Cux1, Nr2f1, Hes1, Rorb, Jun, Zfp667, Lhx3, Nhlh1, Mxd4, Zmiz1, Myt1, Stat3, Barhl1, Tox, Prox1, Nfia, Thrb, Mycl1, Kdm5a, Creb3I4, Etv1, Peg3, Bach2, Isl1, Zbtb38, Lbh, Tub, Hmg20, Rest, Zfp827, Aff3, Pknox2, Arid3b, Mlxip, Zfp532, Ikzf2, Sall1, Six2, Sall3, Lin28b, Pou4f3, Rfx7, Atoh1, an Atoh1 variant comprising a mutation at amino acids 328, 331 and / or 334 (e.g., S328A, S331A, S334A, S328A / S331A, S328A / S334A, S331A / S334A and S328A / S331A / S334, e.g., a variant having the sequence of any one of SEQ ID NOs: 4-10), Gfi1, Sox4, Bdnf, Ntf3, Tead2, Yap1, Tmtc4, Sox11 or a nuclease (e.g., Cas9, TALEN, ZFN or gRNA) or is a microRNA (e.g., miR-183, miR-96 or miR-182).

[0295] E287. The method according to any one of E282-E286, wherein the vestibular dysfunction is vertigo, dizziness or imbalance.

[0296] E288. A method of treating a primate subject having hearing loss (e.g., sensorineural hearing loss, deafness, or auditory neuropathy) or at risk of developing hearing loss by administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide.

[0297] E289. The method of E288, wherein the promoter is an IHC promoter, an OHC promoter, a supporting cell promoter, a cochlear supporting cell subtype promoter, a cochlear hair cell promoter, or an SGN promoter listed in Table 5.

[0298] E290. The method of E288 or E289, wherein the AAV1 vector comprises a polynucleotide for expression in an IHC, an OHC, a cochlear hair cell, a vestibular hair cell, and a cochlear hair cell, a cochlear supporting cell and a vestibular supporting cell, a cochlear supporting cell subtype, or a spiral ganglion neuron selected from the list in Table 2.

[0299] E291. The method of any one of E224 - E290, wherein the hearing loss, deafness, vestibular disorder, vestibular dysfunction, or vestibular hypofunction is associated with a loss of hair cells (e.g., cochlear hair cells and / or vestibular hair cells).

[0300] E292. A method of promoting hair cell regeneration in a primate subject in need thereof by administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide.

[0301] E293. The method of E292, wherein the promoter is a supporting cell promoter, a vestibular supporting cell promoter, or a cochlear supporting cell subtype promoter listed in Table 5.

[0302] E294. The method of E292 or E293, wherein the AAV1 vector contains a polynucleotide for expression in an IHC, an OHC, a cochlear hair cell, a vestibular hair cell, and a cochlear hair cell, a cochlear supporting cell subtype, or a cochlear supporting cell and a vestibular supporting cell selected from the list in Table 2.

[0303] E295. A method of increasing the number of supporting cells (e.g., cochlear and / or vestibular supporting cells, e.g., increasing supporting cell proliferation) in a primate subject in need thereof by administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide.

[0304] E296. The method of E295, wherein the promoter is a supporting cell promoter, a cochlear supporting cell subtype promoter, or a vestibular supporting cell promoter listed in Table 5.

[0305] E297. The method as described in E295 or E296, wherein the AAV1 vector contains a polynucleotide for expression in cochlear supporting cells and vestibular supporting cells or cochlear supporting cell subtypes selected from the list in Table 2.

[0306] E298. A method for preventing or reducing inner ear cell damage or death in a primate subject in need thereof by administering an effective amount of the AAV1 vector of the present invention or the composition of the present invention to the subject.

[0307] E299. The method as described in E298, wherein the inner ear cell damage or death is damage or death caused by ototoxic drugs.

[0308] E300. The method as described in E299, wherein the ototoxic drugs are selected from the group consisting of: aminoglycosides (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), antineoplastic drugs (e.g., platinum-containing chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin), ethacrynic acid, furosemide, salicylates (e.g., aspirin, especially at high doses), and quinine.

[0309] E301. The method as described in E298, wherein the inner ear cell damage or death is caused by acoustic trauma, disease or infection, head trauma, or aging.

[0310] E302. A method for treating a subject by administering an effective amount of the AAV1 vector of the present invention or the composition of the present invention to a subject suffering from tinnitus or at risk of developing tinnitus.

[0311] E303. A method for increasing the survival of inner ear cells in a primate subject in need thereof by administering an effective amount of the AAV1 vector of the present invention or the composition of the present invention to the subject.

[0312] E304. The method as described in any one of E1 - E303, wherein the AAV1 vector is administered locally to the middle ear or inner ear of the subject.

[0313] E305. The method as described in E304, wherein the AAV1 vector is administered through the tympanic membrane or intratympanically.

[0314] E306. The method as described in E304, wherein the AAV1 vector is administered into the perilymph.

[0315] E307. The method as described in E304, wherein the AAV1 vector is administered into the endolymph.

[0316] E308. The method according to E304, wherein the AAV1 vector is administered to or through the oval window.

[0317] E309. The method according to E304, wherein the AAV1 vector is administered to or through the round window.

[0318] E310. The method according to E304, wherein the AAV1 vector is administered to the endolymphatic sac.

[0319] E311. The method according to E304, wherein the AAV1 vector is administered to or through the semicircular canal.

[0320] E312. The method according to any one of E1 - E311, wherein the AAV1 vector comprises inverted terminal repeats (ITRs) from a serotype 2 adeno-associated virus (AAV2) vector.

[0321] E313. The method according to any one of E1 - E312, wherein the AAV1 vector comprises a wild-type AAV1 capsid (e.g., wherein the AAV1 vector contains a capsid protein having the amino acid sequence of SEQ ID NO: 1 - 3).

[0322] E314. The method according to any one of E1 - E313, wherein the AAV1 vector has from about 1 x 10 9 vector genomes (VG) / mL to 1 x 10 15 VG / mL (e.g., 1 x 10 9 VG / mL, 2 x 10 9 VG / mL, 3 x 10 9 VG / mL, 4 x 10 9 VG / mL, 5 x 10 9 VG / mL, 6 x 10 9 VG / mL, 7 x 10 9 VG / mL, 8 x 10 9 VG / mL, 9 x 10 9 VG / mL, 1 x 10 10 VG / mL, 2 x 10 10 VG / mL, 3 x 10 10 VG / mL, 4 x 10 10 VG / mL, 5 x 10 10 VG / mL, 6 x 10 10 VG / mL, 7 x 10 10 VG / mL, 8 x 10 10 VG / mL, 9 x 10 10VG / mL, 1x10 11 VG / mL, 2x10 11 VG / mL, 3x10 11 VG / mL, 4x10 11 VG / mL, 5x10 11 VG / mL, 6x10 11 VG / mL, 7x10 11 VG / mL, 8x10 11 VG / mL, 9x10 11 VG / mL, 1x10 12 VG / mL, 2x10 12 VG / mL, 3x10 12 VG / mL, 4x10 12 VG / mL, 5x10 12 VG / mL, 6x10 12 VG / mL, 7x10 12 VG / mL, 8x10 12 VG / mL, 9x10 12 VG / mL, 1x10 13 VG / mL, 2x10 13 VG / mL, 3x10 13 VG / mL, 4x10 13 VG / mL, 5x10 13 VG / mL, 6x10 13 VG / mL, 7x10 13 VG / mL, 8x10 13 VG / mL, 9x10 13 VG / mL, 1x10 14 VG / mL, 2x10 14 VG / mL, 3x10 14 VG / mL, 4x10 14 VG / mL, 5x10 14 VG / mL, 6x10 14 VG / mL, 7x10 14 VG / mL, 8x10 14 VG / mL, 9x10 14 VG / mL or 1x10 15a titer of VG / mL) and administered 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).

[0323] E315. The method according to E1 - E314, wherein the AAV1 vector is at 1x10 7 vg / ear to 2x10 14 vg / ear (e.g., 1x10 7 vg / ear, 2x10 7 vg / ear, 3x10 7 vg / ear, 4x10 7 vg / ear, 5x10 7 vg / ear, 6x10 7 vg / ear, 7x10 7 vg / ear, 8x10 7 vg / ear, 9x10 7 vg / ear, 1x10 8 vg / ear, 2x10 8 vg / ear, 3x10 8 vg / ear, 4x10 8 vg / ear, 5x10 8 vg / ear, 6x10 8 vg / ear, 7x10 8 vg / ear, 8x10 8 vg / ear, 9x10 8 vg / ear, 1x10 9 vg / ear, 2x10 9 vg / ear, 3x10 9 vg / ear, 4x10 9 vg / ear, 5x10 9 vg / ear, 6x10 9 vg / ear, 7x10 9 vg / ear, 8x10 9 vg / ear, 9x10 9 vg / ear, 1x10 10 vg / ear, 2x10 10 vg / ear, 3x10 10 vg / ear, 4x10 10VG / ear, 5x10 10 VG / ear, 6x10 10 VG / ear, 7x10 10 VG / ear, 8x10 10 VG / ear, 9x10 10 VG / ear, 1x10 11 VG / ear, 2x10 11 VG / ear, 3x10 11 VG / ear, 4x10 11 VG / ear, 5x10 11 VG / ear, 6x10 11 VG / ear, 7x10 11 VG / ear, 8x10 11 VG / ear, 9x10 11 VG / ear, 1x10 12 VG / ear, 2x10 12 VG / ear, 3x10 12 VG / ear, 4x10 12 VG / ear, 5x10 12 VG / ear, 6x10 12 VG / ear, 7x10 12 VG / ear, 8x10 12 VG / ear, 9x10 12 VG / ear, 1x10 13 VG / ear, 2x10 13 VG / ear, 3x10 13 VG / ear, 4x10 13 VG / ear, 5x10 13 VG / ear, 6x10 13 VG / ear, 7x10 13 VG / ear, 8x10 13 VG / ear, 9x10 13 VG / ear, 1x10 14 VG / ear or 2x10 14 administered in a dose of

[0324] E316. The method according to any one of E1 - E315, wherein the primate is a human.

[0325] E317. The method according to any one of E1 - E316, wherein the subject is an adult.

[0326] E318. The method according to any one of E1 - E316, wherein the subject is an adolescent.

[0327] E319. The method according to any one of E1 - E316, wherein the subject is a child.

[0328] E320. The method according to any one of E1 - E316, wherein the subject is an infant or a full - term neonate.

[0329] E321. The method according to any one of E55 - E57 and E134 - E138, wherein the hair cell is a cochlear hair cell.

[0330] E322. The method according to E321, wherein the cochlear hair cell is an IHC.

[0331] E323. The method according to E321, wherein the cochlear hair cell is an OHC.

[0332] E324. The method according to any one of E55 - E57 and E134 - E138, wherein the hair cell is a vestibular hair cell.

[0333] E325. The method according to any one of E48 - E51 and E120 - E130, wherein the supporting cell is a vestibular supporting cell.

[0334] E326. The method according to any one of E48 - E51 and E120 - E130, wherein the supporting cell is a cochlear supporting cell (e.g., Hensen's cell, Deiter's cell, pillar cell, inner phalangeal cell, and / or marginal cell).

[0335] E327. The method according to any one of E1 - E326, wherein the method further comprises evaluating the hearing of the subject before administering the AAV1 vector or composition (e.g., evaluating hearing using standard tests such as audiometry, auditory brainstem response (ABR), electrochocleography (ECOG), or otoacoustic emissions).

[0336] E328. The method according to any one of E1 - E327, wherein the method further comprises evaluating the hearing of the subject after administering the AAV1 vector or composition (e.g., evaluating hearing using standard tests such as audiometry, ABR, ECOG, or otoacoustic emissions).

[0337] E329. The method according to any one of E1-E328, wherein the method further comprises evaluating the vestibular function of the subject before administering the AAV1 vector or composition (e.g., evaluating vestibular function using standard tests such as electronystagmogram (ENG) or videonystagmogram (VNG), posturography, rotary-chair testing, ECOG, vestibular evoked myogenic potential (VEMP), or specialized clinical balance test).

[0338] E330. The method according to any one of E1-E329, wherein the method further comprises evaluating the vestibular function of the subject after administering the AAV1 vector or composition (e.g., evaluating vestibular function using standard tests such as ENG or VNG, posturography, rotary-chair testing, ECOG, VEMP, or specialized clinical balance test).

[0339] E331. The method according to any one of E1 - E330, wherein the AAV1 vector or composition is in an amount sufficient to prevent or mitigate hearing loss, prevent or mitigate vestibular dysfunction, prevent or mitigate tinnitus, delay the development of hearing loss, delay the development of vestibular dysfunction, slow the progression of hearing loss, slow the progression of vestibular dysfunction, improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), promote or induce inner ear cell regeneration (e.g., regeneration of cochlear hair cells, vestibular hair cells or spiral ganglion neurons), increase the number of hair cells (e.g., the number of IHCs, OHCs and / or vestibular hair cells), increase the number of SGNs, increase the number of supporting cells (e.g., cochlear and / or vestibular supporting cells, such as increasing the proliferation of supporting cells), increase the differentiation of supporting cells into hair cells (e.g., causing cochlear supporting cells to differentiate into IHCs and / or OHCs, and / or causing vestibular supporting cells to differentiate into vestibular hair cells), prevent or reduce inner ear cell injury or death (e.g., injury or death of IHCs, OHCs, SGNs, cochlear supporting cells, vestibular supporting cells and / or vestibular hair cells), promote or increase inner ear cell development, promote or increase inner ear cell survival (e.g., increasing the survival of damaged inner ear cells, promoting the repair of damaged inner ear cells, or preserving inner ear cells in a subject at risk of injury or degeneration or loss of inner ear cells), improve inner ear cell function, preserve ribbon synapses, promote or increase ribbon synapse formation, maintain the connection (e.g., synaptic connection) between hair cells and neurons (e.g., SGNs and / or vestibular ganglion neurons) or increase or restore the connection (e.g., synaptic connection) between hair cells and neurons (e.g., SGNs and / or vestibular ganglion neurons).

[0340] E332. The method according to any one of E1 - E331, wherein the subject has hearing loss (e.g., sensorineural hearing loss, deafness or auditory neuropathy) or is at risk of developing hearing loss.

[0341] E333. The method according to any one of E1 - E332, wherein the subject has vestibular dysfunction (e.g., dizziness, vertigo or imbalance) or is at risk of developing vestibular dysfunction.

[0342] E334. The method according to any one of E1 - E333, wherein the subject has previously been diagnosed with vestibular dysfunction (e.g., dizziness, vertigo or imbalance).

[0343] E335. The method according to any one of E1 - E334, wherein the subject has previously been diagnosed with hearing loss (e.g., sensorineural hearing loss, deafness or auditory neuropathy).

[0344] E336. A method according to any one of E234 - E236, E238, E247, E254, E264, E270, E288 - E291, and E331 - E335, wherein the hearing loss is genetic hearing loss.

[0345] E337. A method according to E336, wherein the genetic hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X - linked hearing loss.

[0346] E338. A method according to any one of E234 - E236, E238, E247, E254, E264, E270, E288 - E291, and E331 - E335, wherein the hearing loss is acquired hearing loss.

[0347] E339. A method according to E338, 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.

[0348] E340. A method according to E339, wherein the acquired hearing loss is age - related hearing loss.

[0349] E341. A method according to E339, wherein the hearing loss is noise - induced hearing loss.

[0350] E342. A method according to E339, wherein the hearing loss is ototoxic - drug - induced hearing loss.

[0351] E343. An AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the list of promoters in Table 5.

[0352] E344. The AAV1 vector according to E343, wherein the promoter is the GFAP promoter.

[0353] E345. The AAV1 vector according to E343, wherein the promoter is the SYN promoter.

[0354] The AAV1 vector as described in E343, wherein the promoter is selected from the group consisting of: SLC26A5 promoter, OCM promoter, STRC promoter, MYO15 promoter, OTOF promoter, SLC17A8 promoter, FGF8 promoter, JAG1 promoter, NOTCH1 promoter, MYO7A promoter, GLAST promoter, POU4F3 promoter, GFI1 promoter, KCNQ1 promoter, KCNJ10 promoter, TYR promoter, SOX2 promoter, CALB2 promoter, BHLHE22 promoter, LGR5 promoter, HES1 promoter, HES5 promoter, SOX9 promoter, ATP2B2 promoter, CD44 promoter, FGFR3 promoter, FRZB promoter, SLC26A4 promoter, CEACAM16 promoter, CLDN11 promoter, PMP22 promoter, KCNE1 promoter, POU3F4 promoter, GJB2 promoter and TUBB3 promoter.

[0355] E347. The AAV1 vector as described in E346, wherein the promoter is the SLC26A5 promoter.

[0356] E348. The AAV1 vector as described in E346, wherein the promoter is the OCM promoter.

[0357] E349. The AAV1 vector as described in E346, wherein the promoter is the OTOF promoter.

[0358] E350. The AAV1 vector as described in E346, wherein the promoter is the MYO15 promoter.

[0359] E351. The AAV1 vector as described in E346, wherein the promoter is the ATP2B2 promoter.

[0360] E352. The AAV1 vector as described in E346, wherein the promoter is the GJB2 promoter.

[0361] E353. The AAV1 vector as described in E346, wherein the promoter is the TYR promoter.

[0362] E354. The AAV1 vector as described in any one of E343 - E353, wherein the polynucleotide is selected from the polynucleotides listed in Table 2.

[0363] E355. The AAV1 vector as described in E354, wherein the polynucleotide encodes Otof.

[0364] E356. The AAV1 vector as described in E354, wherein the polynucleotide encodes Strc.

[0365] The AAV1 vector as described in E354, wherein the polynucleotide encodes Atoh1.

[0366] The AAV1 vector as described in E354, wherein the polynucleotide encodes Gjb2.

[0367] The AAV1 vector as described in E354, wherein the polynucleotide encodes Slc17a8.

[0368] The AAV1 vector as described in E354, wherein the polynucleotide encodes Tmc1.

[0369] The AAV1 vector as described in E354, wherein the polynucleotide encodes Ntf3.

[0370] The AAV1 vector as described in E354, wherein the polynucleotide encodes Bdnf.

[0371] The AAV1 vector as described in E354, wherein the polynucleotide encodes Sox4.

[0372] The AAV1 vector as described in E354, wherein the polynucleotide encodes Sox11.

[0373] The AAV1 vector as described in E354, wherein the polynucleotide encodes Ush1c.

[0374] The AAV1 vector as described in E354, wherein the polynucleotide encodes Myo7a.

[0375] The AAV1 vector as described in E354, wherein the polynucleotide encodes Clrn1.

[0376] The AAV1 vector as described in E354, wherein the polynucleotide encodes Chrna9.

[0377] The AAV1 vector as described in E354, wherein the polynucleotide encodes Chrna10.

[0378] The AAV1 vector as described in E354, wherein the polynucleotide encodes Tmtc4.

[0379] The AAV1 vector as described in E354, wherein the polynucleotide encodes Ocm.

[0380] The AAV1 vector as described in E354, wherein the polynucleotide encodes Tyr.

[0381] The AAV1 vector as described in E354, wherein the polynucleotide encodes Tead2.

[0382] The AAV1 vector as described in E354, wherein the polynucleotide encodes Yap1.

[0383] An AAV1 vector comprising a promoter operably linked to a polynucleotide selected from the polynucleotides listed in Table 2.

[0384] The AAV1 vector as described in E375, wherein the polynucleotide encodes Otof.

[0385] The AAV1 vector as described in E375, wherein the polynucleotide encodes Strc.

[0386] The AAV1 vector as described in E375, wherein the polynucleotide encodes Atoh1.

[0387] The AAV1 vector as described in E375, wherein the polynucleotide encodes Gjb2.

[0388] The AAV1 vector as described in E375, wherein the polynucleotide encodes Slc17a8.

[0389] The AAV1 vector as described in E375, wherein the polynucleotide encodes Tmc1.

[0390] The AAV1 vector as described in E375, wherein the polynucleotide encodes Ntf3.

[0391] The AAV1 vector as described in E375, wherein the polynucleotide encodes Bdnf.

[0392] The AAV1 vector as described in E375, wherein the polynucleotide encodes Sox4.

[0393] The AAV1 vector as described in E375, wherein the polynucleotide encodes Sox11.

[0394] The AAV1 vector as described in E375, wherein the polynucleotide encodes Ush1c.

[0395] The AAV1 vector as described in E375, wherein the polynucleotide encodes Myo7a.

[0396] The AAV1 vector as described in E375, wherein the polynucleotide encodes Clrn1.

[0397] The AAV1 vector as described in E375, wherein the polynucleotide encodes Chrna9.

[0398] E390. The AAV1 vector as described in E375, wherein the polynucleotide encodes Chrna10.

[0399] E391. The AAV1 vector as described in E375, wherein the polynucleotide encodes Tmtc4.

[0400] E392. The AAV1 vector as described in E375, wherein the polynucleotide encodes Ocm.

[0401] E393. The AAV1 vector as described in E375, wherein the polynucleotide encodes Tyr.

[0402] E394. The AAV1 vector as described in E375, wherein the polynucleotide encodes Tead2.

[0403] E395. The AAV1 vector as described in E375, wherein the polynucleotide encodes Yap1.

[0404] E396. The AAV1 vector as described in any one of E375 - E395, wherein the promoter is selected from the list of promoters in Table 5.

[0405] E397. The AAV1 vector as described in E396, wherein the promoter is the GFAP promoter.

[0406] E398. The AAV1 vector as described in E396, wherein the promoter is the SYN promoter.

[0407] E399. The AAV1 vector as described in E396, wherein the promoter is selected from the group consisting of: SLC26A5 promoter, OCM promoter, STRC promoter, MYO15 promoter, OTOF promoter, SLC17A8 promoter, FGF8 promoter, JAG1 promoter, NOTCH1 promoter, MYO7A promoter, GLAST promoter, POU4F3 promoter, GFI1 promoter, KCNQ1 promoter, KCNJ10 promoter, TYR promoter, SOX2 promoter, CALB2 promoter, BHLHE22 promoter, LGR5 promoter, HES1 promoter, HES5 promoter, SOX9 promoter, ATP2B2 promoter, CD44 promoter, FGFR3 promoter, FRZB promoter, SLC26A4 promoter, CEACAM16 promoter, CLDN11 promoter, PMP22 promoter, KCNE1 promoter, POU3F4 promoter, GJB2 promoter, and TUBB3 promoter.

[0408] The AAV1 vector as described in E399, wherein the promoter is the SLC26A5 promoter.

[0409] The AAV1 vector as described in E399, wherein the promoter is the OCM promoter.

[0410] The AAV1 vector as described in E399, wherein the promoter is the OTOF promoter.

[0411] The AAV1 vector as described in E399, wherein the promoter is the MYO15 promoter.

[0412] The AAV1 vector as described in E399, wherein the promoter is the ATP2B2 promoter.

[0413] The AAV1 vector as described in E399, wherein the promoter is the GJB2 promoter.

[0414] The AAV1 vector as described in E399, wherein the promoter is the TYR promoter.

[0415] The AAV1 vector as described in any one of E343 - E406, wherein the AAV1 vector comprises AAV2 ITR.

[0416] The AAV1 vector as described in any one of E343 - E407, wherein the AAV1 vector comprises a wild - type AAV1 capsid (for example, wherein the AAV1 vector contains a capsid protein having the amino acid sequence of SEQ ID NO: 1 - 3).

[0417] A pharmaceutical composition, comprising the AAV1 vector as described in any one of E343 - E408.

[0418] The pharmaceutical composition as described in E409, further comprising a pharmaceutically acceptable excipient.

[0419] The pharmaceutical composition as described in E409 or E410, wherein the pharmaceutical composition is formulated for topical administration to the inner ear or middle ear.

[0420] A kit, containing the AAV1 vector as described in any one of E343 - E408 or the pharmaceutical composition as described in any one of E409 - E411.

[0421] In the following description, claims and drawings, these and other aspects of the invention will be apparent to those of ordinary skill in the art.

[0422] Define

[0423] As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0424] As used herein, "administering" means providing or giving a therapeutic agent (e.g., the AAV1 vector described herein) to a subject by any effective route. Exemplary routes of administration are described below.

[0425] As used herein, the terms "adeno-associated virus serotype 1 vector" and "AAV1 vector" refer to adeno-associated virus vectors having an AAV1 capsid. The terms "adeno-associated virus serotype 1 vector" and "AAV1 vector" are used interchangeably herein and refer not only to AAV vectors having wild-type AAV1 capsid proteins (VP1, VP2, and VP3), but also to AAV vectors having AAV1 capsid proteins with sequence modifications that have the broad tropism of wild-type AAV1 in the primate inner ear. AAV1 vectors that can be used in the compositions and methods described herein include those vectors having AAV1 capsid proteins that have at least 90% identity to the amino acid sequences of wild-type AAV1 capsid proteins VP1, VP2, and VP3 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identity to the amino acid sequences of wild-type AAV1 capsid proteins VP1, VP2, and VP3); and those vectors having AAV1 capsid proteins that have one or more conservative amino acid substitutions (e.g., up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 conservative amino acid substitutions) and / or one or more non-conservative amino acid substitutions (e.g., up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 non-conservative amino acid substitutions) relative to the amino acid sequences of wild-type AAV1 capsid proteins VP1, VP2, and VP3. AAV1 vectors that can be used in the compositions and methods described herein do not contain all six of the following amino acid substitutions: L129F, E418D, E531K, F584L, A598V, and N642H. AAV1 vectors that can be used in the compositions and methods described herein can have mutations at one or more surface-exposed tyrosine residues of the capsid protein, such as Tyr252 to Phe272 (Y252F), Tyr272 to Phe272 (Y272F), Tyr444 to Phe444 (Y444F), Tyr500 to Phe500 (Y500F), Tyr700 to Phe700 (Y700F), Tyr704 to Phe704 (Y704F), Tyr730 to Phe730 (Y730F), and Tyr733 to Phe733 (Y733F), which reduce proteasomal degradation.The capsid protein of the AAV1 vector used in the compositions and methods described herein can have the amino acid sequence described in U.S. Patent No. 6,759,237 and be encoded by the polynucleotide sequence described therein, which patent is incorporated herein by reference. The AAV1 capsid protein can also be modified as described in U.S. Patent No. 7,749,492, which patent is incorporated herein by reference. The methods described herein can be used to evaluate tropism in the inner ear.

[0426] As used herein, the term "cell type" refers to a group of cells that share a phenotype that is statistically separable based on gene expression data. For example, cells of a common cell type can share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation feature profiles. Cells of a common cell type can include those cells isolated from a common tissue (e.g., epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those cells isolated from a common organ, tissue system, blood vessel, or other structure and / or region of an organism.

[0427] As used herein, the terms "conservative mutation", "conservative substitution", and "conservative amino acid substitution" refer to the substitution of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, net charge, and steric volume. These properties for each of the 20 naturally occurring amino acids are summarized in Table 1 below.

[0428] Table 1. Representative physicochemical properties of the naturally occurring amino acids

[0429]

[0430] It should be appreciated from this table that the conservative amino acid families include (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Thus, a conservative mutation or substitution is a mutation or substitution in which one amino acid is replaced with a member of the same amino acid family (e.g., Ser replaced with Thr or Lys replaced with Arg).

[0431] As used herein, the term "developed inner ear cell" refers to an inner ear cell that has completed the developmental processes that occur during full-term pregnancy, which is defined as the onset of labor in a primate (e.g., a human) at 37 weeks or later (e.g., 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, or later). The ears of adults, adolescents, children, infants, and full-term neonates all contain developed inner ear cells. Developed inner ear cells express cell-specific markers and can be transduced using the AAV1 vectors described herein (e.g., AAV1 vectors containing wild-type AAV1 capsids).

[0432] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein refer to an amount sufficient to achieve a beneficial or desired result (including a clinical result) when administered to a subject, and thus, the "effective amount" or its synonyms depend on the context in which it is applied. For example, in the context of treating sensorineural hearing loss, tinnitus, or vestibular dysfunction, it is an amount of the composition, vector construct, or viral vector sufficient to achieve a therapeutic response as compared to the response obtained in the absence of administration of the composition, vector construct, or viral vector. In the case of transducing inner ear cells, it is an amount of the composition, vector construct, or viral vector sufficient to transduce one or more inner ear cell types (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more inner ear cell types). The amount of a given composition described herein corresponding to such an amount will vary depending on various factors such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, subject being treated (e.g., age, sex, weight), or identity of the host, etc., but can still be readily determined by one of ordinary skill in the art by conventional methods known in the art. The dosage regimen may be adjusted to provide an optimal therapeutic response.

[0433] As used herein, the term "enhancer" refers to a type of regulatory element that is capable of increasing the efficiency of transcription regardless of the distance or orientation of the enhancer relative to the transcription start site. Thus, an enhancer can be placed upstream or downstream of the transcription start site or at a considerable distance from the promoter. An enhancer can also physically and functionally overlap with a promoter. Many polynucleotides that contain a promoter sequence (e.g., the CMV promoter) also contain an enhancer sequence.

[0434] As used herein, the term "expression" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3′ end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0435] As used herein, the term "heterologous" refers to a combination of elements that are not found in nature. For example, a heterologous transgene refers to a transgene that is not naturally expressed by the promoter to which it is operably linked.

[0436] As used herein, the terms "increase" and "decrease" refer to a modulation that results in a greater or lesser amount of function, expression, or activity, respectively, relative to a reference. For example, after administering a composition by the methods described herein, the amount of a marker of a metric (e.g., transgene expression) as described herein can increase or decrease in a subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more relative to the amount of the marker prior to administration. Typically, after administration, the metric is measured at a time when the administration has achieved the effect, e.g., at least one week, one month, 3 months, or 6 months after the start of a treatment regimen.

[0437] As used herein, the term "inner ear cell type" refers to cell types found in the inner ear (e.g., cochlea and / or vestibular system) of a primate (e.g., human) subject. Inner ear cell types include inner hair cells, outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte cells, Scarpa's ganglion neurons (vestibular ganglion neurons), endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiter's cells, second row of Deiter's cells, third row of Deiter's cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte cells, cells of Reissner's membrane, and glial cells.

[0438] As used herein, "local" or "locally administered" refers to an administration that is intended to have a local rather than a systemic effect on a particular site of the body. Examples of local administration are epidermal, inhalation, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional administration, lymph node administration, intratumoral administration, administration to the inner ear (e.g., into the perilymph or endolymph through the oval window, round window, or horizontal semicircular canal, e.g., intratympanic or transtympanic administration), and administration to the mucosa of a subject, where the administration is intended to produce a local rather than a systemic effect.

[0439] As used herein, the term "operably linked" means that a first molecule is linked to a second molecule, where the molecules are arranged such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, if a promoter regulates the transcription of a target transcribable polynucleotide molecule in a cell, then the promoter is operably linked to the transcribable polynucleotide molecule. Additionally, if two portions of a transcriptional regulatory element are joined together such that the transcriptional activation functionality of one portion is not adversely affected by the presence of the other portion, then the two portions are operably linked to each other. Two transcriptional regulatory elements may be operably linked to each other by a linker nucleic acid (e.g., an intervening non-coding nucleic acid), or may be operably linked to each other in the absence of an intervening nucleotide.

[0440] As used herein, the term "polynucleotide" refers to a polymer of nucleotides. Generally, a polynucleotide is composed of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) that are naturally found in DNA or RNA and are linked by phosphodiester bonds. The term encompasses molecules that contain nucleosides or nucleoside analogs that have chemically or biologically modified bases, modified backbones, etc., whether or not they are present in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to polynucleotides, it should be understood that DNA, RNA, and in each case both single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided. As used herein, a "polynucleotide sequence" may refer to the polynucleotide material itself and / or the sequence information (i.e., the string of letters used as base abbreviations) that biochemically characterizes a particular nucleic acid. Unless otherwise indicated, polynucleotide sequences given herein are presented in the 5' to 3' direction.

[0441] As used herein, the term "promoter" refers to an identification site on DNA to which RNA polymerase binds. The polymerase drives the transcription of a transgene that is operably linked to the promoter.

[0442] The "percent sequence identity (%)" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in the candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. The alignment for determining the percent nucleic acid or amino acid sequence identity can be accomplished in various ways within the capabilities of one of ordinary skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, or Megalign software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. For example, the sequence comparison computer program BLAST can be used to generate percent sequence identity values. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which can alternatively be phrased as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows:

[0443] 100 × (fraction X / Y)

[0444] where X is the number of nucleotides or amino acids scored as identical matches in the alignment of A and B by the sequence alignment program (e.g., BLAST), and where Y is the total number of nucleic acids in B. It should be understood that in the case where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0445] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, to be administered to a subject for preventing, treating, or controlling a particular disease or disorder that affects or may affect the subject.

[0446] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject without undue toxicity, irritation, allergic response, and other problem complications and are commensurate with a reasonable benefit / risk ratio.

[0447] As used herein, the term "sample" refers to a specimen isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., neural tissue, placental tissue, or dermal tissue), pancreatic juice, chorion, and cells (e.g., inner ear cells or stem cells).

[0448] As used herein, the term "transcription regulatory element" refers to a nucleic acid that at least partially controls the transcription of a polynucleotide. Transcription regulatory elements can include promoters, enhancers, and other nucleic acids that control or assist in controlling gene transcription (e.g., polyadenylation signals). Examples of transcription regulatory elements are described in, for example, Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0449] As used herein, the terms "subject" and "patient" refer to a primate (e.g., a human). A subject to be treated according to the methods described herein can be a subject that has been diagnosed with hearing loss (e.g., sensorineural hearing loss, auditory neuropathy, or deafness), tinnitus, or vestibular dysfunction (e.g., dizziness, vertigo, or imbalance), or a subject at risk of developing these disorders (e.g., a subject at risk of developing hearing loss, tinnitus, or vestibular dysfunction due to age, head trauma, acoustic trauma (e.g., exposure to loud noise), disease or infection, treatment with ototoxic drugs, gene mutations, or a family history of hearing loss, tinnitus, or vestibular dysfunction). Diagnosis can be made by any method or technique known in the art. One of ordinary skill in the art will understand that a subject to be treated according to the present disclosure may have already undergone standard testing or may be identified as at risk without examination due to the presence of one or more risk factors associated with the disease or disorder.

[0450] As used herein, the terms “transduction” and “transduce” refer to methods of introducing a vector construct or a portion thereof into a cell. Where the vector construct is contained within a viral vector such as an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid), transduction refers to viral infection of the cell and subsequent transfer and integration of the vector construct or a portion thereof into the cell genome. As used herein, the amount of AAV1 vector required to “transduce inner ear cell types” is defined as the amount that transduces at least 20% of the cells of a given cell type or all inner ear cell types (e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the cells of a given cell type, such as 20%-25%, 20%-30%, 20%-35%, 20%-40%, 20%-45%, 20%-50%, 20%-55%, 20%-60%, 20%-65%, 20%-70%, 20%-75%, 20%-80%, 20%-85%, 20%-90%, 20%-95%, 20%-100%, 25%-30%, 25%-35%, 25%-40%, 25%-45%, 25%-50%, 25%-55%, 25%-60%, 25%-65%, 25%-70%, 25%-75%, 25%-80%, 25%-85%, 25%-90%, 25%-95%, 25%-100%, 30%-35%, 30%-40%, 30%-45%, 30%-50%, 30%-55%, 30%-60%, 30%-65%, 30%-70%, 30%-75%, 30%-80%, 30%-85%, 30%-90%, 30%-95%, 30%-100%, 35%-40%, 35%-45%, 35%-50%, 35%-55%, 35%-60%, 35%-65%, 35%-70%, 35%-75%, 35%-80%, 35%-85%, 35%-90%, 35%-95%, 35%-100%, 40%-45%, 40%-50%, 40%-55%, 40%-60%, 40%-65%, 40%-70%, 40%-75%, 40%-80%, 40%-85%, 40%-90%, 40%-95%, 40%-100%, 45%-50%, 45%-55%, 45%-60%, 45%-65%, 45%-70%, 45%-75%, 45%-80%, 45%-85%, 45%-90%, 45%-95%, 45%-100%, 50%-55%, 50%-60%, 50%-65%, 50%-70%, 50%-75%, 50%-80%, 50%-85%,The amount required for cells of a given cell type or all inner ear cell types that are 50%-90%, 50%-95%, 50%-100%, 55%-60%, 55%-65%, 55%-70%, 55%-75%, 55%-80%, 55%-85%, 55%-90%, 55%-95%, 55%-100%, 60%-65%, 60%-70%, 60%-75%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-70%, 65%-75%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-75%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100% or 95%-100%.

[0451] As used herein, "treatment" and "treating" with respect to a disease or disorder refer to methods for obtaining a beneficial or desired result, such as a clinical result. Beneficial or desired results can include, but are not limited to, alleviation or improvement of one or more symptoms or disorders; reduction in the degree of a disease or disorder; stabilization (i.e., non-worsening) of the state of a disease, condition or disorder; prevention of the spread of a disease or disorder; delay or slowing of the progression of a disease or disorder; amelioration or mitigation of a disease or disorder; and remission (whether partial or complete), whether detectable or not. "Improving" or "mitigating" a disease or disorder means that the degree and / or adverse clinical manifestations of the disease, condition or disorder are reduced and / or the time course of progression is slowed or lengthened compared to the degree or time course without treatment. "Treatment" can also mean prolonging survival compared to the expected survival if treatment is not received. Those in need of treatment include those who already have a disorder or condition, those who are predisposed to having a disorder or condition, or those who intend to prevent a disorder or condition. Brief Description of the Drawings

[0452] Figure 1A - 1B Are a series of fluorescence images of the inner ear of 9-10-week-old mice treated with an AAV1 vector expressing EGFP under the control of the CMV promoter. In CBA / CaJ mice (n = 6), a single injection of AAV1-CMV-GFP (virus titer of 2.94×10 13copies / mL). Two weeks after injection, the inner ear was removed and the surface preparation of the basilar membrane was performed. A representative ear is shown. EGFP expression was observed in the apical, middle, and basal turns of the cochlea ( Figure 1A ; upper left panel: apex of the cochlea; lower panel: middle turn of the cochlea; upper right panel: basal turn of the cochlea). High magnification images showed EGFP expression in inner hair cells (IHC), outer hair cells (OHC) in the apical region, and spiral limbus cells ( Figure 1B ).

[0453] Figure 2A - 2B are a series of fluorescence images of the inner ear of 9-10-week-old mice treated with the AAV7m8 vector expressing EGFP under the control of the CAG promoter. In 9-10-week-old CBA / CaJ mice (n = 8), unilateral injection was performed to administer 1 μL of AAV7m8-CAG-EGFP (viral titer of 9.4 × 10 12 copies / mL) into the posterior semicircular canal of the inner ear at a flow rate of 0.1 μL / min. Two weeks after injection, the inner ear was removed and the surface preparation of the basilar membrane was performed. A representative ear is shown. EGFP expression was observed in the apical, middle, and basal turns of the cochlea ( Figure 2A ). High magnification images showed EGFP expression in multiple cell types, including IHC, OHC, and cells of the spiral ganglion and spiral limbus throughout the basal-to-apical extent of the cochlea ( Figure 2B ; left panel: apex of the cochlea; middle panel: middle turn of the cochlea; right panel: basal turn of the cochlea).

[0454] Figure 3A - 3B are a series of fluorescence images of the inner ear of rhesus monkeys treated with the AAV2 vector expressing GFP under the control of the CMV promoter. Unilateral injection (n = 1 animal) and bilateral injection (n = 2 animals) were used to administer 30 μL of AAV2-CMV-GFP (viral titer of 3.39 × 10 12 copies / mL) into the round window of the inner ear at a flow rate of 15 μL / min. Four weeks after injection, the inner ear was removed and the surface preparation of the basilar membrane was performed. A representative ear is shown. Immunohistochemistry for Myo7A was used to identify hair cells ( Figure 3A , images in the right column), and GFP expression was used to monitor AAV1 infectivity ( Figure 3A , images in the left column). AAV2-CMV-GFP transduction of the rhesus monkey inner ear resulted in GFP expression across the basal-apical axis of the entire cochlea ( Figure 3A ; upper left panel: apex of the cochlea, upper middle panel: middle turn of the cochlea; lower left panel: basal turn of the cochlea). Cochlear GFP expression was observed in some cells within the IHC and spiral limbus ( Figure 3A; (images of the left column). GFP expression was quantified in IHCs along the cochlear frequency map, demonstrating that GFP was expressed in 40%-80% of IHCs ( Figure 3B ).

[0455] Figure 4A - 4F are a series of images of paraffin sections of the inner ear of cynomolgus monkeys treated with an adeno-associated virus serotype 7m8 (AAV7m8) vector expressing green fluorescent protein (GFP) under the control of the CAG promoter. Bilateral injections were used to administer 30 μL of AAV7m8-CAG-EGFP (viral titer of 9.4×10 12 genomic copies / mL) to the round window of the inner ear at a flow rate of 15 μL / min. Two weeks (n = 1 animal) or four weeks (n = 1 animal) after injection, the inner ears were removed and embedded in paraffin for sectioning, fixation, immunostaining with an anti-GFP antibody, and histological analysis. In one animal, two weeks after transduction of the inner ear with AAV7m8-CAG-EGFP, EGFP expression was observed at the basal turn of the cochlea ( Figure 4A , upper panel: the dashed square indicates the basal turn of the cochlea), as visualized by immunostaining with EGFP. EGFP was expressed in IHCs, OHCs, and fibrocytes of the spiral ligament ( Figure 4B , Figure 4A magnified view of the dashed square in; upper left inset: magnified view of the organ of Corti). In another animal, four weeks after injection, EGFP expression was observed across the basal-apical axis of the cochlea ( Figure 4C , the dashed square indicates the basal turn of the cochlea and is magnified in Figure 4D ). EGFP was expressed in IHCs, OHCs, and fibrocytes of the spiral ligament ( Figure 4D , the organ of Corti is magnified in the inset in the upper left corner of the figure). As Figure 4E shown, in the absence of the primary anti-GFP antibody, the secondary antibody did not stain the cochlea (the dashed square indicates the basal turn of the cochlea). No staining was observed at the base ( Figure 4F , Figure 4E magnification of the square in; the organ of Corti is magnified in the upper left inset).

[0456] Figure 5A - 5E are a series of images of paraffin sections of the inner ear of cynomolgus monkeys treated with an AAV1 vector expressing green fluorescent protein (GFP) under the control of the CAG promoter. Bilateral injections were used to administer 30 μL of AAV1-CAG-GFP (viral titer of 9.9×10 12 genomic copies / mL) to the round window of the inner ear. Four weeks after injection, the inner ears were removed and embedded in paraffin for sectioning, fixation, immunostaining with an anti-GFP antibody, and histological analysis. As Figure 5AAs shown, staining with only the secondary antibody in the absence of anti-GFP antibody did not stain the cochlea, thus confirming the specificity of the antibody. AAV1-CAG-GFP transduction of the inner ear resulted in robust GFP expression throughout the cochlea and saccule of the inner ear ( Figure 5B , the large circle in the upper left indicates the apical turn of the cochlea, the small oval on the left indicates the stria vascularis, and the small circle in the lower right corner indicates the saccule), as visualized using immunostaining for GFP. In the apical turn of the cochlea ( Figure 5C , magnification of the upper left circle from Figure 5B ) and all layers of the saccule ( Figure 5E , magnification of the lower right circle from Figure 5B ), immunostaining for GFP was detected. Robust GFP expression was detected in the spiral ligament and in marginal cells and intermediate cells of the stria vascularis. In this section, the basal cells of the stria vascularis did not show GFP expression, but GFP expression was visible in basal cells in other turns of the cochlea ( Figure 5D ).

[0457] Figure 6A - 6C are a series of fluorescence images of the inner ear of rhesus monkeys treated with an AAV1 vector expressing GFP under the control of the CMV promoter. Unilateral injection (n = 1 animal) and bilateral injection (n = 2 animals) were used to administer 30 μL of AAV1-CMV-GFP (viral titer of 2.01×10 13 genome copies / mL) into the round window of the inner ear at a flow rate of 15 μL / min. Four weeks after injection, the inner ear was removed and surface preparations of the basilar membrane, utricle, and cristae were performed. One representative inner ear is shown. Immunohistochemistry for Myo7A was used to visualize hair cells, and GFP expression was used to monitor AAV1 infectivity ( Figure 6A - 6C ). AAV1-CMV-GFP transduction of the inner ear of rhesus monkeys resulted in strong pan-pyotropic expression of GFP across the entire basal-apical axis of the cochlea ( Figure 6A ). Cochlear GFP expression was observed in inner hair cells (IHC), outer hair cells (OHC), and supporting cells, as well as in the non-sensory spiral ligament and spiral limbus ( Figure 6B , Figure 6A , magnification of the boxed area in the middle turn of Figure 6C ). Vestibular structures such as the utricle and cristae also showed significant GFP expression in sensory and non-sensory regions (

[0458] Figure 7A - 7F are a series of images of paraffin sections of the inner ear of cynomolgus monkeys treated with an AAV1 vector expressing GFP under the control of the CAG promoter. Bilateral injection was used to administer 30 μL of AAV1-CAG-GFP (3.15×10 10 or 3.15×10 11Genome copies / ear were administered to the round window of the inner ear. Four weeks after injection, the inner ear was removed and embedded in paraffin for sectioning, fixation, immunostaining with anti-GFP antibody, and histological analysis. As Figure 7A shown, the anti-GFP antibody did not stain the cochlea of the ears that were not injected with AAV, thus confirming the specificity of the antibody. Figure 7B shows that transduction of the inner ear with AAV1-CAG-GFP (3.15×10 10 genome copies / ear) resulted in robust GFP expression throughout the cochlea, as visualized by immunostaining with GFP. Immunostaining for GFP was detected in hair cells, supporting cells, inner sulcus cells, outer sulcus cells, and cells of the spiral ligament. As Figure 7C shown, in these cell types, a higher dose of AAV-CAG-GFP (3.15×10 11 genome copies / ear) showed stronger GFP expression. Figure 7D - 7F shows the same images as Figure 7A - 7C respectively, in which the nuclear staining was removed (i.e., these images show only the GFP labeling). All scale bars are 100 μm. DETAILED DESCRIPTION

[0459] Compositions and methods for transducing inner ear cells of developing primates (e.g., humans), such as cells of the cochlea and / or vestibular system, such as inner hair cells, outer hair cells, vestibular hair cells, cochlear supporting cells, and vestibular supporting cells, are described herein. The present invention features methods for transducing inner ear cells of a developing primate (e.g., human) subject (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more different types of developing inner ear cells) using a serotype 1 adeno-associated virus (AAV1) vector. The present invention further features methods for expressing a polynucleotide in a specific inner ear cell type (e.g., outer hair cells, vestibular hair cells, or vestibular supporting cells) in a primate (e.g., human) subject using an AAV1 vector, and AAV1 vectors containing a promoter (e.g., a promoter that induces gene expression in one or more inner ear cell types) and / or a polynucleotide (e.g., a polynucleotide corresponding to a gene encoding a therapeutic protein, such as a gene expressed in normal inner ear cells, or a polynucleotide encoding a protein that regulates inner ear cell survival, regeneration, cell fate, and / or cell proliferation). The compositions and methods described herein can be used to express one or more polynucleotides in all or a subset of inner ear cells of a primate (e.g., human), and thus the compositions described herein can be administered to a primate (e.g., human) subject to treat conditions caused by damage, degeneration, loss, and / or dysfunction of inner ear cells, such as hearing loss (e.g., sensorineural hearing loss), tinnitus, or vestibular dysfunction.

[0460] Inner ear cells

[0461] The inner ear consists of many specialized cells. Both the cochlea and the vestibular system contain hair cells, which are the main sensory cells of the inner ear. Cochlear hair cells are composed of two main cell types: inner hair cells (IHCs) that are responsible for sensing sound and outer hair cells (OHCs) that are thought to amplify low-volume sounds. Vestibular hair cells are located in the semicircular canals and otolith organs (e.g., utricle and saccule) of the vestibular apparatus and are involved in the sense of movement, which contributes to the sense of balance and spatial orientation. Spiral ganglion neurons innervate the cochlear hair cells and send axons into the central nervous system, while neurons of the vestibular ganglion innervate the vestibular hair cells. Non-sensory cells called supporting cells are located between the hair cells of the cochlea and the vestibular system and perform many important functions, such as providing a structural scaffold to allow mechanical stimulation of the hair cells, maintaining the ionic composition of endolymph and perilymph, and regulating synaptogenesis at ribbon synapses. Within the cochlea, supporting cells can be divided into five different types: 1) Hensen's cells, 2) Deiters' cells, 3) pillar cells; 4) inner phalangeal cells; and 5) border cells, all of which have different morphologies and gene expression patterns. Mutations in genes expressed in cochlear hair cells, cochlear supporting cells, and / or spiral ganglion neurons have been associated with hearing loss (e.g., sensorineural hearing loss), auditory neuropathy, deafness, and tinnitus due to damage, injury, degeneration, or loss (e.g., death) of these cells. Similarly, mutations in genes expressed in vestibular system cells (e.g., vestibular hair cells, vestibular supporting cells, and / or vestibular ganglion neurons) and damage, injury, degeneration, or loss (e.g., death) of vestibular system cells have been associated with vestibular dysfunction (e.g., vertigo, dizziness, and / or imbalance). Gene therapy has recently emerged as an attractive treatment for hearing loss and vestibular dysfunction; however, given the potential need to target a large number of cell types to address the various causes of hearing loss and vestibular dysfunction, there is a need for viral vectors with broad tropism to transduce many cell types in the inner ear of primates (e.g., humans).

[0462] The present invention is in part based on the discovery that administration of an AAV1 vector containing a ubiquitous promoter operably linked to green fluorescent protein (GFP) to the inner ear of adult non-human primates results in pan-tropic transduction and strong GFP expression in most or all cell types of the inner ear cells, in contrast to the tropism observed with other AAV serotypes. Without wishing to be bound by theory, the high similarity between humans and non-human primates suggests that AAV1 vectors can be used to transduce most or all developed human inner ear cells (e.g., most or all inner ear cells in human adults, adolescents, children, or full-term neonates). Thus, the compositions and methods described herein can be used to induce the expression of polynucleotides in all cells of the inner ear of a developing primate (e.g., human), or they can be used to induce the expression of polynucleotides in a specific region (e.g., the cochlea or vestibular system) or cell type (e.g., one or more inner ear cell types, such as inner hair cells, outer hair cells, vestibular hair cells, cochlear supporting cells, and / or vestibular supporting cells) of the inner ear based on the promoter contained in the AAV1 vector. Accordingly, the methods and compositions described herein can be administered to a primate (e.g., human) subject to treat a disorder caused by a genetic mutation or damage, degeneration, loss, and / or dysfunction in all cell types of the inner ear, or to treat a disorder caused by a genetic mutation or damage, degeneration, loss, and / or dysfunction in one or a subset of inner ear cell types.

[0463] In some embodiments, an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid) is administered to the developing inner ear of a primate (e.g., human) subject in an amount sufficient to transduce three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more) inner ear cell types selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa's ganglion neurons (vestibular ganglion neurons), endothelial cells of the vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiter's cells, second row of Deiter's cells, third row of Deiter's cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, strial basal cells, strial intermediate cells, strial marginal cells, spiral ganglion neurons, endothelial cells of the cochlear capillaries, fibrocyte, cells and glial cells of Reissner's membrane. Pan-tropic transduction of inner ear cells by the AAV1 vector can be used to induce expression of a polynucleotide throughout the inner ear (e.g., in an AAV1 vector in which the polynucleotide is operably linked to a ubiquitous promoter). The ability to transduce three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) different inner ear cell types is beneficial for therapeutic methods in which it is desired to express a polynucleotide in many or most cell types of the inner ear, e.g., to deliver a wild-type form of a gene mutated in all inner ear cell types or to produce a large amount of a secreted protein that has a therapeutic effect at high concentrations.

[0464] In some embodiments, an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid) is administered to a primate (e.g., human) subject to express a polynucleotide in a specific cell type (e.g., a supporting cell such as a cochlear and / or vestibular supporting cell; OHC; vestibular hair cell; or spiral ganglion neuron) or region (e.g., the cochlear or vestibular system). A cell type-specific promoter can be included in the AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid) to induce gene expression in one or more cell types. Cell type-specific expression can be used to mimic the endogenous expression pattern of a polynucleotide that is naturally expressed only in one or a few cell types. This approach is beneficial because it can be used to avoid ectopic expression of the polynucleotide in cells in which it is not normally expressed.

[0465] Expression of exogenous polynucleotides in human cells

[0466] Mutations in multiple genes, such as myosin 7A (MYO7A), POU class 4 homeobox 3 (POU4F3), solute carrier family 17 member 8 (SLC17A8), gap junction protein beta 2 (GJB2), claudin 14 (CLDN14), cochlin (COCH), protocadherin related 15 (PCDH15), and transmembrane channel-like 1 (TMC1), have been associated with sensorineural hearing loss and / or deafness, and some of these mutations (such as mutations in MYO7A, POU4F3, and COCH) are also associated with vestibular dysfunction. The compositions and methods described herein can be used to induce or increase the expression of a protein encoded by a polynucleotide in developing human inner ear cells (e.g., a nucleic acid corresponding to a gene expressed in healthy inner ear cells, such as the wild-type form of a gene involved in hearing loss and / or vestibular dysfunction, or a gene involved in inner ear cell development, function, cell fate determination, regeneration, survival, proliferation, and / or maintenance). An AAV1 vector containing a polynucleotide (e.g., an AAV1 vector containing a wild-type AAV1 capsid) can be administered to a human subject (e.g., to the inner ear of a subject) to induce or increase the expression of a protein encoded by the polynucleotide in one or more inner ear cell types. Extensive methods have been established for delivering proteins to human cells and for stably expressing a polynucleotide encoding a protein in human cells.

[0467] The AAV1 vectors described herein can be used to express a polynucleotide in one or more inner ear cell types. In some embodiments, the AAV1 vectors described herein can be used to express two or more (e.g., 2, 3, 4, or more) polynucleotides in one or more cell types. A list of inner ear cell types and polynucleotides that can be expressed in each cell type is provided in Table 2 below. Accession numbers for the polynucleotides of Table 2 are provided in Table 3.

[0468] Table 2: Polynucleotides That Can Be Expressed in One or More Inner Ear Cell Types

[0469]

[0470]

[0471]

[0472] Table 3: Accession Numbers for the Polynucleotides Listed in Table 2

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482] Table 4: Amino acid sequences of Atoh1 variants

[0483]

[0484]

[0485] In some embodiments, the polynucleotide is or encodes a component of a gene editing system. An AAV1 vector comprising a component of a gene editing system operably linked to a cell type-specific promoter (e.g., a cell type-specific promoter listed in Table 5) can be used for cell type-specific gene editing. For example, a component of a gene editing system can be used to introduce alterations (e.g., insertions, deletions (e.g., knockouts), translocations, inversions, single point mutations, or other mutations) into genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction. Exemplary gene editing systems include zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) systems. ZFN-, TALEN-, and CRISPR-based methods are described, for example, in Gaj et al., Trends Biotechnol. 31:397-405, 2013.

[0486] CRISPR refers to a set (or includes a set) of clustered regularly interspaced short palindromic repeats (system). A CRISPR system refers to a system derived from CRISPR and Cas (a CRISPR-associated protein) or another nuclease, which can be used to silence or mutate the genes described herein. The CRISPR system is a natural system found in bacterial and archaeal genomes. The CRISPR locus consists of alternating repeat sequences and spacer sequences. In a naturally occurring CRISPR system, the spacers are typically sequences foreign to the bacterium (e.g., plasmid or phage sequences). The CRISPR system has been modified for gene editing in eukaryotes (e.g., altering, silencing, and / or enhancing certain genes). See, e.g., Wiedenheft et al., Nature 482:331, 2012. For example, such modification of the system includes introducing a plasmid containing a specifically designed CRISPR and one or more appropriate Cas proteins into a eukaryotic cell. The CRISPR locus is transcribed into RNA and processed by Cas proteins into small RNAs that contain repeat sequences flanking the spacer. Depending on the spacer sequence, the RNA acts as a guide to direct the Cas protein to silence a specific DNA / RNA sequence. See, e.g., Horvath et al., Science 327:167, 2010; Makarova et al., Biology Direct 1:7, 2006; Pennisi, Science 341:833, 2013. In some instances, the CRISPR system includes the Cas9 protein, a nuclease that cuts both strands of DNA. See, e.g., supra.

[0487] In some embodiments, in the CRISPR systems described herein for use, e.g., according to one or more of the methods described herein, the spacer of the CRISPR is derived from a target gene sequence, e.g., from a gene known to regulate the function of inner ear cells, such as a gene implicated in sensorineural hearing loss or vestibular dysfunction.

[0488] In some embodiments, the polynucleotide comprises a guide RNA (gRNA) in a clustered regularly interspaced short palindromic repeats (CRISPR) system for gene editing. In some embodiments, the polynucleotide comprises or encodes a zinc finger nuclease (ZFN) or an mRNA encoding a ZFN that targets (e.g., cleaves) a nucleic acid sequence (e.g., a DNA sequence) of a gene known to regulate inner ear cell function, such as a gene associated with sensorineural hearing loss or vestibular dysfunction. In some embodiments, the polynucleotide comprises or encodes a transcription activator-like effector nuclease (TALEN) or an mRNA encoding a TALEN that targets (e.g., cleaves) a nucleic acid sequence (e.g., a DNA sequence) of a gene known to regulate inner ear cell function, such as a gene associated with sensorineural hearing loss or vestibular dysfunction.

[0489] For example, a gRNA can be used in a CRISPR system to engineer an alteration of a gene (e.g., a gene known to regulate inner ear cell function, such as a gene involved in sensorineural hearing loss or vestibular dysfunction). In other examples, ZFNs and / or TALENs can be used to engineer an alteration of a gene (e.g., a gene known to regulate inner ear cell function, such as a gene involved in sensorineural hearing loss or vestibular dysfunction). Exemplary alterations include insertions, deletions (e.g., knockouts), translocations, inversions, single point mutations, or other mutations. The alteration can be introduced into the gene in a cell, for example, in vitro, ex vivo, or in vivo. In some embodiments, the alteration reduces (e.g., knockdowns or knocks out) the level and / or activity of a gene known to regulate inner ear cell function, such as a gene involved in sensorineural hearing loss or vestibular dysfunction, e.g., the alteration is a negative regulator of function. In another example, the alteration corrects a defect (e.g., a mutation causing the defect) in a gene known to regulate inner ear cell function, such as a gene involved in sensorineural hearing loss or vestibular dysfunction.

[0490] In certain embodiments, the CRISPR system is used to edit (e.g., add or delete base pairs) a target gene, such as a gene known to regulate inner ear cell function, such as a gene involved in sensorineural hearing loss or vestibular dysfunction. In other embodiments, the CRISPR system is used to introduce a premature stop codon, e.g., thereby reducing the expression of the target gene. In other embodiments, the CRISPR system is used to reversibly turn off a target gene, e.g., similar to RNA interference. In some embodiments, the CRISPR system is used to direct Cas to the promoter of a target gene, such as a gene known to regulate inner ear cell function, such as a gene involved in sensorineural hearing loss or vestibular dysfunction, thereby spatially blocking RNA polymerase.

[0491] In some embodiments, techniques such as those described in U.S. Publication No. 20140068797; Cong, Science 339:819, 2013; Tsai, Nature Biotechnol., 32:569, 2014; and U.S. Patent Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359 can be used to generate CRISPR systems to edit genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction.

[0492] In some embodiments, CRISPR interference (CRISPRi) technology can be used for transcriptional repression of specific genes, such as genes encoding genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction. In CRISPRi, an engineered Cas9 protein (e.g., nuclease - free dCas9 or a dCas9 fusion protein, e.g., dCas9–KRAB or dCas9–SID4X fusion protein) can be paired with a sequence - specific guide RNA (sgRNA). The Cas9 - gRNA complex can block RNA polymerase, thereby interfering with transcription elongation. The complex can also block transcription initiation by interfering with transcription factor binding. The CRISPRi method is specific, has minimal off - target effects, and is reusable, e.g., more than one gene can be repressed simultaneously (e.g., using multiple gRNAs). Also, the CRISPRi method allows reversible gene repression.

[0493] In some embodiments, CRISPR-mediated gene activation (CRISPRa) can be used for transcriptional activation of one or more genes as described herein, such as genes known to regulate inner ear cell function, e.g., genes involved in sensorineural hearing loss or vestibular dysfunction. In CRISPRa technology, the dCas9 fusion protein recruits transcriptional activators. For example, dCas9 can be used to recruit polypeptides (e.g., activation domains), such as the VP64 or p65 activation domain (p65D), and used in conjunction with sgRNA (e.g., a single sgRNA or multiple sgRNAs) to activate one or more genes, such as one or more endogenous genes. Activation efficiency can be increased by using multiple sgRNAs to recruit multiple activators - multiple activation domains and single or multiple activation domains can be used. In addition to engineering dCas9 to recruit activators, sgRNAs can also be engineered to recruit activators. For example, RNA aptamers can be incorporated into sgRNAs to recruit proteins (e.g., activation domains), such as VP64. In some instances, the synergistic activation mediator (SAM) system can be used for transcriptional activation. In SAM, the MS2 aptamer is added to the sgRNA. The MS2 recruits the MS2 coat protein (MCP) fused to p65AD and heat shock factor 1 (HSF1). CRISPRi and CRISPRa technologies are described in detail, e.g., in Dominguez et al., Nat. Rev. Mol. Cell Biol. 17:5, 2016, which is incorporated herein by reference.

[0494] A polynucleotide encoding a target protein

[0495] One platform that can be used to achieve therapeutically effective intracellular target protein concentrations in primate (e.g., human) cells is through stable expression of a nucleic acid encoding the target protein (e.g., by integration into the nuclear or mitochondrial genome of a primate (e.g., human) cell, or by formation of an episomal concatemer in the nucleus of a primate (e.g., human) cell). The nucleic acid is a polynucleotide encoding the primary amino acid sequence of the corresponding protein. To introduce a foreign gene into a primate (e.g., human) cell, the gene can be incorporated into a vector, such as an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid). To introduce into inner ear cells, the AAV1 vector can be locally administered to the inner ear of a primate (e.g., human) subject.

[0496] The recognition and binding of a primate (e.g., human) RNA polymerase to a polynucleotide encoding a target protein is important for gene expression. Accordingly, sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and facilitate the assembly of the transcription complex at the transcription start site can be included within the polynucleotide. Such sequence elements include, for example, promoters, the sequences of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of promoters have been described in the online publication by Smith et al., Mol. Sys. Biol., 3:73, the disclosure of which is incorporated herein by reference. The promoters used in the methods and compositions described herein can be ubiquitous promoters (e.g., to induce or increase the expression of the polynucleotide in all cells of the inner ear) or cell type-specific promoters (e.g., to induce or increase the expression of the polynucleotide in one or more inner ear cell types). Ubiquitous promoters include the CAG promoter, the cytomegalovirus (CMV) promoter, the smCBA promoter (described in Haire et al., Invest. Opthalmol. Vis. Sci. 47:3745-3753, 2006), the dihydrofolate reductase (DHFR) promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1α promoter. Alternatively, promoters derived from viral genomes can also be used for stable expression of polynucleotides in primate (e.g., human) cells. Examples of functional viral promoters that can be used for expressing polynucleotides in primate (e.g., human) cells include the adenovirus late promoter, the vaccinia virus 7.5K promoter, the tk promoter of HSV, the mouse mammary tumor virus (MMTV) promoter, the LTR promoter of HIV, the promoter of Moloney virus, the Epstein-Barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter.

[0497] Table 5 below provides cell type-specific promoters of nucleic acids that can be included in the AAV1 vectors described herein for expressing a polynucleotide encoding a target protein (e.g., the polynucleotides listed in Table 2) in one or more inner ear cell types.

[0498] Table 5: Inner ear cell type-specific promoters

[0499]

[0500] Once a polynucleotide encoding a target protein is incorporated into a primate (e.g., human) cell, transcription of this polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing a primate (e.g., human) cell to an external chemical reagent, such as an agent that modulates the binding of transcription factors and / or RNA polymerase to a promoter and thus regulates gene expression. Chemical reagents can be used to promote the binding of RNA polymerase and / or transcription factors to a promoter, for example, by removing a repressor protein that has bound to the promoter. Alternatively, chemical reagents can be used to enhance the affinity of a promoter for RNA polymerase and / or transcription factors such that, in the presence of the chemical reagent, the transcription rate of a gene located downstream of the promoter is increased. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to primate (e.g., human) cells according to established protocols to promote gene expression.

[0501] Other DNA sequence elements that can be included in the polynucleotides used in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in a polynucleotide containing the polynucleotide such that the DNA adopts a three-dimensional orientation that is favorable for the binding of transcription factors and RNA polymerase at the transcription start site. Thus, the polynucleotides used in the compositions and methods described herein include those encoding a target protein and additionally include enhancer sequences. Many enhancer sequences are now known, and examples include enhancers from genes encoding globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers used in the compositions and methods described herein also include those derived from genetic material of viruses capable of infecting eukaryotic cells. Examples include the SV40 enhancer (bp 100 - 270) behind the origin of replication, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer behind the origin of replication, and the adenovirus enhancer. Additional enhancer sequences that induce activation of eukaryotic gene transcription include the CMV enhancer and the RSV enhancer. The enhancer can be spliced into a vector containing a polynucleotide encoding a target protein, for example, at the 5' or 3' position of the gene. In a preferred orientation, the enhancer is located 5' of a promoter, which in turn is located 5' of a polynucleotide encoding a target protein.

[0502] The AAV1 vectors described herein can include the woodchuck post-transcriptional regulatory element (WPRE). WPRE acts at the transcriptional level by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of nascent transcripts, thereby increasing the total amount of mRNA in the cell. Addition of WPRE to a vector can result in a significant improvement in the level of transgene expression from several different promoters in vitro and in vivo.

[0503] In some embodiments, the AAV1 vectors described herein contain a reporter-based sequence that can be used to verify the expression of nucleic acids contained in the AAV1 vectors, for example, in cells and tissues (such as in the cochlea and / or vestibular system, or in specific inner ear cell types, such as IHCs, OHCs, cochlear supporting cells, vestibular hair cells, and / or vestibular supporting cells). Reporter gene sequences that can be provided in the transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (Cat), luciferase, and other substances well known in the art. When associated with regulatory elements that drive their expression, such as the ubiquitous or cell type-specific promoters described herein, the reporter gene sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunoassays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, in the case where the marker sequence is the LacZ gene, the presence of the signal-carrying vector is detected by an assay for β-galactosidase activity. In the case where the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visually detected by color or light production in a photometer.

[0504] In some embodiments, the AAV1 vectors described herein are used to express two or more polynucleotides (e.g., two or more polynucleotides listed in Table 2, e.g., the AAV1 vector encodes polynucleotides for two different target proteins). In some embodiments, a bicistronic or polycistronic expression cassette is used to express two or more polynucleotides. In some embodiments, the polycistronic expression cassette contains an internal ribosome entry site (IRES) located between two or more polynucleotides (e.g., an IRES located between polynucleotides encoding two different target proteins). In some embodiments, the polycistronic expression cassette contains a foot-and-mouth disease virus 2A (FMDV2A) polynucleotide located between two or more polynucleotides (e.g., an FMDV 2A polynucleotide located between each nucleic acid encoding a target protein).

[0505] In some embodiments, two or more AAV1 vectors as described herein (e.g., 2, 3, 4, or more AAV1 vectors) can be used to express a single polynucleotide (e.g., the single polynucleotides listed in Table 2), such as a polynucleotide having a coding sequence that is 3 kb or longer (e.g., 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, or longer). For example, two or more AAV1 vectors can be used to express a polynucleotide encoding otogelin, which polynucleotide has a coding sequence of approximately 6 kb. In embodiments where two or more AAV1 vectors are used to express a single polynucleotide, the coding sequence of the polynucleotide is apportioned among the vectors such that the full-length coding sequence can be reconstituted in vivo. In some embodiments, a dual-vector system comprising two AAV1 vectors can be used to express a single polynucleotide (e.g., the single polynucleotides listed in Table 2). A portion of the coding sequence of the polynucleotide (e.g., a polynucleotide having a coding sequence that is 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, or longer) can be included in each AAV1 vector (e.g., one AAV1 vector can contain the N-terminal portion of the coding sequence and the other AAV1 vector can contain the C-terminal portion of the coding sequence). Exemplary dual-vector systems include fragmented dual-vectors, overlapping dual-vectors, trans-splicing dual-vectors, and two-hybrid vectors. These systems are described in McClements and MacLaren, Yale J Biol Med. 90:611-623, 2017, the disclosure of which is incorporated herein by reference.

[0506] In some embodiments, the AAV1 vectors described herein encompass polynucleotide sequences that enhance the translation rate of a polynucleotide or improve the stability or nuclear export of the mRNA produced from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, IRESs, and polyadenylation signal sites to direct efficient transcription of the nucleic acid carried in the vector. Vectors suitable for the compositions and methods described herein can also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or hygromycin.

[0507] AAV1 Vectors for Nucleic Acid Delivery

[0508] In some embodiments, polynucleotides (e.g., polynucleotides corresponding to genes expressed in healthy inner ear cells, such as the wild-type forms of genes involved in hearing loss and / or vestibular dysfunction, or genes involved in inner ear cell development, function, cell fate determination, regeneration, survival, proliferation, and / or maintenance) are incorporated into AAV1 vectors and / or virions (e.g., AAV1 vectors containing wild-type AAV1 capsids) to facilitate their introduction into cells. In some embodiments, the AAV1 vectors useful in the compositions and methods described herein have wild-type AAV1 capsid proteins having the amino acid sequences SEQ ID NO: 1-3, as shown below.

[0509] In some embodiments, the wild-type AAV1 capsid protein VP1 has the amino acid sequence of SEQ ID NO: 1.

[0510] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL(SEQ ID NO:1)

[0511] In some embodiments, the wild-type AAV1 capsid protein VP2 has the amino acid sequence of SEQ ID NO:2.

[0512] TAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL(SEQ ID NO:2)

[0513] In some embodiments, the wild-type AAV1 capsid protein VP3 has the amino acid sequence of SEQ ID NO:3.

[0514] MASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0515] (SEQ ID NO:3)

[0516] The AAV1 vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that include one or more of the following: (1) a promoter described herein (e.g., a ubiquitous promoter or a cell type-specific promoter), (2) a heterologous sequence to be expressed (e.g., a polynucleotide listed in Table 2), and (3) viral sequences that facilitate integration and expression of the heterologous gene. The viral sequences can include those sequences of AAV that are required (cis) for replication and packaging of DNA (e.g., functional ITRs) into virions. In typical applications, the heterologous transgene encodes a protein that is expressed in healthy inner ear cells and that can promote inner ear cell development, function, cell fate determination, regeneration, survival, proliferation, and / or maintenance, or the wild-type form of a hair cell protein that is mutated in a subject with genetic or hereditary hearing loss, deafness, and / or vestibular dysfunction (e.g., dizziness, vertigo, or imbalance). Such AAV1 vectors can also contain a marker or reporter gene. The available AAV1 vectors have one or more deletions, in whole or in part, of the AAV WT gene but still retain functional flanking ITR sequences. The AAV ITRs contained in the AAV1 vectors described herein can have any serotype suitable for a particular application (e.g., the ITR can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITR). For use in the methods and compositions described herein, the ITR can be an AAV2 ITR. Methods of using AAV 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 regarding AAV vectors for gene delivery are incorporated herein by reference.

[0517] The promoters and polynucleotides described herein can be incorporated into AAV1 virions to facilitate introduction of the promoter and / or polynucleotide into inner ear cells of a primate (e.g., human). The capsid protein of AAV1 forms the outer non-nucleic acid portion of the virion and is encoded by the AAV1 cap gene. The cap gene encodes three viral capsid proteins, VP1, VP2, and VP3, required for virion assembly. The construction of AAV virions has been described, for example, in US 5,173,414; US 5,139,941; US 5,863,541; US 5,869,305; US 6,057,152; and US 6,376,237; as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which regarding AAV vectors for gene delivery are incorporated herein by reference. The capsid protein of the AAV1 vector for use in the compositions and methods described herein can have the amino acid sequence described in U.S. Patent No. 6,759,237 (and is encoded by the polynucleotide sequence described therein, the patent being incorporated herein by reference).

[0518] AAV virions that can be used in conjunction with the compositions and methods described herein include those AAV virions derived from AAV serotype 1 (e.g., AAV1 vectors containing wild-type AAV1 capsids). The construction and use of AAV vectors and AAV proteins of different serotypes have been described, for example, 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 regarding AAV vectors for gene delivery are incorporated herein by reference.

[0519] Also useful in combination with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV8) pseudotyped with a capsid gene derived from AAV1 (e.g., wild-type AAV1 capsid). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and described, for example, 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).

[0520] Pharmaceutical composition

[0521] The AAV1 vectors described herein (e.g., AAV1 vectors comprising wild-type AAV1 capsid) can be incorporated into a vehicle for administration to a patient, such as a primate (e.g., human) patient suffering from sensorineural hearing loss, deafness, auditory neuropathy, tinnitus, and / or vestibular dysfunction. Pharmaceutical compositions containing a vector (such as an AAV1 vector) can be prepared using methods known in the art. For example, such compositions can be prepared using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology, 22nd ed., Allen, L. ed. (2013); incorporated herein by reference) and in the desired form, for example, as a lyophilized preparation or an aqueous solution.

[0522] A mixture of AAV1 vectors can be prepared in water, suitably mixed with one or more excipients, carriers or diluents. The dispersion can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent the growth of microorganisms. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the formulation should be sterile and fluid to the extent that easy injectability exists. The formulation can be stable under the conditions of manufacture and storage and can be preserved under conditions that prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof and / or vegetable oils. The proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents, for example sugars or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using agents that delay absorption in the composition (e.g., aluminum monostearate and gelatin).

[0523] For example, if desired, the solution containing the pharmaceutical composition described herein can be suitably buffered and first diluted the liquid diluent isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site. Depending on the disorder of the subject being treated, certain dosage variations will necessarily occur. For topical administration to the inner ear, the composition can be formulated to contain synthetic perilymph. Exemplary synthetic perilymph includes 20 - 200 mM NaCl, 1 - 5 mM KCl, 0.1 - 10 mM CaCl2, 1 - 10 mM glucose and 2 - 50 mM HEPES, having a pH between about 6 and 9 and an osmolarity of about 300 mOsm / kg. The person responsible for administration will in any case determine the appropriate dose for the individual subject. In addition, for administration to primates (e.g., humans), the formulation can meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biologics standard.

[0524] Method of treatment

[0525] The AAV1 vectors described herein (e.g., AAV1 vectors containing wild-type AAV1 capsids) can be administered to a subject having sensorineural hearing loss, auditory neuropathy, deafness, tinnitus, and / or vestibular dysfunction by a variety of routes, such as local administration to the inner ear or middle ear (e.g., e.g., administered into the perilymph or endolymph through the oval window, endolymphatic sac, round window, or semicircular canals, e.g., trans-tympanic or intra-tympanic administration), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. In any given case, the most suitable route of administration will depend on the particular composition being administered, the patient, the method of drug formulation, the method of administration (e.g., time of administration and route of administration), the age, weight, sex of the patient, the severity of the disease being treated, the diet of the patient, and the excretion rate of the patient. The composition can be administered one or more times (e.g., once a year, twice a year, three times a year, once every two months, or once a month).

[0526] Subjects who can be treated as described herein are subjects suffering from sensorineural hearing loss, deafness, auditory neuropathy, tinnitus, and / or vestibular dysfunction or at risk of developing sensorineural hearing loss, deafness, auditory neuropathy, tinnitus, and / or vestibular dysfunction (e.g., subjects suffering from hearing loss, vestibular dysfunction, or both, or at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods described herein can be used to treat subjects suffering from inner ear cell damage (e.g., cochlear hair cells, vestibular hair cells, cochlear supporting cells, vestibular supporting cells, and / or spiral ganglion neurons, e.g., damage associated with acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging) or at risk of developing inner ear cell damage, subjects suffering from sensorineural hearing loss, deafness, tinnitus, or auditory neuropathy or at risk of developing sensorineural hearing loss, deafness, tinnitus, or auditory neuropathy, subjects suffering from vestibular dysfunction (e.g., dizziness, vertigo, or imbalance) or at risk of developing vestibular dysfunction, subjects suffering from tinnitus (e.g., tinnitus alone, or tinnitus associated with sensorineural hearing loss or vestibular dysfunction), subjects having gene mutations associated with hearing loss and / or vestibular dysfunction, or subjects having a family history of hereditary hearing loss, deafness, auditory neuropathy, tinnitus, or vestibular dysfunction. In some embodiments, the subject suffers from hearing loss and / or vestibular dysfunction associated with or caused by damage, degeneration, dysfunction, or loss of inner ear cells (e.g., cochlear hair cells, vestibular hair cells, cochlear supporting cells, vestibular supporting cells, and / or spiral ganglion neurons). The methods described herein can include the step of screening the subject for one or more mutations in genes known to be associated with hearing loss or vestibular dysfunction prior to treatment or administration of the compositions described herein. The subject can be screened for gene mutations using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein can also include the step of assessing the hearing and / or vestibular function of the subject prior to treatment 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. Vestibular function can be assessed using standard tests such as oculomotor tests (e.g., electro-oculography (ENG) or video-oculography (VNG)), posturography, rotatory chair testing, ECOG, vestibular evoked myogenic potentials (VEMP), and specialized clinical balance tests such as those described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010). These tests can also be used to assess the hearing and / or vestibular function of the subject after treatment or administration of the compositions described herein.The compositions and methods described herein can also be administered as a prophylactic treatment to patients at risk of developing hearing loss and / or vestibular dysfunction, e.g., patients with a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), patients carrying a gene mutation associated with hearing loss or vestibular dysfunction but who have not yet manifested hearing impairment or vestibular dysfunction, or patients exposed to risk factors for acquired hearing loss (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging) or vestibular dysfunction (e.g., disease or infection, head trauma, ototoxic drugs, or aging). In some embodiments, the compositions and methods described herein are used to treat hearing loss or vestibular dysfunction caused by autoimmune inner ear disease, inflammation (e.g., labyrinthitis or vestibular neuritis), or Meniere's disease.

[0527] The compositions and methods described herein can be used to promote or induce the regeneration of inner ear cells (e.g., cochlear hair cells, vestibular hair cells, or spiral ganglion neurons) and / or increase the number of hair cells (e.g., IHCs, OHCs, and / or vestibular hair cells) and / or SGNs in a primate (e.g., human) subject. The compositions and methods described herein can also be used to increase the number of supporting cells (e.g., cochlear and / or vestibular supporting cells, e.g., by inducing or increasing supporting cell proliferation), or induce the differentiation of supporting cells into hair cells (e.g., to induce cochlear supporting cells to differentiate into IHCs and / or OHCs, and / or induce vestibular supporting cells to differentiate into vestibular hair cells). Subjects who can benefit from compositions that promote or induce the regeneration of inner ear cells, increase the number of hair cells, SGNs, and / or supporting cells (e.g., cochlear and / or vestibular supporting cells), and / or induce the differentiation of supporting cells (e.g., cochlear and / or vestibular supporting cells) into hair cells include subjects with hearing loss or vestibular dysfunction caused by damage, degeneration, dysfunction, or loss of inner ear cells (e.g., IHCs, OHCs, SGNs, vestibular hair cells, or supporting cells (e.g., cochlear and / or vestibular supporting cells), e.g., damage, degeneration, or loss of inner ear cells associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), and subjects with abnormal inner ear cells (e.g., inner ear cells that do not function properly compared to normal healthy inner ear cells) or a reduced number of inner ear cells due to genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase hair cell survival (e.g., increase the survival of damaged hair cells, promote the repair of damaged inner ear cells, or preserve inner ear cells in subjects at risk of damage, degeneration, or loss of inner ear cells (e.g., inner ear cell loss due to age, exposure to loud noise, disease or infection, head trauma, or ototoxic drugs)).

[0528] The compositions and methods described herein can also be used to prevent or reduce inner ear cell damage or death (e.g., IHC, OHC, SGN, cochlear supporting cells, vestibular supporting cells, and / or vestibular hair cell damage or death) caused by ototoxic drugs in subjects who have been treated with ototoxic drugs or are being treated with or about to start treatment with ototoxic drugs. Ototoxic drugs are toxic to cells in the inner ear and can cause sensorineural hearing loss, vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), tinnitus, or a combination of these symptoms. Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamicin, 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, especially at high doses), and quinine. In some embodiments, the methods described herein prevent or reduce inner ear cell damage or death (e.g., IHC, OHC, SGN, cochlear supporting cells, vestibular supporting cells, and / or vestibular hair cell damage or death) associated with acoustic trauma, disease or infection, head trauma, or aging.

[0529] The compositions and methods described herein can also be used to maintain or improve the connection between hair cells and neurons in the inner ear (e.g., the synaptic connection between cochlear hair cells (IHC and / or OHC) and SGN or between vestibular hair cells and vestibular ganglion neurons). In some embodiments, the compositions and methods described herein preserve ribbon synapses or promote or increase ribbon synapse formation. In some embodiments, the compositions and methods described herein maintain or increase the innervation of hair cells (e.g., cochlear hair cells (IHC and / or OHC) or vestibular hair cells) by neurons in the inner ear (e.g., SGN or vestibular ganglion neurons).

[0530] Treatment can include administering a composition comprising the AAV1 vector described herein (e.g., an AAV1 vector comprising a wild-type AAV1 capsid) in various unit doses. Each unit dose will typically comprise a predetermined amount of the therapeutic composition. The amount to be administered and the specific route of administration and formulation are within the skill of the artisan in the clinical field. The unit dose need not be administered as a single injection but can include a continuous infusion over a set period of time. An infusion pump can be used for administration to control the infusion rate so as to minimize damage to the inner ear (e.g., the cochlea). The AAV1 vector can have, for example, from about 1x10 9 vector genomes (VG) / mL to about 1x10 15 VG / mL (e.g., 1x10 9 VG / mL, 2x10 9 VG / mL, 3x10 9 VG / mL, 4x109 VG / mL, 5x10 9 VG / mL, 6x10 9 VG / mL, 7x10 9 VG / mL, 8x10 9 VG / mL, 9x10 9 VG / mL, 1x10 10 VG / mL, 2x10 10 VG / mL, 3x10 10 VG / mL, 4x10 10 VG / mL, 5x10 10 VG / mL, 6x10 10 VG / mL, 7x10 10 VG / mL, 8x10 10 VG / mL, 9x10 10 VG / mL, 1x10 11 VG / mL, 2x10 11 VG / mL, 3x10 11 VG / mL, 4x10 11 VG / mL, 5x10 11 VG / mL, 6x10 11 VG / mL, 7x10 11 VG / mL, 8x10 11 VG / mL, 9x10 11 VG / mL, 1x10 12 VG / mL, 2x10 12 VG / mL, 3x10 12 VG / mL, 4x10 12 VG / mL, 5x10 12 VG / mL, 6x10 12 VG / mL, 7x10 12 VG / mL, 8x10 12 VG / mL, 9x10 12 VG / mL, 1x10 13 VG / mL, 2x10 13 VG / mL, 3x10 13 VG / mL, 4x10 13 VG / mL, 5x10 13 VG / mL, 6x10 13 VG / mL, 7x10 13 VG / mL, 8x10 13 VG / mL, 9x10 13VG / mL, 1x10 14 VG / mL, 2x10 14 VG / mL, 3x10 14 VG / mL, 4x10 14 VG / mL, 5x10 14 VG / mL, 6x10 14 VG / mL, 7x10 14 VG / mL, 8x10 14 VG / mL, 9x10 14 VG / mL or 1x10 15 VG / mL) and can be administered 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 AAV1 vector can be 1x10 7 VG / ear to 2x10 14 VG / ear (e.g., 1x10 7 VG / ear, 2x10 7 VG / ear, 3x10 7 VG / ear, 4x10 7 VG / ear, 5x10 7 VG / ear, 6x10 7 VG / ear, 7x10 7 VG / ear, 8x10 7 VG / ear, 9x10 7 VG / ear, 1x10 8 VG / ear, 2x10 8 VG / ear, 3x10 8 VG / ear, 4x10 8 VG / ear, 5x10 8 VG / ear, 6x10 8 VG / ear, 7x10 8 VG / ear, 8x10 8 VG / ear, 9x10 8 VG / ear, 1x10 9 VG / ear, 2x10 9 VG / ear, 3x10 9 VG / ear, 4x10 9 VG / ear, 5x10 9VG / ear, 6x10 9 VG / ear, 7x10 9 VG / ear, 8x10 9 VG / ear, 9x10 9 VG / ear, 1x10 10 VG / ear, 2x10 10 VG / ear, 3x10 10 VG / ear, 4x10 10 VG / ear, 5x10 10 VG / ear, 6x10 10 VG / ear, 7x10 10 VG / ear, 8x10 10 VG / ear, 9x10 10 VG / ear, 1x10 11 VG / ear, 2x10 11 VG / ear, 3x10 11 VG / ear, 4x10 11 VG / ear, 5x10 11 VG / ear, 6x10 11 VG / ear, 7x10 11 VG / ear, 8x10 11 VG / ear, 9x10 11 VG / ear, 1x10 12 VG / ear, 2x10 12 VG / ear, 3x10 12 VG / ear, 4x10 12 VG / ear, 5x10 12 VG / ear, 6x10 12 VG / ear, 7x10 12 VG / ear, 8x10 12 VG / ear, 9x10 12 VG / ear, 1x10 13 VG / ear, 2x10 13 VG / ear, 3x10 13 VG / ear, 4x10 13 VG / ear, 5x10 13 VG / ear, 6x10 13 VG / ear, 7x10 13 VG / ear, 8x10 13 VG / ear, 9x10 13 VG / ear, 1x10 14 VG / ear or 2x1014 A dose of VG / ear) is administered to a subject.

[0531] In some embodiments, the compositions described herein are administered in an amount sufficient to transduce three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) types of developing inner ear cells selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocytes, Scarpa's ganglion neurons (vestibular ganglion neurons), endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiters' cells, second row of Deiters' cells, third row of Deiters' cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocytes, cells and glia of Reissner's membrane. In some embodiments, the compositions described herein are administered in an amount sufficient to transduce all or the most highly developed types of inner ear cells.

[0532] The compositions described herein are administered in an amount sufficient to achieve the following: improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), reduce tinnitus, increase the expression of the polynucleotide contained in the AAV1 vector, increase the function of the protein encoded by the polynucleotide contained in the AAV1 vector, promote or induce inner ear cell regeneration (e.g., regeneration of cochlear hair cells, vestibular hair cells, or spiral ganglion neurons), increase the number of hair cells (e.g., IHC, OHC, and / or vestibular hair cell numbers), increase the number of SGNs, increase the number of supporting cells (e.g., cochlear and / or vestibular supporting cells, such as by increasing the proliferation of supporting cells), increase the differentiation of supporting cells into hair cells (e.g., cause cochlear supporting cells to differentiate into IHCs and / or OHCs, and / or cause vestibular supporting cells to differentiate into vestibular hair cells), prevent or reduce inner ear cell injury or death (e.g., IHC, OHC, SGN, cochlear supporting cell, vestibular supporting cell, and / or vestibular hair cell injury or death associated with acoustic trauma, head trauma, ototoxic drugs, disease or infection, or aging), promote or increase inner ear cell development, promote or increase inner ear cell survival (e.g., increase the survival of damaged inner ear cells, promote the repair of damaged inner ear cells, or preserve inner ear cells in a subject at risk of inner ear cell injury or degeneration or loss (e.g., due to age, exposure to loud noise, disease or infection, head trauma, or ototoxic drugs)), improve inner ear cell function, preserve ribbon synapses, promote or increase ribbon synapse formation, maintain the connection (e.g., synaptic connection) between hair cells and neurons (e.g., SGNs and / or vestibular ganglion neurons), or increase or restore the connection (e.g., synaptic connection) between hair cells and neurons (e.g., SGNs and / or vestibular ganglion neurons). Hearing can be evaluated using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions), and can 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 the hearing measurements obtained prior to treatment. Vestibular function can be evaluated using standard tests for balance and dizziness (e.g., oculomotor tests (e.g., ENG or VNG), posturography, chair tests, ECOG, VEMP, and specialized clinical balance tests), and can 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 the measurements obtained prior to treatment. In some embodiments, the composition is administered in an amount sufficient to improve the subject's ability to understand conversation.The compositions described herein can also be administered in an amount sufficient to slow or prevent the development or progression of sensorineural hearing loss and / or vestibular dysfunction (e.g., in a subject who carries a gene mutation associated with hearing loss or vestibular dysfunction, has a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), or has been exposed to a risk factor associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, disease or infection, or acoustic trauma), but does not exhibit hearing impairment or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), or in a subject who exhibits mild to moderate hearing loss or vestibular dysfunction). The expression of the protein encoded by the polynucleotide can be evaluated using immunohistochemistry, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detecting proteins or mRNAs, and can 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 the expression before administration of the compositions described herein. The number of inner ear cells, inner ear cell function, hair cell or SGN regeneration, or the function of the protein encoded by the polynucleotide contained in the AAV1 vector can be indirectly evaluated based on hearing tests or vestibular function tests, and can 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 the number of inner ear cells, inner ear cell function, hair cell or SGN regeneration, or the function of the protein encoded by the polynucleotide before administration of the compositions described herein. Inner ear cell injury or death can 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 the inner ear cell injury and death typically observed in untreated subjects. These effects can 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 longer after administration of the compositions described herein. Depending on the dose and route of administration used for treatment, the patient can be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer after administration of the composition. Based on the evaluation results, the patient may receive additional treatment.

[0533] Kit

[0534] The compositions described herein can be provided in a kit for the treatment of sensorineural hearing loss or vestibular dysfunction. The composition can comprise an AAV1 vector that contains a nucleic acid sequence of a promoter and / or polynucleotide, and can be provided in unit dosage form, optionally in a pharmaceutically acceptable excipient (e.g., saline or artificial lymph), in an amount sufficient to transduce 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) types of developing inner ear cells selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte cells, Scarpa ganglion neurons (vestibular ganglion neurons), endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiter cells, second row of Deiter cells, third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte cells, cells of Reissner's membrane, and glial cells. The kit can also include package instructions that direct the user of the kit, such as a physician, to perform the methods described herein. The kit can optionally include a syringe or other device for administering the composition.

[0535] Examples

[0536] The following examples are provided to describe for those of ordinary skill in the art how the compositions and methods described herein can be used, prepared, and evaluated, and are intended merely to illustrate the invention and not to limit the scope of the invention as determined by the inventors.

[0537] The examples below illustrate the unexpectedly superior tropism of AAV1 viral vectors in the inner ear of non-human primates compared to the tropism of AAV2 and AAV7m8 serotype vectors. Different from the AAV1 tropism in mice, which exhibits limited transduction of several cell types in the inner ear, AAV1-mediated transgenic delivery to the inner ear of non-human primates exhibits extensive transduction of multiple cell types. This is in contrast to serotypes such as AAV7m8, which exhibits extensive transduction of multiple inner ear cell types in mice but shows a more restricted tropism in the inner ear of non-human primates.

[0538] In all examples described below, the AAV1 vector contains a capsid protein having the amino acid sequences of SEQ ID NO: 1-3.

[0539] Example 1. In Vivo Administration of a Composition Containing an AAV1 Viral Vector Carrying a Transgene to the Cochlea of Mice

[0540] To determine the transduction efficacy of the AAV1 viral vector in the inner ear of mice, a composition containing the AAV1 vector carrying the EGFP transgene under the control of the CMV promoter (AAV1-CMV-EGFP) was unilaterally delivered into the inner ear of 9-10-week-old CBA / CaJ mice (n = 6 mice) by injection into the posterior semicircular canal of the inner ear. An injection (1 μL) with a virus titer of 2.94×10 13 genomic copies / mL was performed at an injection rate of 0.1 μL / min. Two weeks after the injection, the inner ear was surgically removed and the surface preparation of the basilar membrane was carried out. To evaluate AAV-1-mediated inner ear transduction, EGFP expression was directly observed using fluorescence microscopy. EGFP expression was observed throughout the entire basal-apical axis of the cochlea, but differed between cell types along the length of the frequency map. ( Figure 1A ; upper left panel: apex of the cochlea; lower panel: middle turn of the cochlea; upper right panel: basal turn of the cochlea). Inner hair cells (IHCs) expressed EGFP across the basal-apical axis of the cochlea, but outer hair cells (OHCs) mainly showed GFP expression in the apical region of the cochlea. EGFP expression was also observed in some cells of the spiral limbus ( Figure 1B ).

[0541] Example 2. In vivo administration of a composition containing the AAV7m8 viral vector carrying a transgene to the cochlea of mice

[0542] To determine the transduction efficacy of the AAV7m8 viral vector in the inner ear of mice, a composition containing the AAV7m8 vector carrying the EGFP transgene under the control of the CAG promoter (AAV7m8-CAG-EGFP) was unilaterally delivered into the inner ear of 9-10-week-old CBA / CaJ mice (n = 8 mice) by injection into the posterior semicircular canal of the inner ear. An injection (1 μL) with a virus titer of 9.4×10 12 genomic copies / mL was performed at an injection rate of 0.1 μL / min. Two weeks after the injection, the inner ear was surgically removed and the surface preparation of the basilar membrane was carried out. EGFP expression was observed throughout the cochlea ( Figure 2A ). High-magnification images showed EGFP expression in multiple cell types, including IHCs, OHCs, and cells of the spiral ganglion and spiral limbus throughout the basal-to-apical extent of the cochlea ( Figure 2B ; left panel: apex of the cochlea; middle panel: middle turn of the cochlea; right panel: basal turn of the cochlea).

[0543] Example 3. In vivo administration of a composition containing the AAV2 vector carrying a transgene to the inner ear of adult non-human primates

[0544] To determine the transduction efficacy of the AAV2 viral vector in the inner ear of adult non-human primates, a composition containing the AAV2 vector carrying the GFP transgene under the control of the CMV promoter was delivered unilaterally (n = 1 animal) and bilaterally (n = 2 animals) to the inner ear of rhesus monkeys by round window membrane injection. An injection (30 μL) with a virus titer of 3.39×10 12 genomic copies / mL was performed at an injection rate of 15 μL / min. Four weeks after injection, the inner ear was surgically removed and the surface preparation of the basilar membrane was carried out. Immunolabeling of Myo7A was used to identify the hair cells of the inner ear ( Figure 3A , figures in the right column), and AAV2-mediated inner ear cell transduction was measured by direct GFP fluorescence ( Figure 3A , figures in the left column). GFP expression was observed along the basal-apical axis of the cochlea, particularly in some cells of the IHC and spiral limbus ( Figure 3A , figures in the left column). To determine the transduction efficacy of the AAV2-CMV-GFP vector in the IHC of the inner ear of rhesus monkeys, GFP expression in the IHC was quantified along the frequency map of the cochlea, demonstrating GFP expression in 40%-80% of the cells ( Figure 3B )

[0545] Example 4. In vivo administration of a composition containing the AAV7m8 vector carrying a transgene to the inner ear of adult non-human primates

[0546] To determine the transduction efficacy of the AAV7m8 viral vector in the inner ear of adult non-human primates, a composition containing the AAV7m8 vector carrying the EGFP transgene under the control of the CAG promoter was delivered bilaterally to the inner ear of cynomolgus monkeys by round window membrane injection. An injection (30 μL) with a virus titer of 9.4×10 12 genomic copies / mL was performed at an injection rate of 15 μL / min. Two weeks (n = 1 animal) or four weeks (n = 1 animal) after injection, the inner ear was surgically removed and embedded in paraffin for sectioning, fixation and immunostaining with an anti-GFP antibody for histological analysis. In one animal, two weeks after transduction of the inner ear with AAV7m8-CAG-EGFP, EGFP expression was observed at the basal turn of the cochlea ( Figure 4A , upper figure: the dashed box indicates the basal turn of the cochlea). EGFP was observed in the IHC, OHC and fibrocyte of the spiral ligament ( Figure 4B , Figure 4A , magnified view of the dashed box in Figure 4C , left upper inset: magnified view of the organ of Corti). In the other animal, four weeks after injection, EGFP expression was observed along the basal-apical axis of the cochlea ( Figure 4D(Magnified in the middle). EGFP was expressed in the spiral ligament, OHCs, and fibroblasts ( Figure 4D , and the organ of Corti was magnified in the inset in the upper left corner of the figure). As a negative control, immunostaining with the secondary antibody in the absence of the anti-GFP primary antibody showed no staining in the cochlea ( Figure 4E -F).

[0547] Example 5. In Vivo Administration of a Composition Containing an AAV1 Viral Vector Carrying a Transgene to the Inner Ear of Adult Non-Human Primates

[0548] To determine the transduction efficacy of the AAV1 viral vector in the inner ear of adult non-human primates, a composition containing an AAV1 vector carrying the GFP transgene under the control of the CAG promoter (AAV1-CAG-GFP) was bilaterally delivered to the inner ear of cynomolgus monkeys by injection through the round window membrane. Injections (30 μL) were performed at a viral titer of 9.9×10 12 GC / mL at an injection rate of 15 μL / min. Four weeks after injection, the inner ears were surgically removed and embedded in paraffin for sectioning and histological analysis. To evaluate AAV1-mediated inner ear transduction, the paraffin sections were immunolabeled with a rabbit monoclonal anti-GFP antibody (Abcam EPR14104). GFP expression was observed in multiple inner ear structures including the cochlea ( Figure 5B and 5C ) and the saccule ( Figure 5B and 5E ). Robust GFP expression was detected in the spiral ligament and the marginal and intermediate cells of the stria vascularis. In the sections shown in the images, the basal cells of the stria vascularis did not show GFP expression, but GFP expression was visible in the basal cells in other turns of the cochlea ( Figure 5D ). Staining with only the secondary antibody in the absence of the anti-GFP antibody was used as a negative control to confirm the specificity of the antibody ( Figure 5A ).

[0549] To determine whether the extensive transduction of the AAV1 vector observed in the inner ear of cynomolgus monkeys could be observed in other primate species, another set of experiments was performed in rhesus monkeys using injections of an AAV1 vector carrying the GFP transgene under the control of the CMV promoter (AAV1-CMV-GFP). Unilateral (n = 1 animal) and bilateral (n = 2 animals) injections of 2.01×10 13The viral titer of GC / mL, and 30 μL of the composition was delivered at an injection rate of 15 μL / min. Four weeks after injection, the inner ear was surgically removed and the surfaces of the basilar membrane, utricle, and cristae were prepared to determine the tropism of the AAV1 vector. AAV1-mediated GFP expression was directly monitored by GFP fluorescence, and hair cells in the inner ear were identified by immunolabeling with an antibody against Myo7a. Fluorescence imaging revealed robust GFP expression along the entire base-to-apex axis of the cochlea ( Figure 6A ), and this GFP expression was strongly evident in the inner hair cell (IHC) and outer hair cell (OHC) layers ( Figure 6B ). GFP expression was also observed in supporting cells and in the non-sensory spiral ligament and spiral limbus ( Figure 6B ). In addition, the vestibular structures of the inner ear showed strong GFP expression in the sensory and non-sensory cells of the utricle and cristae ( Figure 6C ).

[0550] Thus, as shown in FIGS. 5 and 6, AAV1 exhibits robust pan-tropic transduction within the inner ear of non-human primates. This broad tropism in the sensory and non-sensory cells of the cochlea and vestibule can be exploited to obtain therapeutic benefits. Table 6 below provides a summary of the tropisms observed using different AAV capsids in the inner ear of non-human primates.

[0551] Table 6: Tropisms of Different Capsids in the Inner Ear of Non-Human Primates

[0552]

[0553] Strong = strong expression; Moderate = moderate expression; Weak = weak expression; Little / None = little or no expression

[0554] Example 6. In Vivo Administration of a Composition Containing an AAV1 Viral Vector Carrying a Transgene to the Inner Ear of Adult Non-Human Primates Using Low and High Viral Titers

[0555] To determine the effect of viral titer on the transduction efficacy of the AAV1 viral vector in the inner ear of adult non-human primates, a composition containing an AAV1 vector carrying a GFP transgene under the control of the CAG promoter (AAV1-CAG-GFP) was bilaterally delivered to the inner ear of cynomolgus monkeys by injection through the round window membrane. The viral titers were 1.05×10 12 GC / mL (3.15×10 10 GC / ear; "AAV1 low") or 1.05×10 13 GC / mL (3.15×10 11Injection (30 μL) into one GC / ear; (“AAV1 high”). Four weeks after injection, the inner ear was surgically removed and embedded in paraffin for sectioning and histological analysis. To evaluate AAV1-mediated inner ear transduction, paraffin sections were immunolabeled with a rabbit monoclonal anti-GFP antibody (Abcam EPR14104). Figure 7A - 7C Shown in the non-injected AAV ear ( Figure 7A ), in the AAV low-injected ear ( Figure 7B ), and in the AAV high-injected ear ( Figure 7C ) of GFP labeling. Figure 7D to 7F Shown in the case of no nuclear staining (i.e., only GFP staining) respectively with Figure 7A - 7C the same images. The cochlea of the non-injected AAV ear was not labeled with the anti-GFP antibody, thus confirming the specificity of the antibody ( Figure 7A and 7D ). The AAV1 low-injected ear showed robust GFP expression from the base to the apex of the cochlea in a variety of cell types, including hair cells, supporting cells, inner sulcus cells, outer sulcus cells, and cells in the spiral ligament ( Figure 7B and 7E ). Increasing the virus titer produced stronger GFP expression in the aforementioned cell types, as well as GFP expression in additional cells and cell types ( Figure 7C and 7F ). Thus, AAV1 exhibits robust pan-tropic transduction within the inner ear of non-human primates, and such transduction can be enhanced by increasing the virus titer.

[0556] Example 7. Expression of polynucleotides in outer hair cells of the inner ear of human subjects

[0557] According to the methods and compositions disclosed herein, one of ordinary skill in the art can administer to a human subject a composition containing an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid) to induce polynucleotide expression in OHCs. To this end, one of ordinary skill in the art can locally administer the composition containing the AAV1 vector to the inner ear of a human subject (e.g., administer to the endolymph or perilymph, such as administer to the round window, oval window, or horizontal semicircular canal or administer through the round window, oval window, or horizontal semicircular canal, or by transtympanic or intratympanic injection), the AAV1 vector containing a polynucleotide (e.g., a polynucleotide encoding Strc, Chrna9, Chrna10, Ocm, Tmc1, Myo7a, or Ush1c) operably linked to an OHC-specific promoter (e.g., the SLC26A5 promoter, the OCM promoter, the STRC promoter, or the ATP2B2 promoter). To induce the expression of polynucleotides in OHCs, for example, 1x10 7 vg / ear to 2x10 14VG / ear (e.g., 1x10 7 VG / ears, 2x10 7 VG / ears, 3x10 7 VG / ears, 4x10 7 VG / ears, 5x10 7 VG / ears, 6x10 7 VG / ears, 7x10 7 VG / ears, 8x10 7 VG / ears, 9x10 7 VG / ears, 1x10 8 VG / ears, 2x10 8 VG / ears, 3x10 8 VG / ears, 4x10 8 VG / ears, 5x10 8 VG / ears, 6x10 8 VG / ears, 7x10 8 VG / ears, 8x10 8 VG / ears, 9x10 8 VG / ears, 1x10 9 VG / ears, 2x10 9 VG / ears, 3x10 9 VG / ears, 4x10 9 VG / ears, 5x10 9 VG / ears, 6x10 9 VG / ears, 7x10 9 VG / ears, 8x10 9 VG / ears, 9x10 9 VG / ears, 1x10 10 VG / ears, 2x10 10 VG / ears, 3x10 10 VG / ears, 4x10 10 VG / ears, 5x10 10 VG / ears, 6x10 10 VG / ears, 7x10 10 VG / ears, 8x10 10 VG / ears, 9x10 10 VG / ears, 1x10 11 VG / ears, 2x10 11 VG / ears, 3x10 11 VG / ears, 4x10 11 VG / ears, 5x10 11 VG / ears, 6x1011 VG / ear, 7x10 11 VG / ear, 8x10 11 VG / ear, 9x10 11 VG / ear, 1x10 12 VG / ear, 2x10 12 VG / ear, 3x10 12 VG / ear, 4x10 12 VG / ear, 5x10 12 VG / ear, 6x10 12 VG / ear, 7x10 12 VG / ear, 8x10 12 VG / ear, 9x10 12 VG / ear, 1x10 13 VG / ear, 2x10 13 VG / ear, 3x10 13 VG / ear, 4x10 13 VG / ear, 5x10 13 VG / ear, 6x10 13 VG / ear, 7x10 13 VG / ear, 8x10 13 VG / ear, 9x10 13 VG / ear, 1x10 14 VG / ear or 2x10 14 administer the AAV1 vector in a dose of (number of VG / ear)

[0558] After administering the composition to a human subject, a skilled practitioner in the art can monitor gene expression and / or the expression of the protein encoded by the polynucleotide, as well as the improvement of the patient in response to the therapy, by a variety of methods. For example, after administering the composition, a physician can indirectly assess gene expression and / or protein production and monitor the patient's hearing by performing standard tests such as audiometry, ABR, electrocochleography (ECOG), and distortion product otoacoustic emissions (DPOAE). The hearing measurements collected after treatment can be compared with the measurements obtained before treatment. Compared with the hearing test results before administering the composition, it is found that the patient shows an improvement in hearing in one or more tests after administering the composition (e.g., improved OHC function as indicated by DPOAE measurements), indicating that the patient has a good response to the treatment. Subsequent doses can be determined and administered as needed.

[0559] Example 8. Treatment of vestibular dysfunction in human subjects by administering an AAV1 vector

[0560] According to the methods disclosed herein, one of ordinary skill in the art can treat a subject, such as a human patient, suffering from vestibular dysfunction (e.g., vertigo, dizziness, or imbalance) to improve or restore vestibular function (e.g., balance, spatial orientation, righting, gait, and / or vestibulo-ocular reflex). To this end, a physician in the art can administer to a human patient a composition comprising an AAV1 vector (e.g., an AAV1 vector comprising a wild-type AAV1 capsid), the AAV1 vector comprising a polynucleotide encoding a transgene operably linked to a vestibular cell type-specific promoter (e.g., an utricular supporting cell-specific promoter such as a GFAP promoter, a GLAST promoter, a HES1 promoter, a JAG1 promoter, a NOTCH1 promoter, a LGR5 promoter, a SOX2 promoter, a HES5 promoter, or a SOX9 promoter; or a vestibular hair cell-specific promoter such as a MYOSIN15 promoter, a GFI1 promoter, a POU4F3 promoter, or a MYOSIN7A promoter). The polynucleotide can encode, for example, Atoh1, Gfi1, Sox11, Ntf3, Bdnf, Whirlin, Sox11, Tmtc4, or Pou4f3. The composition comprising the AAV vector can be administered to the patient, for example, by local administration to the inner ear (e.g., endolymph or perilymph), such as by administration to the round window, oval window, or horizontal semicircular canal or by injection through the round window, oval window, or horizontal semicircular canal, or by administration to the semicircular canal, to treat vestibular dysfunction. To treat vestibular dysfunction, for example, from 1x10 7 vg / ear to 2x10 14 vg / ear (e.g., 1x10 7 vg / ear, 2x10 7 vg / ear, 3x10 7 vg / ear, 4x10 7 vg / ear, 5x10 7 vg / ear, 6x10 7 vg / ear, 7x10 7 vg / ear, 8x10 7 vg / ear, 9x10 7 vg / ear, 1x10 8 vg / ear, 2x10 8 vg / ear, 3x10 8 vg / ear, 4x10 8 vg / ear, 5x10 8 vg / ear, 6x10 8 vg / ear, 7x10 8 vg / ear, 8x10 8 vg / ear, 9x10 8VG / ear, 1x10 9 VG / ear, 2x10 9 VG / ear, 3x10 9 VG / ear, 4x10 9 VG / ear, 5x10 9 VG / ear, 6x10 9 VG / ear, 7x10 9 VG / ear, 8x10 9 VG / ear, 9x10 9 VG / ear, 1x10 10 VG / ear, 2x10 10 VG / ear, 3x10 10 VG / ear, 4x10 10 VG / ear, 5x10 10 VG / ear, 6x10 10 VG / ear, 7x10 10 VG / ear, 8x10 10 VG / ear, 9x10 10 VG / ear, 1x10 11 VG / ear, 2x10 11 VG / ear, 3x10 11 VG / ear, 4x10 11 VG / ear, 5x10 11 VG / ear, 6x10 11 VG / ear, 7x10 11 VG / ear, 8x10 11 VG / ear, 9x10 11 VG / ear, 1x10 12 VG / ear, 2x10 12 VG / ear, 3x10 12 VG / ear, 4x10 12 VG / ear, 5x10 12 VG / ear, 6x10 12 VG / ear, 7x10 12 VG / ear, 8x10 12 VG / ear, 9x10 12 VG / ear, 1x10 13 VG / ear, 2x10 13 VG / ear, 3x10 13 VG / ear, 4x10 13 VG / ear, 5x10 13 VG / ear, 6x1013 VG / ear, 7x10 13 VG / ear, 8x10 13 VG / ear, 9x10 13 VG / ear, 1x10 14 VG / ear or 2x10 14 administering an AAV1 vector in a dose of (number of VG / ear) of (7x10, 8x10, 9x10, 1x10 or 2x10).

[0561] After administering the composition to a patient, those skilled in the art can monitor the improvement of the patient in response to the treatment by various methods. For example, a physician can monitor the patient's vestibular function by performing standard tests such as electrooculogram, video-oculogram, rotary test, vestibular evoked myogenic potential or computerized dynamic posturography. Compared with the test results before administering the composition, it is found that the patient shows improved balance, gait, posture and / or vestibulo-ocular reflex in one or more tests after administering the composition, indicating that the patient has a good response to the treatment. Subsequent doses can be determined and administered as needed.

[0562] Other embodiments

[0563] Without departing from the scope and spirit of the present invention, various modifications and changes of the present invention will be apparent to those skilled in the art. Although the present invention has been described in connection with specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the ways described for practicing the present invention that are apparent to those skilled in the art are intended to be within the scope of the present invention. Other embodiments are in the claims.

[0564] For example, the present application provides the following embodiments.

[0565] 1. A method of transducing developing inner ear cells in a human subject, the method comprising administering to the subject an effective amount of a serotype 1 adeno-associated virus (AAV1) vector that transduces three or more inner ear cell types selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, the first row of Deiter cells, the second row of Deiter cells, the third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane and glial cells.

[0566] 2. The method according to embodiment 1, wherein the AAV1 vector transduces five or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibroblast cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiter cells, second row of Deiter cells, third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibroblast cells, cells of Reissner's membrane, and glial cells.

[0567] 3. The method according to embodiment 2, wherein the AAV1 vector transduces ten or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibroblast cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiter cells, second row of Deiter cells, third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibroblast cells, cells of Reissner's membrane, and glial cells.

[0568] 4. The method according to embodiment 3, wherein the AAV1 vector transduces fifteen or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibroblast cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiter cells, second row of Deiter cells, third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibroblast cells, cells of Reissner's membrane, and glial cells.

[0569] 5. The method according to embodiment 4, wherein the AAV1 vector transduces twenty or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte cells, Scarpa's ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiter cells, second row of Deiter cells, third row of Deiter's cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, basal cells of the stria vascularis, intermediate cells of the stria vascularis, marginal cells of the stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte cells, cells of Reissner's membrane, and glial cells.

[0570] 6. The method according to any one of embodiments 1-5, wherein the AAV1 vector transduces at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cells of a given cell type or all inner ear cell types.

[0571] 7. The method according to any one of embodiments 1-6, wherein the AAV1 vector is administered locally to the middle ear or inner ear of the subject.

[0572] 8. The method according to embodiment 7, wherein the AAV1 vector is administered through or into the tympanic membrane.

[0573] 9. The method according to any one of embodiments 1-8, wherein the AAV1 vector comprises a promoter selected from the list of promoters in Table 5.

[0574] 10. The method according to any one of embodiments 1-8, wherein the AAV1 vector comprises a ubiquitous promoter.

[0575] 11. The method according to any one of embodiments 1-10, wherein the AAV1 vector comprises a polynucleotide selected from the list in Table 2.

[0576] 12. A method of expressing a polynucleotide in the supporting cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: GFAP promoter, GLAST promoter, HES1 promoter, JAG1 promoter, NOTCH1 promoter, LGR5 promoter, SOX2 promoter, HES5 promoter, and SOX9 promoter.

[0577] 13. The method according to embodiment 12,wherein the polynucleotide encodes Sox9, Spalt-like transcription factor 2 (Sall2), calmodulin-binding transcription activator 1 (Camta1), Hes-related family BHLH transcription factor with YRPW motif 2 (Hey2), Gata-binding protein 2 (Gata2), Hes-related family BHLH transcription factor with YRPW motif 1 (Hey1), ceramide synthase 2 (Lass2), SRY-box 10 (Sox10), GATA-binding protein 3 (Gata3), cut-like homeobox 1 (Cux1), nuclear receptor subfamily 2F member (Nr2f1), Hes family BHLH transcription factor 1 (Hes1), RAR-related orphan receptor B (Rorb), Jun proto-oncogene AP-1 transcription factor subunit (Jun), zinc finger protein 667 (Zfp667), LIM homeobox 3 (Lhx3), Nescient helix-loop-helix 1 (Nhlh1), MAX dimerization protein 4 (Mxd4), zinc finger MIZ type 1-containing (Zmiz1), myelin transcription factor 1 (Myt1), signal transducer and activator of transcription 3 (Stat3), BarH-like homeobox 1 (Barhl1), thymocyte selection-associated high mobility group box (Tox), Prospero homeobox 1 (Prox1), nuclear factor I A (Nfia), thyroid hormone receptor beta (Thrb), MYCL proto-oncogene BHLH transcription factor (Mycl1), lysine demethylase 5A (Kdm5a), CAMP response element-binding protein 3-like 4 (Creb3I4), ETS variant 1 (Etv1), paternally expressed 3 (Peg3), BTB domain and CNC homolog 2 (Bach2), ISL LIM homeobox (Isl1), zinc finger and BTB domain-containing 38 (Zbtb38), limb bud and heart development (Lbh), Tubby bipartite transcription factor (Tub), ubiquitin C (Hmg20), RE1-silencing transcription factor (Rest), zinc finger protein 827 (Zfp827), AF4 / FMR2 family member 3 (Aff3), PBX / knotted 1 homeobox 2 (Pknox2), AT-rich interactive domain 3B (Arid3b), MLX interacting protein (Mlxip), zinc finger protein (Zfp532), IKAROS family zinc finger 2 (Ikzf2), Spalt-like transcription factor 1 (Sall1), SIX homeobox 2 (Six2), Spalt-like transcription factor 3 (Sall3), Lin-28 homolog B (Lin28b), Pou4f3, regulatory factor X7 (Rfx7), Atoh1, an Atoh1 variant containing mutations at amino acids 328, 331, and / or 334, Gfi1, Sox4, Bdnf, Ntf3, Sox11, Tead2, Yap1, or a nuclease,or a microRNA.

[0578] 14. A method of expressing a polynucleotide in hair cells of the inner ear of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, and MYO7A promoter.

[0579] 15. The method of embodiment 14, wherein the polynucleotide encodes Bdnf, Ntf3, transmembrane and tetratricopeptide repeat containing 4 (Tmtc4), or a nuclease, or is a microRNA.

[0580] 16. A method of expressing a polynucleotide in outer hair cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: SLC26A5 promoter, OCM promoter, STRC promoter, and ATP2B2 promoter.

[0581] 17. The method of embodiment 16, wherein the polynucleotide encodes Strc, Tmc1, Myo7a, Ush1c, Atoh1, Pou4f3, Gfi1, ISL LIM homeobox 1 (Isl1), Clrn1, protocadherin related 15 (Pcdh15), protocadherin related 23 (Cdh23), Chrna9, Chrna10, Ocm, Bdnf, Ntf3, Tmtc4, or a nuclease, or is a microRNA.

[0582] 18. A method of expressing a polynucleotide in cochlear hair cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, and MYO7A promoter.

[0583] 19. The method of embodiment 18, wherein the polynucleotide encodes Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, otoferlin (Otof), solute carrier family 17 member 8 (Vglut3), Strc, Chrna9, Chrna10, Ocm, Tmc1, Myo7a, Ush1c, Whirlin, Bdnf, Ntf3, Tmtc4, or a nuclease, or is a microRNA.

[0584] 20. A method of expressing a polynucleotide in inner hair cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: OTOF promoter, fibroblast growth factor 8 (FGF8) promoter, and solute carrier family 17 member 8 (SLC17A8) promoter.

[0585] 21. The method of embodiment 20, wherein the polynucleotide encodes Otof, Vglut3, Whirlin, Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, Myo7a, Tmc1, Ush1c, Bdnf, Ntf3, Tmtc4, or a nuclease, or is a microRNA.

[0586] 22. A method of expressing a polynucleotide in vestibular hair cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, MYO7A promoter, ATP2B2 promoter, and CALB2 promoter.

[0587] 23. The method of embodiment 22, wherein the polynucleotide encodes Whirlin, Bdnf, Ntf3, Tmtc4, or a nuclease, or is a microRNA.

[0588] 24. A method of expressing a polynucleotide in pillar cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a CD44 promoter or a GJB2 promoter.

[0589] 25. The method of embodiment 24, wherein the polynucleotide encodes nerve growth factor receptor (Ngfr), Bdnf, Ntf3, Tectorin β (Tectb), Tectorin α (Tecta), Gjb2, or connexin β6 (Gjb6).

[0590] 26. A method of expressing a polynucleotide in spiral ganglion neurons of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: BHLHE22 promoter, SYN promoter, and CALB2 promoter.

[0591] 27. The method according to embodiment 26, wherein the polynucleotide encodes Bdnf, Ntf3, or a nuclease, or is a microRNA or shRNA against RGMA.

[0592] 28. A method for expressing a polynucleotide in the marginal cells of the stria vascularis of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of the KCNQ1 promoter, the KCNE1 promoter, and the GJB2 promoter.

[0593] 29. A method for expressing a polynucleotide in the basal cells of the stria vascularis of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to the CLDN11 promoter or the GJB2 promoter.

[0594] 30. A method for expressing a polynucleotide in the intermediate cells of the stria vascularis of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of the KCNJ10 promoter, the GJB2 promoter, and the TYR promoter.

[0595] 31. The method according to any one of embodiments 28 - 30, wherein the polynucleotide encodes Kcnq1, Kcne1, Tyr, Gjb2, Gjb6, or a nuclease, or is a microRNA.

[0596] 32. A method for expressing a polynucleotide in the marginal cells and / or inner phalangeal cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to the GLAST promoter or the GJB2 promoter.

[0597] 33. The method according to embodiment 32, wherein the polynucleotide encodes Bdnf, Ntf3, Tectb, Tecta, transmembrane protein 16A (Tmem16a), Gjb2, or Gjb6.

[0598] 34. A method for expressing a polynucleotide in Dieter's cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to the FGFR3 promoter or the GJB2 promoter.

[0599] 35. The method according to embodiment 34, wherein the polynucleotide encodes Bdnf, Ntf3, Tectb, Tecta, IKAROS family zinc finger 2 (Ikzf2), Gjb2 or Gjb6.

[0600] 36. A method of expressing a polynucleotide in Hensen's cells and / or Claudius cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to an FRZB promoter or a GJB2 promoter.

[0601] 37. The method according to embodiment 36, wherein the polynucleotide encodes Gjb2 or Gjb6.

[0602] 38. A method of expressing a polynucleotide in spiral prominence cells and / or root cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to an SLC26A4 promoter.

[0603] 39. The method according to embodiment 38, wherein the polynucleotide is Slc26a4.

[0604] 40. A method of expressing a polynucleotide in interdental cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a CEACAM16 promoter or a GJB2 promoter.

[0605] 41. The method according to embodiment 40, wherein the polynucleotide encodes Ceacam16, otoancorin (Otoa), Gjb2 or Gjb6.

[0606] 42. A method of expressing a polynucleotide in glial cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a PMP22 promoter.

[0607] 43. The method according to embodiment 42, wherein the polynucleotide encodes Pmp22, Bdnf, Ntf3 or myelin protein zero (Mpz).

[0608] 44. A method of expressing a polynucleotide in vestibular dark cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a KCNE1 promoter.

[0609] 45. The method according to embodiment 44, wherein the polynucleotide encodes Kcnq1, Kcne1 or Slc26a4.

[0610] 46. A method of expressing a polynucleotide in fibroblasts of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a POU3F4 promoter or a GJB2 promoter.

[0611] 47. The method according to embodiment 46, wherein the polynucleotide encodes Gjb2, Gjb6 or collagen.

[0612] 48. A method of expressing a polynucleotide in Scarpa ganglion neurons of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of a TUBB3 promoter and a SYN promoter.

[0613] 49. The method according to embodiment 48, wherein the polynucleotide encodes Bdnf or Ntf3, or is an shRNA directed against repulsive guidance molecule BMP coreceptor A (RGMA).

[0614] 50. A method of expressing a polynucleotide in supporting cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Sox9, Sall2, Camta1, Hey2, Gata2, Hey1, Lass2, Sox10, Gata3, Cux1, Nr2f1, Hes1, Rorb, Jun, Zfp667, Lhx3, Nhlh1, Mxd4, Zmiz1, Myt1, Stat3, Barhl1, Tox, Prox1, Nfia, Thrb, Mycl1, Kdm5a, Creb3I4, Etv1, Peg3, Bach2, Isl1, Zbtb38, Lbh, Tub, Hmg20, Rest, Zfp827, Aff3, Pknox2, Arid3b, Mlxip, Zfp532, Ikzf2, Sall1, Six2, Sall3, Lin28b, Pou4f3, Rfx7, Atoh1, an Atoh1 variant comprising mutations at amino acids 328, 331 and / or 334, Gfi1, Sox4, Bdnf, Ntf3, Sox11, Tead2, Yap1 or a nuclease, or is a microRNA.

[0615] 51. The method according to any one of embodiment 50, wherein the promoter is selected from the group consisting of: GFAP promoter, GLAST promoter, HES1 promoter, JAG1 promoter, NOTCH1 promoter, LGR5 promoter, SOX2 promoter, HES5 promoter, and SOX9 promoter.

[0616] 52. A method for expressing a polynucleotide in hair cells of the inner ear of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Bdnf, Ntf3, Tmtc4, or a nuclease, or being a microRNA.

[0617] 53. The method according to embodiment 52, wherein the promoter is selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, and MYO7A promoter.

[0618] 54. A method for expressing a polynucleotide in outer hair cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Strc, Tmc1, Myo7a, Ush1c, Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, Chrna9, Chrna10, Ocm, Bdnf, Ntf3, Tmtc4, or a nuclease, or being a microRNA.

[0619] 55. The method according to embodiment 54, wherein the promoter is selected from the group consisting of: SLC26A5 promoter, OCM promoter, STRC promoter, and ATP2B2 promoter.

[0620] 56. A method for expressing a polynucleotide in cochlear hair cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, Otof, Vglut3, Strc, Chrna9, Chrna10, Ocm, Tmc1, Myo7a, Ush1c, Whirlin, Bdnf, Ntf3, Tmtc4, or a nuclease, or being a microRNA.

[0621] 57. The method according to embodiment 56, wherein the promoter is selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, and MYO7a promoter.

[0622] 58. A method of expressing a polynucleotide in inner hair cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Otof, Vglut3, Whirlin, Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh14, Cdh23, Myo7a, Ush1c, Tmc1, Bdnf, Ntf3, Tmtc4, or a nuclease, or being a microRNA.

[0623] 59. The method according to embodiment 58, wherein the promoter is selected from the group consisting of: OTOF promoter, FGF8 promoter, and SLC17A8 promoter.

[0624] 60. A method of expressing a polynucleotide in vestibular hair cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Whirlin, Bdnf, Ntf3, Tmtc4, or a nuclease, or being a microRNA.

[0625] 61. The method according to embodiment 60, wherein the promoter is selected from the group consisting of: MYO15 promoter, GFI1 promoter, POU4F3 promoter, MYO7A promoter, ATP2B2 promoter, and CALB2 promoter.

[0626] 62. A method of expressing a polynucleotide in pillar cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Ngfr, Bdnf, Ntf3, Tectb, Tecta, Gjb2, or Gjb6.

[0627] 63. The method according to embodiment 62, wherein the promoter is the CD44 promoter or the GJB2 promoter.

[0628] 64. A method of expressing a polynucleotide in spiral ganglion neurons of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf, Ntf3, or a nuclease, or linked to a microRNA or an shRNA against RGMA.

[0629] 65. The method according to embodiment 64, wherein the promoter is selected from the group consisting of: BHLHE22 promoter, SYN promoter, and CALB2 promoter.

[0630] 66. A method of expressing a polynucleotide in cells of the stria vascularis of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Kcnq1, Kcne1, Gjb2, Gjb6, Tyr, or a nuclease, or being a microRNA.

[0631] 67. The method according to embodiment 66, wherein the cells are marginal cells of the stria vascularis, and the promoter is selected from the group consisting of: KCNQ1 promoter, GJB2 promoter, or KCNE1 promoter.

[0632] 68. The method according to embodiment 66, wherein the cells are basal cells of the stria vascularis, and the promoter is CLDN11 promoter or GJB2 promoter.

[0633] 69. The method according to embodiment 66, wherein the cells are intermediate cells of the stria vascularis, and the promoter is selected from the group consisting of: KCNJ10 promoter, GJB2 promoter, and TYR promoter.

[0634] 70. A method of expressing a polynucleotide in marginal cells and / or inner phalangeal cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf, Ntf3, Tectb, Tecta, Tmem16a, Gjb2, or Gjb6.

[0635] 71. The method according to embodiment 70, wherein the promoter is GLAST promoter or GJB2 promoter.

[0636] 72. A method of expressing a polynucleotide in Dieter's cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf, Ntf3, Tectb, Tecta, Ikzf2, Gjb2, or Gjb6.

[0637] 73. The method according to embodiment 72, wherein the promoter is FGFR3 promoter or GJB2 promoter.

[0638] 74. A method of expressing a polynucleotide in Hensen's cells and / or Claudius cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Gjb2 or Gjb6.

[0639] 75. The method of embodiment 74, wherein the promoter is an FRZB promoter or a GJB2 promoter.

[0640] 76. A method of expressing a polynucleotide in spiral prominence cells and / or root cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Slc26a4.

[0641] 77. The method of embodiment 76, wherein the promoter is an SLC26A4 promoter.

[0642] 78. A method of expressing a polynucleotide in interdental cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ceacam16, Otoa, Gjb2 or Gjb6.

[0643] 79. The method of embodiment 78, wherein the promoter is a CEACAM16 promoter or a GJB2 promoter.

[0644] 80. A method of expressing a polynucleotide in glial cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Pmp22, Bdnf, Ntf3 or Mpz.

[0645] 81. The method of embodiment 80, wherein the promoter is a PMP22 promoter.

[0646] 82. A method of expressing a polynucleotide in vestibular dark cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Kcnq1, Kcne1 or Slc26a4.

[0647] 83. The method of embodiment 82, wherein the promoter is a KCNE1 promoter.

[0648] 84. A method of expressing a polynucleotide in fibroblast cells of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding Gjb2, Gjb6 or collagen.

[0649] 85. The method according to embodiment 84, wherein the promoter is a POU3F4 promoter or a GJB2 promoter.

[0650] 86. A method of expressing a polynucleotide in Scarpa ganglion neurons of a human subject, the method comprising administering to the subject an effective amount of an AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf or Ntf3 or linked to an shRNA against RGMA.

[0651] 87. The method according to embodiment 86, wherein the promoter is selected from the group consisting of the TUBB3 promoter and the SYN promoter.

[0652] 88. The method according to any one of embodiments 1-87, wherein the AAV1 vector comprises a wild-type AAV1 capsid.

[0653] 89. The method according to any one of embodiments 1-88, wherein the AAV1 vector comprises AAV2 inverted terminal repeats (ITRs).

[0654] 90. An AAV1 vector, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the list of promoters in Table 5.

[0655] 91. The AAV1 vector according to embodiment 90, wherein the polynucleotide is selected from the polynucleotides listed in Table 2.

[0656] 92. An AAV1 vector, the AAV1 vector comprising a promoter operably linked to a polynucleotide selected from the polynucleotides listed in Table 2.

[0657] 93. The AAV1 vector according to embodiment 92, wherein the promoter is selected from the list of promoters in Table 5.

[0658] 94. The AAV1 vector according to any one of embodiments 90-93, wherein the AAV1 vector comprises AAV2 ITRs.

[0659] 95. The AAV1 vector according to any one of embodiments 90-94, wherein the AAV1 vector comprises a wild-type AAV1 capsid.

[0660] 96. A pharmaceutical composition, the pharmaceutical composition comprising the AAV1 vector as described in any one of embodiments 88-93 and a pharmaceutically acceptable excipient.

[0661] 97. The pharmaceutical composition as described in any one of embodiments 96, wherein the pharmaceutical composition is formulated for topical administration to the inner ear or middle ear.

Claims

1. A method of transducing developing inner ear cells in a human subject, the method comprising administering to the subject an effective amount of a serotype 1 adeno-associated virus (AAV1) vector that transduces three or more inner ear cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa's ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiters' cells, second row of Deiters' cells, third row of Deiters' cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane, and glial cells.

2. The method of claim 1, wherein the AAV1 vector transduces five or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa's ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiters' cells, second row of Deiters' cells, third row of Deiters' cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane, and glial cells.

3. The method of claim 2, wherein the AAV1 vector transduces ten or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa's ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row of Deiters' cells, second row of Deiters' cells, third row of Deiters' cells, Hensen's cells, Claudius cells, spiral prominence cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane, and glial cells.

4. The method according to claim 3, wherein the AAV1 vector transduces fifteen or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, the first row of Deiter cells, the second row of Deiter cells, the third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, basal cells of the stria vascularis, intermediate cells of the stria vascularis, marginal cells of the stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane, and glial cells.

5. The method according to claim 4, wherein the AAV1 vector transduces twenty or more cell types selected from the group consisting of: outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fibrocyte, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner phalangeal cells, inner pillar cells, outer pillar cells, the first row of Deiter cells, the second row of Deiter cells, the third row of Deiter cells, Hensen cells, Claudius cells, spiral prominence cells, root cells, interdental cells, basal cells of the stria vascularis, intermediate cells of the stria vascularis, marginal cells of the stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fibrocyte, cells of Reissner's membrane, and glial cells.

6. The method according to any one of claims 1-5, wherein the AAV1 vector transduces at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of a given cell type or all inner ear cell types.

7. The method according to any one of claims 1-6, wherein the AAV1 vector is locally administered to the middle ear or inner ear of the subject.

8. The method according to claim 7, wherein the AAV1 vector is administered through or within the tympanic membrane.

9. The method according to any one of claims 1-8, wherein the AAV1 vector comprises a promoter selected from the list of promoters in Table 5.

10. The method according to any one of claims 1-8, wherein the AAV1 vector comprises a ubiquitous promoter.

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