AAV1 vector and its use for treating ear indications

Abstract

The present invention provides AAV1 vectors that can be used to transduce multiple inner ear cell types and their use in treating hearing loss, deafness, tinnitus and vestibular dysfunction.

Description

[0001] Sequence Listing

[0002] 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

[0003] The present invention provides AAV1 vectors that can be used to transduce a variety of inner ear cell types and their use for treating hearing loss, tinnitus and vestibular dysfunction. Background of the Invention

[0005] Gene therapy has recently emerged as a promising approach for treating inner ear disorders such as hearing loss and vestibular dysfunction because it can be used to treat the genetic causes of these disorders, induce expression of genes encoding therapeutic proteins, and may result in preservation or restoration of hearing with more natural sound perception compared to cochlear implants. However, mutations that cause hearing loss and / or vestibular dysfunction have been identified in multiple different cell types of the inner ear, and preclinical studies in rodent models have yet to identify a single viral vector that can be used to transduce most or all inner ear cell types. Furthermore, few studies have been conducted in larger animal models that are more predictive of human treatment. Therefore, there is a need for viral vectors that demonstrate pantropic transduction of inner ear cells in clinically relevant animal models. Summary of the invention

[0006] The present disclosure is based on the inventors' discovery that adeno-associated virus (AAV) vectors (e.g., AAV1 capsids) having the AAV1 serotype are unexpectedly pan-tropic in the inner ear of non-human primates. AAV1 vectors exhibit strong expression and unexpectedly superior tropism in multiple cell types of the inner ear of non-human primates, compared to the tropism of AAV1 in the mouse inner ear and also compared to the tropism of other AAV serotypes (e.g., AAV2 and AAV7m8) in non-human primates. Thus, 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 primate (e.g., human) subjects to promote expression of polynucleotides, such as polynucleotides corresponding to genes that promote or improve inner ear cell function, regeneration, maintenance, development, proliferation, or survival of one or more primate (e.g., human) inner ear cells. The compositions and methods described herein can be administered to primate (eg, human) patients to treat or prevent hearing loss (eg, sensorineural hearing loss) and / or vestibular dysfunction (eg, vertigo, dizziness, or imbalance).

[0007] Exemplary embodiments of the invention are described in the following enumerated paragraphs.

[0008] E1. A method of transducing developing inner ear cells in a primate subject, the method comprising administering to the subject an effective amount of an adeno-associated virus serotype 1 (AAV1) vector in an amount that transduces 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 fiber cells, Scarpa ganglion neurons Aneurysms, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitating cells, inner column cells, outer column cells, first row of Deiter cells, second row of Deiter cells, third row of Deiter cells, Hensen cells, Claudius cells, spiral convex cells, root cells, interdental cells, basal cells of stria vascularis, intermediate cells of stria vascularis, marginal cells of stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fiber cells, cells of Reissner's membrane and glial cells.

[0009] E2. A method as described in 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 fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate 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 convex 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, fiber cells, cells of Reissner's membrane and glial cells.

[0010] E3. A method as described in 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 fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate 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 convex cells, root cells, interdental cells, basal cells of stria vascularis, intermediate cells of stria vascularis, marginal cells of stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fiber cells, cells of Reissner's membrane and glial cells.

[0011] E4. A method as described in 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 fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate 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 convex 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, fiber cells, cells of Reissner's membrane and glial cells.

[0012] E5. A method as described in 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 fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate 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 convex cells, root cells, interdental cells, basal cells of stria vascularis, intermediate cells of stria vascularis, marginal cells of stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fiber cells, cells of Reissner's membrane and glial cells.

[0013] E6. A method as described in 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 fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate 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 convex 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, fiber cells, cells of Reissner's membrane and glial cells.

[0014] E7. A method as described in any of E1-E6, wherein the AAV1 vector transduces at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of cells of a given cell type or all inner ear cell types.

[0015] E8. The method of any one of E1-E7, wherein the AAV1 vector transduces all cells of a given cell type or all inner ear cell types.

[0016] E9. The method of any one of E1-E8, wherein transduction occurs throughout the entire length of the cochlea (eg, the basal to apical axis of the cochlea).

[0017] E10. The method of 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 cells of a given cell type or all inner ear cell types across the basal to apical axis length of the cochlea.

[0018] E11. A method as described in any of E1-E10, 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 cells of a given cell type or all inner ear cell types in the base of the cochlea.

[0019] E12. A method as described in any of E1-E11, 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 cells of a given cell type or all inner ear cell types in the middle portion of the cochlea.

[0020] E13. A method as described in any of E1-E12, 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 cells of a given cell type or all inner ear cell types in the apex of the cochlea.

[0021] E14. The method of any one of E1-E13, wherein the AAV1 vector comprises a promoter selected from the list of promoters in Table 5.

[0022] E15. The method of E14, wherein the promoter is the glial fibrillary acidic protein (GFAP) promoter.

[0023] E16. The method of E14, wherein the promoter is the synapsin (SYN) promoter.

[0024] E17. The method of E14, wherein the promoter is selected from the group consisting of: Jagged 1 (JAG1) promoter, Notch 1 (NOTCH1 promoter), myosin 7A (MYO7A) promoter, solute transporter family 1 member 3 (GLAST) promoter, and POU class 4 homeobox 3 (POU4F3) promoter.

[0025] E18. A method as described in 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, calcium binding protein 2 (CALB2) promoter, basic helix-loop-helix family member E22 (BHLHE22) promoter, leucine-rich repeat-containing sequence The promoters of G protein-coupled receptor 5 (LGR5), Hes family BHLH transcription factor 1 (HES1), Hes family BHLH transcription factor 5 (HES5), SRY-box 9 (SOX9), ATPase plasma membrane Ca2+ transporter 2 (ATP2B2), CD44 molecule (CD44), fibroblast growth factor receptor 3 (FGFR3), frizzled-related protein (FRZB), solute carrier family 26 member 4 (SLC26A4), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), claudin 11 (CLDN11), peripheral myelin protein 22 (PMP22), potassium voltage-gated channel subfamily E regulatory subunit 1 (KCNE1), POU class 3 homeobox 4 (POU3F4) promoter, gap junction protein β2 (GJB2) promoter, and tubulin β3 class III (TUBB3) promoter.

[0026] E19. The method of E18, wherein the promoter is the SLC26A5 promoter.

[0027] E20. The method of E18, wherein the promoter is an OCM promoter.

[0028] E21. The method of E18, wherein the promoter is the MYO15 promoter.

[0029] E22. The method of E18, wherein the promoter is the ATP2B2 promoter.

[0030] E23. The method of E18, wherein the promoter is a GJB2 promoter.

[0031] E24. The method of E18, wherein the promoter is a TYR promoter.

[0032] E25. The method of any one of E1-E13, wherein the promoter is a ubiquitous promoter.

[0033] E26. The method of E25, wherein the promoter is a cytomegalovirus (CMV) promoter.

[0034] E27. The method of E25, wherein the promoter is a hybrid promoter derived from a CMV early enhancer element, a promoter, the first exon and the first intron of a chicken β-actin gene, and a splice acceptor of a rabbit β-globin gene (CAG promoter).

[0035] E28. The method of E25, wherein the promoter is a truncated CMV-chicken β-actin (smCBA) promoter.

[0036] E29. The method of any one of E1-E28, wherein the AAV1 vector comprises a polynucleotide selected from the list in Table 2.

[0037] E30. The method of E29, wherein the polynucleotide encodes Strc.

[0038] E31. The method of E29, wherein the polynucleotide encodes transmembrane channel-like 1 (Tmc1).

[0039] E32. The method of E29, wherein the polynucleotide encodes Harmonin (Ush1c).

[0040] E33. The method of E29, wherein the polynucleotide encodes Atonal BHLH transcription factor 1 (Atoh1).

[0041] E34. The method of E29, wherein the polynucleotide encodes Clarin 1 (Clrn1).

[0042] E35. The method of E29, wherein the polynucleotide encodes SRY-box 4 (Sox4).

[0043] E36. The method of E29, wherein the polynucleotide encodes brain-derived neurotrophic factor (Bdnf).

[0044] E37. The method of E29, wherein the polynucleotide encodes neurotrophin 3 (Ntf3).

[0045] E38. The method of E29, wherein the polynucleotide encodes SRY-box 11 (Sox11).

[0046] E39. The method of E29, wherein the polynucleotide encodes myosin 7A (Myo7a).

[0047] E40. The method of E29, wherein the polynucleotide encodes Gjb2.

[0048] E41. The method of E29, wherein the polynucleotide encodes the cholinergic receptor nicotinic α9 subunit (Chrna9).

[0049] E42. The method of E29, wherein the polynucleotide encodes the cholinergic receptor nicotinic alpha 10 subunit (Chrna10).

[0050] E43. A method as described in E29, wherein the polynucleotide encodes Ocm.

[0051] E44. The method of E29, wherein the polynucleotide encodes Tyr.

[0052] E45. The method of E29, wherein the polynucleotide encodes Tmtc4 containing transmembrane and tetratricopeptide repeat sequences.

[0053] E46. The method of E29, wherein the polynucleotide encodes TEA domain transcription factor 2 (Tead2).

[0054] E47. The method of E29, wherein the polynucleotide encodes Yes-associated protein 1 (Yap1).

[0055] E48. A method for 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.

[0056] E49. The method of E48, wherein the promoter is the GFAP promoter.

[0057] E50. The method of E48, wherein the promoter is selected from the group consisting of: a HES1 promoter, a LGR5 promoter, a SOX2 promoter, a HES5 promoter, and a SOX9 promoter.

[0058] E51. A method as described in 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), cleavage-like homeobox 1 (Cux1), nuclear receptor subfamily 2F group 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 (Zfp6 67), LIM homeobox 3 (Lhx3), Nescient helix-loop-helix 1 (Nhlh1), MAX dimerization protein 4 (Mxd4), zinc finger-containing MIZ type 1 (Zmiz1), myelin transcription factor 1 (Myt1), signal transducer and activator of transcription 3 (Stat3), BarH-like homeobox 1 (Barhl1), thymocyte selection-related high-mobility group box (Tox), Prospero homeobox 1 (Prox1), nuclear factor IA (Nfia), thyroid hormone receptor β (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), parental 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 phase 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, Atoh1 variants 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, such as variants having a sequence of any one of SEQ ID NOs: 4-10), Gfi1, Sox4, Bdnf, Ntf3, Sox11, Tead2, Yap1, or a nuclease (e.g., CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA (miRNA, such as miR-183, miR-96, or miR-182). ,

[0059] E52. The method of any one of E48-E51, wherein the supporting cells are vestibular supporting cells.

[0060] E53. The method of E52, wherein the vestibular supporting cells are located in the utricle.

[0061] E54. The method of any one of E48-E51, wherein the supporting cells are cochlear supporting cells and the promoter is SOX2.

[0062] 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: a MYO15 promoter, a GFI1 promoter, a POU4F3 promoter, and a MYO7A promoter.

[0063] E56. The method of E55, wherein the promoter is the MYO15 promoter.

[0064] E57. A method as described in E55 or E56, wherein the polynucleotide encodes Bdnf, Ntf3, Tmtc4 or a nuclease (such as Cas9, TALEN, ZFN or gRNA), or is a microRNA (miRNA, such as miR-183, miR-96 or miR-182).

[0065] E58. A method as described in any of E55-E57, wherein the hair cells are cochlear hair cells.

[0066] E59. The method of E58, wherein the cochlear hair cells are inner hair cells.

[0067] E60. The method of E58, wherein the cochlear hair cells are outer hair cells.

[0068] E61. The method of any one of E55-E57, wherein the hair cells are vestibular hair cells.

[0069] 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, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: an SLC26A5 promoter, an OCM promoter, a STRC promoter, and an ATP2B2 promoter.

[0070] E63. The method of E62, wherein the promoter is the SLC26A5 promoter.

[0071] E64. The method of E62, wherein the promoter is an OCM promoter.

[0072] E65. The method of E62, wherein the promoter is the ATP2B2 promoter.

[0073] E66. A method as described in 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).

[0074] 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, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: a MYO15 promoter, a GFI1 promoter, a POU4F3 promoter, and a MYO7A promoter.

[0075] E68. The method of E67, wherein the promoter is the GFI1 promoter.

[0076] E69. The method of E67, wherein the promoter is the MYO15 promoter.

[0077] E70. A method as described in any one of E67-E69, wherein the polynucleotide encodes Atoh1, Pou4f3, Gfi1, Isl1, Clrn1, Pcdh15, Cdh23, otoferlin (Otof), solute transporter 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).

[0078] 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: an OTOF promoter, a fibroblast growth factor 8 (FGF8) promoter, and a solute carrier protein family 17, member 8 (SLC17A8) promoter.

[0079] E72. The method of E71, wherein the promoter is OTOF promoter.

[0080] E73. A method as described in 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).

[0081] 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: a MYO15 promoter, a GFI1 promoter, a POU4F3 promoter, a MYO7A promoter, an ATP2B2 promoter, and a CALB2 promoter.

[0082] E75. A method as described in E74, wherein the promoter is the MYO15 promoter.

[0083] E76. A method as described in E74, wherein the promoter is the ATP2B2 promoter.

[0084] E77. The method of E74, wherein the vestibular hair cells are type I vestibular hair cells and the promoter is the ATP2B2 promoter.

[0085] E78. The method of E74, wherein the vestibular hair cells are type II vestibular hair cells and the promoter is the CALB2 promoter.

[0086] E79. A method as described in any 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).

[0087] E80. A method of expressing a polynucleotide in mast 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 CD44 promoter or a GJB2 promoter.

[0088] E81. The method of E80, wherein the promoter is a GJB2 promoter.

[0089] E82. A method as described in E80 or E81, wherein the polynucleotide encodes nerve growth factor receptor (Ngfr), Bdnf, Ntf3, Tectorin β (Tectb), Tectorin α (Tecta), Gjb2 or gap junction protein β6 (Gjb6).

[0090] 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, the AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the group consisting of: a BHLHE22 promoter, a SYN promoter, and a CALB2 promoter.

[0091] E84. The method of E83, wherein the promoter is the SYN promoter.

[0092] E85. The method of E83, wherein the spiral ganglion neurons have a high spontaneous firing rate and the promoter is the CALB2 promoter.

[0093] E86. The method of E83, wherein the spiral ganglion neurons are afferent spiral ganglion neurons and the promoter is the BHLHE22 promoter.

[0094] E87. A method as described in 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, such as miR-183, miR-96 or miR-182) or an shRNA against RGMA.

[0095] E88. A method for expressing a polynucleotide in marginal cells of 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 polynucleotide operably linked to a promoter selected from the group consisting of: a KCNQ1 promoter, a KCNE1 promoter, and a GJB2 promoter.

[0096] E89. The method of E88, wherein the promoter is the GJB2 promoter.

[0097] E90. A method for expressing a polynucleotide in basal cells of 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 polynucleotide operably linked to a CLDN11 promoter or a GJB2 promoter.

[0098] E91. The method of E90, wherein the promoter is the GJB2 promoter.

[0099] E92. A method for expressing a polynucleotide in intermediate cells of 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 polynucleotide operably linked to a promoter selected from the group consisting of: a KCNJ10 promoter, a GJB2 promoter, and a TYR promoter.

[0100] E93. The method of E92, wherein the promoter is the GJB2 promoter.

[0101] E94. The method of E92, wherein the promoter is a TYR promoter.

[0102] E95. A method as described in 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).

[0103] E96. A method for expressing a polynucleotide in marginal cells and / or interdigital 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 GLAST promoter or a GJB2 promoter.

[0104] E97. The method of E96, wherein the promoter is the GJB2 promoter.

[0105] E98. A method as described in E96 or E97, wherein the polynucleotide encodes Bdnf, Ntf3, Tectb, Tecta, transmembrane protein 16A (Tmem16a), Gjb2 or Gjb6.

[0106] E99. 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 polynucleotide operably linked to a FGFR3 promoter or a GJB2 promoter.

[0107] E100. The method of E99, wherein the promoter is a GJB2 promoter.

[0108] E101. The method of E99 or E100, wherein the polynucleotide encodes Bdnf, Ntf3, Tectb, Tecta, IKAROS family zinc finger 2 (Ikzf2), Gjb2 or Gjb6.

[0109] E102. 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 polynucleotide operably linked to a FRZB promoter or a GJB2 promoter.

[0110] E103. The method as described in E102, wherein the promoter is GJB2 promoter

[0111] E104. The method of E102 or E103, wherein the polynucleotide encodes Gjb2 or Gjb6.

[0112] E105. A method for expressing a polynucleotide in spiral lobule 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 a SLC26A4 promoter.

[0113] E106. The method of E105, wherein the polynucleotide is Slc26a4.

[0114] E107. 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 polynucleotide operably linked to a CEACAM16 promoter or a GJB2 promoter.

[0115] E108. A method as described in E107, wherein the promoter is a GJB2 promoter

[0116] E109. The method of E107 or E108, wherein the polynucleotide encodes Ceacam16, Otoa, Gjb2 or Gjb6.

[0117] E110. A method of 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 polynucleotide operably linked to a PMP22 promoter.

[0118] E111. The method of E110, wherein the polynucleotide encodes Pmp22, Bdnf, Ntf3 or myelin protein zero (Mpz).

[0119] E112. A method of 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 the polynucleotide operably linked to a KCNE1 promoter.

[0120] E113. The method of E112, wherein the polynucleotide encodes Kcnq1, Kcne1 or Slc26a4.

[0121] E114. A method of expressing a polynucleotide in a fiber cell 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.

[0122] E115. A method as described in E114, wherein the promoter is a GJB2 promoter

[0123] E116. The method of E114 or E115, wherein the polynucleotide encodes Gjb2, Gjb6 or collagen.

[0124] E117. 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 polynucleotide operably linked to a promoter selected from the group consisting of: a TUBB3 promoter and a SYN promoter.

[0125] E118. The method of E117, wherein the promoter is a SYN promoter.

[0126] E119. The method of E117 or E118, wherein the polynucleotide encodes Bdnf or Ntf3, or is a shRNA against the repulsive guidance molecule BMP co-receptor A (RGMA).

[0127] E120. A method for expressing a polynucleotide in a supporting cell 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, Camtal, 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, Cre b3I4, Etv1, Peg3, Bach2, Isl1, Zbtb38, Lbh, Tub, Hmg20, Rest, Zfp827, Aff3, Pknox2, Arid3b, Mlxip, Zfp532, Ikzf2, Sall1, Six2, Sall3, Lin28b, Pou4f3, Rfx7, Atoh1, Atoh1 variants 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., having SEQ ID NO: 1 ID NO: 4-10), Gfi1, Sox4, Bdnf, Ntf3, Sox11, Tead2, Yap1, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or a microRNA (e.g., miR-183, miR-96, or miR-182).

[0128] E121. The method of E120, wherein the polynucleotide encodes Atoh1.

[0129] E122. The method of E120, wherein the polynucleotide encodes Sox4.

[0130] E123. The method of E120, wherein the polynucleotide encodes Sox11.

[0131] E124. The method of E120, wherein the polynucleotide encodes Bdnf.

[0132] E125. The method of E120, wherein the polynucleotide encodes Ntf3.

[0133] E126. The method of E120, wherein the polynucleotide encodes Tead2.

[0134] E127. The method of E120, wherein the polynucleotide encodes Yap1.

[0135] E128. The method of 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.

[0136] E129. The method of E128, wherein the promoter is the GFAP promoter.

[0137] E130. The method of E128, wherein the promoter is selected from the group consisting of: a HES1 promoter, a LGR5 promoter, a SOX2 promoter, a HES5 promoter, and a SOX9 promoter.

[0138] E131. The method of any one of E120-E130, wherein the supporting cells are vestibular supporting cells.

[0139] E132. The method of E131, wherein the vestibular supporting cells are located in the utricle.

[0140] E133. The method of any one of E120-E130, wherein the supporting cells are cochlear supporting cells and the promoter is SOX2.

[0141] E134. A method for 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 comprising a promoter operably linked to a 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).

[0142] E135. The method of E134, wherein the polynucleotide encodes Bdnf.

[0143] E136. The method of E134, wherein the polynucleotide encodes Ntf3.

[0144] E137. The method of E134, wherein the polynucleotide encodes Tmtc4.

[0145] E138. The method of any one of E134-E137, wherein the promoter is selected from the group consisting of: a MYO15 promoter, a GFI1 promoter, a POU4F3 promoter, and a MYO7A promoter.

[0146] E139. The method of any one of E134-E138, wherein the hair cells are cochlear hair cells.

[0147] E140. The method of E139, wherein the cochlear hair cells are inner hair cells.

[0148] E141. The method of E139, wherein the cochlear hair cells are outer hair cells.

[0149] E142. The method of any one of E134-E138, wherein the hair cells are vestibular hair cells.

[0150] E143. 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, 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).

[0151] E144. The method of E143, wherein the polynucleotide encodes Strc.

[0152] E145. A method as described in E143, wherein the polynucleotide encodes Chrna9.

[0153] E146. The method of E143, wherein the polynucleotide encodes Chrna10.

[0154] E147. A method as described in E143, wherein the polynucleotide encodes Ocm.

[0155] E148. The method of E143, wherein said polynucleotide encodes Tmc1.

[0156] E149. The method of E143, wherein the polynucleotide encodes Myo7a.

[0157] E150. The method of E143, wherein the polynucleotide encodes Ush1c.

[0158] E151. The method of E143, wherein the polynucleotide encodes Tmtc4.

[0159] E152. The method of any one of E143-E151, wherein the promoter is selected from the group consisting of: a SLC26A5 promoter, an OCM promoter, a STRC promoter, and an ATP2B2 promoter.

[0160] 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).

[0161] E154. The method of E153, wherein the polynucleotide encodes Atoh1.

[0162] E155. The method of E153, wherein said polynucleotide encodes Clrn1.

[0163] E156. The method of E153, wherein said polynucleotide encodes Otof.

[0164] E157. The method of E153, wherein said polynucleotide encodes Tmc1.

[0165] E158. The method of E153, wherein the polynucleotide encodes Ush1c.

[0166] E159. The method of E153, wherein the polynucleotide encodes Myo7a.

[0167] E160. The method of E153, wherein the polynucleotide encodes Vglut3.

[0168] E161. The method of E153, wherein the polynucleotide encodes Strc.

[0169] E162. A method as described in E153, wherein the polynucleotide encodes Chrna9.

[0170] E163. A method as described in E153, wherein the polynucleotide encodes Chrna10.

[0171] E164. The method of E153, wherein the polynucleotide encodes Tmtc4.

[0172] E165. A method as described in E153, wherein the polynucleotide encodes Ocm.

[0173] E166. The method of any one of E153-E165, wherein the promoter is selected from the group consisting of: a MYO15 promoter, a GFI1 promoter, a POU4F3 promoter, and a MYO7a promoter.

[0174] E167. The method of E166, wherein the promoter is the GFI1 promoter.

[0175] E168. The method of E166, wherein the promoter is a MYO15 promoter.

[0176] E169. 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 comprising a promoter operably linked to a 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 a microRNA (e.g., miR-183, miR-96 or miR-182).

[0177] E170. The method of E169, wherein said polynucleotide encodes Otof.

[0178] E171. The method of E169, wherein the polynucleotide encodes Atoh1.

[0179] E172. The method of E169, wherein the polynucleotide encodes Vglut3.

[0180] E173. The method of E169, wherein said polynucleotide encodes Clrn1.

[0181] E174. The method of any one of E169-E173, wherein the promoter is selected from the group consisting of: an OTOF promoter, a FGF8 promoter, and a SLC17A8 promoter.

[0182] E175. 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 comprising a promoter operably linked to a polynucleotide encoding 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).

[0183] E176. The method of E175, wherein said polynucleotide encodes Bdnf.

[0184] E177. The method of E175, wherein said polynucleotide encodes Ntf3.

[0185] E178. The method of E175, wherein said polynucleotide encodes Tmtc4.

[0186] E179. The method of any one of E175-E178, wherein said promoter is selected from the group consisting of: a MYO15 promoter, a GFI1 promoter, a POU4F3 promoter, a MYO7A promoter, an ATP2B2 promoter, and a CALB2 promoter.

[0187] E180. The method of any one of E175-E179, wherein the vestibular hair cells are type I vestibular hair cells and the promoter is ATP2B2.

[0188] E181. The method of any one of E175-E179, wherein the vestibular hair cells are type II vestibular hair cells and the promoter is the CALB2 promoter.

[0189] E182. A method for expressing a polynucleotide in column 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 Ngfr, Bdnf, Ntf3, Tectb, Tecta, Gjb2 or Gjb6.

[0190] E183. The method of E182, wherein the polynucleotide encodes Gjb2.

[0191] E184. The method of E182 or E183, wherein the promoter is CD44 promoter or GJB2 promoter.

[0192] 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 comprising a promoter operably linked to a polynucleotide encoding Bdnf, Ntf3 or a nuclease (e.g., Cas9, TALEN, ZFN or gRNA) or a microRNA (e.g., miR-183, miR-96 or miR-182) or an shRNA against RGMA.

[0193] E186. The method of E185, wherein said polynucleotide encodes Bdnf.

[0194] E187. The method of E185, wherein said polynucleotide encodes Ntf3.

[0195] E188. The method of any one of E185-E187, wherein said promoter is selected from the group consisting of: a BHLHE22 promoter, a SYN promoter, and a CALB2 promoter.

[0196] E189. The method of any one of E185-E188, wherein the spiral ganglion neurons have a high spontaneous firing rate and the promoter is a CALB2 promoter.

[0197] E190. The method of any one of E185-E188, wherein the vestibular hair cells are afferent spiral ganglion neurons and the promoter is the BLHLE22 promoter.

[0198] 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 encoding Kcnq1, Kcne1, Gjb2, Gjb6, Tyr or a nuclease (e.g., Cas9, TALEN, ZFN or gRNA) or a microRNA (e.g., miR-183, miR-96 or miR-182).

[0199] E192. The method of E191, wherein the polynucleotide encodes Gjb2.

[0200] E193. The method of E191, wherein said polynucleotide encodes Tyr.

[0201] E194. The method of any one of E191-E193, wherein said cell is a stria vascularis marginal cell, and said promoter is selected from the group consisting of: a KCNQ1 promoter, a GJB2 promoter, and a KCNE1 promoter.

[0202] E195. The method of any one of E191-E193, wherein the cell is a stria vascularis basal cell, and the promoter is a CLDN11 promoter or a GJB2 promoter.

[0203] E196. The method of any one of E191-E193, wherein the cell is a stria vascularis intermediate cell, and the promoter is selected from the group consisting of: a KCNJ10 promoter, a GJB2 promoter, and a TYR promoter.

[0204] E197. A method for expressing a polynucleotide in marginal cells and / or inner finger 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 Bdnf, Ntf3, Tectb, Tecta, Tmem16a, Gjb2 or Gjb6.

[0205] E198. The method of E197, wherein the polynucleotide encodes Gjb2.

[0206] E199. The method of E197 or E196, wherein the promoter is a GLAST promoter or a GJB2 promoter.

[0207] 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 encoding Bdnf, Ntf3, Tectb, Tecta, Ikzf2, Gjb2 or Gjb6.

[0208] E201. The method of E200, wherein the polynucleotide encodes Gjb2.

[0209] E202. The method of E200 or E201, wherein the promoter is a FGFR3 promoter or a GJB2 promoter.

[0210] 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.

[0211] E204. The method of E203, wherein the polynucleotide encodes Gjb2.

[0212] E205. The method of E203 or E204, wherein the promoter is a FRZB promoter or a GJB2 promoter.

[0213] E206. A method for expressing a polynucleotide in spiral lobule 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 promoter operably linked to a polynucleotide encoding Slc26a4.

[0214] E207. The method of E206, wherein the promoter is SLC26A4 promoter.

[0215] 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 encoding Ceacam16, Otoa, Gjb2 or Gjb6.

[0216] E209. The method of E208, wherein the polynucleotide encodes Gjb2.

[0217] E210. The method of E208 or E209, wherein the promoter is CEACAM16 promoter or GJB2 promoter.

[0218] 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 encoding Pmp22, Bdnf, Ntf3 or Mpz.

[0219] E212. The method of E211, wherein the polynucleotide encodes Bdnf.

[0220] E213. The method of E211, wherein the polynucleotide encodes Ntf3.

[0221] E214. The method of any one of E211-E213, wherein the promoter is a PMP22 promoter.

[0222] 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 encoding Kcnq1, Kcne1 or Slc26a4.

[0223] E216. The method of E215, wherein the promoter is KCNE1 promoter.

[0224] E217. A method for expressing a polynucleotide in a primate subject's fiber cells, 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, Gjb6 or collagen.

[0225] E218. The method of E217, wherein the polynucleotide encodes Gjb2.

[0226] E219. The method of E217 or E218, wherein the promoter is a POU3F4 promoter or a GJB2 promoter.

[0227] E220. A method for 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 comprising a promoter operably linked to a polynucleotide encoding Bdnf or Ntf3 or an shRNA against RGMA.

[0228] E221. The method of E220, wherein the polynucleotide encodes Bdnf.

[0229] E222. The method of E220, wherein the polynucleotide encodes Ntf3.

[0230] E223. The method of any one of E220-E222, wherein the promoter is selected from the group consisting of a TUBB3 promoter and a SYN promoter.

[0231] E224. A method of treating a primate subject having or at risk of developing monogenic inherited deafness associated with a mutation in OTOF, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Otof.

[0232] E225. The method of E224, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, a smCBA promoter, a MYO15 promoter, and an OTOF promoter.

[0233] E226. A method as described in E225, wherein the promoter is a MYO15 promoter or an OTOF promoter

[0234] E227. A method for treating a primate subject having 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 comprising a promoter operably linked to a polynucleotide encoding Strc.

[0235] E228. The method of E227, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, a smCBA promoter, a MYO15 promoter, a SLC26A5 promoter, an OCM promoter, and an ATP2B2 promoter.

[0236] E229. The method of E228, wherein the promoter is selected from the group consisting of: MYO15 promoter, SLC26A5 promoter, OCM promoter and ATP2B2 promoter.

[0237] E230. A method for treating a primate subject having 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.

[0238] E231. A method of treating a primate subject having 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.

[0239] E232. A method of treating a primate subject having 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.

[0240] E233. A method of treating a primate subject having or at risk of developing a 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] E234. A method of treating a primate subject having 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.

[0242] E235. The method of any one of E230-E234, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, a smCBA promoter, and a GFAP promoter.

[0243] E236. The method of E235, wherein the promoter is the GFAP promoter.

[0244] E237. A method for treating a primate subject having or at risk of developing monogenic hereditary deafness associated with a mutation in GJB2, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Gjb2.

[0245] E238. A method of treating a primate subject having, or at risk of developing, age-related hearing loss associated with a mutation in GJB2, 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] E239. The method of E237 or E238, wherein the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, a smCBA promoter, and a GJB2 promoter.

[0247] E240. The method of E239, wherein the promoter is a GJB2 promoter.

[0248] E241. A method of treating a primate subject having or at risk of developing monogenic hereditary deafness associated with a mutation in SLC17A8, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Slc17a8.

[0249] E242. The method of E241, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, a smCBA promoter, a MYO15 promoter, and an OTOF promoter.

[0250] E243. The method of E242, wherein the promoter is a MYO15 promoter or an OTOF promoter.

[0251] E244. A method of treating a primate subject having or at risk of developing monogenic inherited deafness associated with a mutation in TMC1, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Tmc1.

[0252] E245. The method of E244, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, and a smCBA promoter.

[0253] E246. A method as described in E244, wherein the promoter is the MYO15 promoter.

[0254] E247. A method of treating a primate subject having 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 Ntf3.

[0255] E248. A method of treating a primate subject having or at risk of developing tinnitus, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0256] E249. A method for treating a primate subject having or at risk of developing difficulty understanding speech in noise, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0257] E250. A method of treating a primate subject having or at risk of developing Charcot-Marie-Tooth disease, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0258] E251. A method of treating a primate subject having or at risk of developing Friedreich's ataxia, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ntf3.

[0259] E252. The method of any one of E247-E251, wherein the promoter is a SYN promoter.

[0260] E253. The method of any one of E247-E251, wherein the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, and a smCBA promoter.

[0261] E254. A method of treating a primate subject having 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 Bdnf.

[0262] E255. A method of treating a primate subject having or at risk of developing tinnitus, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0263] E256. A method of treating a primate subject having or at risk of developing difficulty understanding speech in noise, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0264] E257. A method of treating a primate subject having or at risk of developing Charcot-Marie-Tooth disease, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0265] E258. A method of treating a primate subject having or at risk of developing Friedreich's ataxia, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Bdnf.

[0266] E259. The method of any one of E254-E258, wherein the promoter is a SYN promoter.

[0267] E260. The method of any one of E254-E258, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, and a smCBA promoter.

[0268] E261. A method of treating a primate subject having 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 Sox4.

[0269] E262. A method of treating a primate subject having 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 Sox4.

[0270] E263. A method of treating a primate subject having or at risk of developing a 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 Sox4.

[0271] E264. A method of treating a primate subject having 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 Sox4.

[0272] E265. The method of any one of E261-E264, wherein the promoter is the GFAP promoter.

[0273] E266. The method of any one of E261-E264, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, and a smCBA promoter.

[0274] E267. A method of treating a primate subject having 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 Sox11.

[0275] E268. A method of treating a primate subject having 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 Sox11.

[0276] E269. A method of treating a primate subject having or at risk of developing a 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 Sox11.

[0277] E270. A method of treating a primate subject having 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 Sox11.

[0278] E271. The method of any one of E267-E270, wherein the promoter is the GFAP promoter.

[0279] E272. The method of any one of E267-E270, wherein the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, and a smCBA promoter.

[0280] E273. A method of treating a primate subject having or at risk of developing monogenic hereditary deafness associated with a mutation in USH1C, comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Ush1c.

[0281] E274. A method as described in E273, wherein the promoter is the MYO15 promoter.

[0282] E275. The method of E273, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, and a smCBA promoter.

[0283] E276. A method of treating a primate subject having or at risk of developing monogenic inherited deafness associated with a mutation in MYO7A, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Myo7a.

[0284] E277. A method as described in E276, wherein the promoter is the MYO15 promoter.

[0285] E278. The method of E276, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, and a smCBA promoter.

[0286] E279. A method of treating a primate subject having or at risk of developing monogenic inherited deafness associated with a mutation in CLRN1, the method comprising administering to the subject an effective amount of an AAV1 vector comprising a promoter operably linked to a polynucleotide encoding Clrn1.

[0287] E280. The method of E279, wherein the promoter is the MYO15 promoter.

[0288] E281. The method of E279, wherein the promoter is selected from the group consisting of: a CMV promoter, a CAG promoter, and a smCBA promoter.

[0289] 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 comprising a promoter operably linked to a polynucleotide.

[0290] E283. The method of E282, wherein the promoter is a supporting cell promoter, a Scarpa ganglion promoter, a vestibular hair cell promoter, or a vestibular supporting cell promoter listed in Table 5.

[0291] E284. A method as described in E283, wherein the promoter is MYO15.

[0292] E285. The method of 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 the ganglion of Scarpa selected from the list in Table 2.

[0293] E286. A method as described in any of E282-E285, wherein the polynucleotide encodes Whirlin, Sox9, Sall2, Camtal, 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, Isl 1, Zbtb38, Lbh, Tub, Hmg20, Rest, Zfp827, Aff3, Pknox2, Arid3b, Mlxip, Zfp532, Ikzf2, Sall1, Six2, Sall3, Lin28b, Pou4f3, Rfx7, Atoh1, Atoh1 variants 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., having SEQ ID NO: 4-10), Gfi1, Sox4, Bdnf, Ntf3, Tead2, Yap1, Tmtc4, Sox11, or a nuclease (e.g., Cas9, TALEN, ZFN, or gRNA), or a microRNA (e.g., miR-183, miR-96, or miR-182).

[0294] E287. The method of any one of E282-E286, wherein the vestibular dysfunction is vertigo, dizziness or imbalance.

[0295] 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 comprising a promoter operably linked to a polynucleotide.

[0296] 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.

[0297] E290. The method of E288 or E289, wherein the AAV1 vector comprises a polynucleotide for expression in IHCs, OHCs, cochlear hair cells, vestibular hair cells and cochlear hair cells, cochlear supporting cells and vestibular supporting cells, cochlear supporting cell subtypes or spiral ganglion neurons selected from the list in Table 2.

[0298] E291. A method as described in any 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).

[0299] 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 comprising a promoter operably linked to a polynucleotide.

[0300] 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.

[0301] E294. A method as described in E292 or E293, wherein the AAV1 vector contains a polynucleotide for expression in IHCs, OHCs, cochlear hair cells, vestibular hair cells and cochlear hair cells, cochlear supporting cell subtypes or cochlear supporting cells and vestibular supporting cells selected from the list in Table 2.

[0302] E295. A method for 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 comprising a promoter operably linked to a polynucleotide.

[0303] E296. The method of E295, wherein the promoter is a supporting cell promoter listed in Table 5, a cochlear supporting cell subtype promoter, or a vestibular supporting cell promoter.

[0304] E297. The method of E295 or E296, wherein the AAV1 vector contains a polynucleotide for expression in cochlear supporting cells and vestibular supporting cells or a subtype of cochlear supporting cells selected from the list in Table 2.

[0305] 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 an AAV1 vector of the invention or a composition of the invention to the subject.

[0306] E299. A method as described in E298, wherein the inner ear cell damage or death is damage or death caused by ototoxic drugs.

[0307] E300. A method as described in E299, wherein the ototoxic drug is selected from the group comprising: aminoglycosides (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin and amikacin), anti-tumor drugs (e.g., platinum-containing chemotherapeutics such as cisplatin, carboplatin and oxaliplatin), ethacrynic acid, furosemide, salicylates (e.g., aspirin, especially at high doses) and quinine.

[0308] E301. The method of E298, wherein the inner ear cell damage or death is due to acoustic trauma, disease or infection, head trauma or aging.

[0309] E302. A method of treating a subject suffering from or at risk of developing tinnitus by administering to the subject an effective amount of an AAV1 vector of the invention or a composition of the invention.

[0310] E303. A method of increasing inner ear cell survival in a primate subject in need thereof by administering to the subject an effective amount of an AAV1 vector of the invention or a composition of the invention.

[0311] E304. The method of any one of E1-E303, wherein the AAV1 vector is topically administered to the middle ear or inner ear of the subject.

[0312] E305. The method of E304, wherein the AAV1 vector is administered transtympanically or intratympanically.

[0313] E306. The method of E304, wherein the AAV1 vector is administered into the perilymph.

[0314] E307. The method of E304, wherein the AAV1 vector is administered into the endolymph.

[0315] E308. The method of E304, wherein the AAV1 vector is administered to or through the oval window.

[0316] E309. The method of E304, wherein the AAV1 vector is administered to or through the round window.

[0317] E310. The method of E304, wherein the AAV1 vector is administered to the endolymphatic sac.

[0318] E311. The method of E304, wherein the AAV1 vector is administered to or through the semicircular canals.

[0319] E312. The method of any one of E1-E311, wherein the AAV1 vector comprises an inverted terminal repeat sequence (ITR) from a serotype 2 adeno-associated virus (AAV2) vector.

[0320] E313. A method as described in 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 an amino acid sequence of SEQ ID NO: 1-3).

[0321] E314. The method of any one of E1-E313, wherein the AAV1 vector has about 1x10 9 Vector genomes (VG) / mL to 1x10 15 VG / mL (e.g., 1x10 9 VG / mL, 2x10 9 VG / mL, 3x10 9 VG / mL, 4x10 9 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 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 15VG / mL) and are 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).

[0322] E315. A method as described in E1-E314, wherein the AAV1 vector is 1x10 7 VG / ear up 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 VG / ear) dose was administered.

[0323] E316. The method of any one of E1-E315, wherein the primate is a human.

[0324] E317. The method of any one of E1-E316, wherein the subject is an adult.

[0325] E318. A method as described in any of E1-E316, wherein the subject is a teenager.

[0326] E319. The method of any one of E1-E316, wherein the subject is a child.

[0327] E320. The method of any one of E1-E316, wherein the subject is an infant or a full-term newborn.

[0328] E321. The method of any one of E55-E57 and E134-E138, wherein the hair cells are cochlear hair cells.

[0329] E322. The method of E321, wherein the cochlear hair cells are IHCs.

[0330] E323. The method of E321, wherein the cochlear hair cells are OHCs.

[0331] E324. The method of any one of E55-E57 and E134-E138, wherein the hair cells are vestibular hair cells.

[0332] E325. The method of any one of E48-E51 and E120-E130, wherein the supporting cells are vestibular supporting cells.

[0333] E326. The method of any one of E48-E51 and E120-E130, wherein the supporting cells are cochlear supporting cells (eg, Hensen's cells, Deiter's cells, pillar cells, inner digitate cells, and / or marginal cells).

[0334] E327. The method of any one of E1-E326, wherein the method further comprises assessing the subject's hearing (e.g., assessing hearing using a standard test such as audiometry, auditory brainstem response (ABR), electrochocleography (ECOG), or otoacoustic emissions) prior to administering the AAV1 vector or composition.

[0335] E328. The method of any one of E1-E327, wherein the method further comprises assessing the subject's hearing after administering the AAV1 vector or composition (e.g., assessing hearing using a standard test such as audiometry, ABR, ECOG, or otoacoustic emissions).

[0336] E329. The method of any one of E1-E328, wherein the method further comprises assessing the subject's vestibular function prior to administering the AAV1 vector or composition (e.g., assessing vestibular function using a standard test such as an electronystagmogram (ENG) or videonystagmogram (VNG), posturography, rotary-chair testing, ECOG, vestibular evoked myogenic potential (VEMP), or a specialized clinical balance test).

[0337] E330. The method of any one of E1-E329, wherein the method further comprises assessing the vestibular function of the subject after administration of the AAV1 vector or composition (e.g., assessing vestibular function using a standard test such as ENG or VNG, posturography, rotation chair test, ECOG, VEMP, or a specialist clinical balance test).

[0338] E331. The method of any one of E1-E330, wherein the AAV1 vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, prevent or reduce vestibular dysfunction, prevent or reduce 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), or C and / or OHC, and / or causing vestibular supporting cells to differentiate into vestibular hair cells), preventing or reducing 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), promoting or increasing inner ear cell development, promoting or increasing 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 damage or degeneration or loss of inner ear cells), improving inner ear cell function, preserving ribbon synapses, promoting or increasing ribbon synapse formation, maintaining connections (e.g., synaptic connections) between hair cells and neurons (e.g., SGNs and / or vestibular ganglion neurons), or increasing or restoring connections (e.g., synaptic connections) between hair cells and neurons (e.g., SGNs and / or vestibular ganglion neurons).

[0339] E332. The method of any one of E1-E331, wherein the subject has or is at risk of developing hearing loss (e.g., sensorineural hearing loss, deafness, or auditory neuropathy).

[0340] E333. A method as described in any of E1-E332, wherein the subject suffers from or is at risk of developing vestibular dysfunction (e.g., dizziness, vertigo, or imbalance).

[0341] E334. A method as described in any of E1-E333, wherein the subject has been previously diagnosed as suffering from vestibular dysfunction (e.g., dizziness, vertigo, or imbalance).

[0342] E335. A method as described in any of E1-E334, wherein the subject has been previously diagnosed with hearing loss (e.g., sensorineural hearing loss, deafness, or auditory neuropathy).

[0343] E336. The method of any one of E234-E236, E238, E247, E254, E264, E270, E288-E291 and E331-E335, wherein the hearing loss is hereditary hearing loss.

[0344] E337. The method of E336, wherein the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss or X-linked hearing loss.

[0345] E338. The method of any one of E234-E236, E238, E247, E254, E264, E270, E288-E291 and E331-E335, wherein the hearing loss is acquired hearing loss.

[0346] E339. A method as described in 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 hearing loss caused by ototoxic drugs.

[0347] E340. The method of E339, wherein the acquired hearing loss is age-related hearing loss.

[0348] E341. A method as described in E339, wherein the hearing loss is noise-induced hearing loss.

[0349] E342. The method of E339, wherein the hearing loss is hearing loss caused by ototoxic drugs.

[0350] E343. An AAV1 vector comprising a polynucleotide operably linked to a promoter selected from the list of promoters in Table 5.

[0351] E344. The AAV1 vector of E343, wherein the promoter is the GFAP promoter.

[0352] E345. The AAV1 vector of E343, wherein the promoter is the SYN promoter.

[0353] E346. An 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. 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.

[0354] E347. The AAV1 vector of E346, wherein the promoter is the SLC26A5 promoter.

[0355] E348. The AAV1 vector of E346, wherein the promoter is an OCM promoter.

[0356] E349. The AAV1 vector of E346, wherein the promoter is the OTOF promoter.

[0357] E350. The AAV1 vector of E346, wherein the promoter is a MYO15 promoter.

[0358] E351. The AAV1 vector of E346, wherein the promoter is the ATP2B2 promoter.

[0359] E352. The AAV1 vector of E346, wherein the promoter is a GJB2 promoter.

[0360] E353. The AAV1 vector of E346, wherein the promoter is a TYR promoter.

[0361] E354. The AAV1 vector of any one of E343-E353, wherein the polynucleotide is selected from the polynucleotides listed in Table 2.

[0362] E355. The AAV1 vector of E354, wherein the polynucleotide encodes Otof.

[0363] E356. The AAV1 vector of E354, wherein the polynucleotide encodes Strc.

[0364] E357. The AAV1 vector of E354, wherein the polynucleotide encodes Atoh1.

[0365] E358. The AAV1 vector of E354, wherein the polynucleotide encodes Gjb2.

[0366] E359. The AAV1 vector of E354, wherein the polynucleotide encodes Slc17a8.

[0367] E360. The AAV1 vector of E354, wherein the polynucleotide encodes Tmc1.

[0368] E361. The AAV1 vector of E354, wherein the polynucleotide encodes Ntf3.

[0369] E362. The AAV1 vector of E354, wherein the polynucleotide encodes Bdnf.

[0370] E363. The AAV1 vector of E354, wherein the polynucleotide encodes Sox4.

[0371] E364. The AAV1 vector of E354, wherein the polynucleotide encodes Sox11.

[0372] E365. The AAV1 vector of E354, wherein the polynucleotide encodes Ush1c.

[0373] E366. The AAV1 vector of E354, wherein the polynucleotide encodes Myo7a.

[0374] E367. The AAV1 vector of E354, wherein the polynucleotide encodes Clrn1.

[0375] E368. The AAV1 vector as described in E354, wherein the polynucleotide encodes Chrna9.

[0376] E369. The AAV1 vector as described in E354, wherein the polynucleotide encodes Chrna10.

[0377] E370. The AAV1 vector of E354, wherein the polynucleotide encodes Tmtc4.

[0378] E371. The AAV1 vector of E354, wherein the polynucleotide encodes Ocm.

[0379] E372. The AAV1 vector of E354, wherein the polynucleotide encodes Tyr.

[0380] E373. The AAV1 vector of E354, wherein the polynucleotide encodes Tead2.

[0381] E374. The AAV1 vector of E354, wherein the polynucleotide encodes Yap1.

[0382] E375. An AAV1 vector comprising a promoter operably linked to a polynucleotide selected from the polynucleotides listed in Table 2.

[0383] E376. The AAV1 vector as described in E375, wherein the polynucleotide encodes Otof.

[0384] E377. The AAV1 vector as described in E375, wherein the polynucleotide encodes Strc.

[0385] E378. The AAV1 vector as described in E375, wherein the polynucleotide encodes Atoh1.

[0386] E379. The AAV1 vector of E375, wherein the polynucleotide encodes Gjb2.

[0387] E380. The AAV1 vector of E375, wherein the polynucleotide encodes Slc17a8.

[0388] E381. The AAV1 vector of E375, wherein the polynucleotide encodes Tmc1.

[0389] E382. The AAV1 vector of E375, wherein the polynucleotide encodes Ntf3.

[0390] E383. The AAV1 vector of E375, wherein the polynucleotide encodes Bdnf.

[0391] E384. The AAV1 vector of E375, wherein the polynucleotide encodes Sox4.

[0392] E385. The AAV1 vector of E375, wherein the polynucleotide encodes Sox11.

[0393] E386. The AAV1 vector of E375, wherein the polynucleotide encodes Ush1c.

[0394] E387. The AAV1 vector of E375, wherein the polynucleotide encodes Myo7a.

[0395] E388. The AAV1 vector of E375, wherein the polynucleotide encodes Clrn1.

[0396] E389. The AAV1 vector as described in E375, wherein the polynucleotide encodes Chrna9.

[0397] E390. The AAV1 vector as described in E375, wherein the polynucleotide encodes Chrna10.

[0398] E391. The AAV1 vector of E375, wherein the polynucleotide encodes Tmtc4.

[0399] E392. The AAV1 vector of E375, wherein the polynucleotide encodes Ocm.

[0400] E393. The AAV1 vector of E375, wherein the polynucleotide encodes Tyr.

[0401] E394. The AAV1 vector as described in E375, wherein the polynucleotide encodes Tead2.

[0402] E395. The AAV1 vector of E375, wherein the polynucleotide encodes Yap1.

[0403] E396. The AAV1 vector of any one of E375-E395, wherein the promoter is selected from the list of promoters in Table 5.

[0404] E397. The AAV1 vector as described in E396, wherein the promoter is the GFAP promoter.

[0405] E398. The AAV1 vector as described in E396, wherein the promoter is the SYN promoter.

[0406] E399. An 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 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.

[0407] E400. The AAV1 vector as described in E399, wherein the promoter is the SLC26A5 promoter.

[0408] E401. The AAV1 vector of E399, wherein the promoter is an OCM promoter.

[0409] E402. The AAV1 vector of E399, wherein the promoter is an OTOF promoter.

[0410] E403. The AAV1 vector as described in E399, wherein the promoter is the MYO15 promoter.

[0411] E404. The AAV1 vector as described in E399, wherein the promoter is the ATP2B2 promoter.

[0412] E405. The AAV1 vector as described in E399, wherein the promoter is the GJB2 promoter.

[0413] E406. The AAV1 vector as described in E399, wherein the promoter is a TYR promoter.

[0414] E407. The AAV1 vector of any one of E343-E406, wherein the AAV1 vector comprises AAV2 ITRs.

[0415] E408. The AAV1 vector of any one of E343-E407, wherein the AAV1 vector comprises a wild-type AAV1 capsid (eg, wherein the AAV1 vector contains a capsid protein having an amino acid sequence of SEQ ID NOs: 1-3).

[0416] E409. A pharmaceutical composition comprising the AAV1 vector as described in any one of E343-E408.

[0417] E410. The pharmaceutical composition as described in E409, further comprising a pharmaceutically acceptable excipient.

[0418] E411. The pharmaceutical composition of E409 or E410, wherein the pharmaceutical composition is formulated for topical administration to the inner ear or the middle ear.

[0419] E412. A pharmaceutical kit comprising the AAV1 vector of any one of E343-E408 or the pharmaceutical composition of any one of E409-E411.

[0420] These and other aspects of the invention will be apparent to those of ordinary skill in the art from the following description, claims and drawings.

[0421] definition

[0422] As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0423] As used herein, "administering" refers to providing or administering a therapeutic agent (eg, an AAV1 vector described herein) to a subject by any effective route. Exemplary routes of administration are described below.

[0424] As used herein, the terms "adeno-associated virus serotype 1 vector" and "AAV1 vector" refer to an adeno-associated virus vector having an AAV1 capsid. The terms "adeno-associated virus serotype 1 vector" and "AAV1 vector" are used interchangeably herein and refer not only to an AAV vector having wild-type AAV1 capsid proteins (VP1, VP2, and VP3), but also to an AAV vector having an AAV1 capsid protein having a sequence modification of the broad tropism of wild-type AAV1 in the inner ear of primates. AAV1 vectors useful in the compositions and methods described herein include those having an AAV1 capsid protein having an amino acid sequence that is at least 90% identical to the amino acid sequence 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% identical to the amino acid sequence of wild-type AAV1 capsid proteins VP1, VP2, and VP3); As well as those vectors having AAV1 capsid proteins with one or more conservative amino acid substitutions (e.g., at most 5, at most 10, at most 15, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 conservative amino acid substitutions) and / or one or more non-conservative amino acid substitutions (e.g., at most 5, at most 10, at most 15, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 non-conservative amino acid substitutions) relative to the amino acid sequences of wild-type AAV1 capsid proteins VP1, VP2, and VP3. The AAV1 vectors useful 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. The AAV1 vectors useful in the compositions and methods described herein may 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 Tyr 733 to Phe733 (Y733F), which reduce proteomic degradation.The capsid protein of the AAV1 vector used in the compositions and methods described herein may have an amino acid sequence described in U.S. Pat. No. 6,759,237 and be encoded by a polynucleotide sequence described therein, which is incorporated herein by reference. The AAV1 capsid protein may also be modified as described in U.S. Pat. No. 7,749,492, which is incorporated herein by reference. The methods described herein may be used to assess tropism in the inner ear.

[0425] 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 common cell types may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of common cell types may include those cells separated from common tissues (e.g., epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those cells separated from other structures and / or regions of common organs, tissue systems, blood vessels, or organisms.

[0426] As used herein, the terms "conservative mutation", "conservative substitution" and "conservative amino acid substitution" refer to substitution of one or more amino acids into one or more different amino acids that exhibit similar physicochemical properties (such as polarity, electrostatic charge and steric bulk). These properties for each of the 20 naturally occurring amino acids are summarized in Table 1 below.

[0427] Table 1. Representative physicochemical properties of naturally occurring amino acids

[0428]

[0429] It will be appreciated from this table that the conservative amino acid family includes (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 one in which an amino acid is substituted for a member of the same amino acid family (e.g., Ser for Thr or Lys for Arg).

[0430] As used herein, the term "developed inner ear cells" refers to inner ear cells that have completed the developmental process that occurs during full-term pregnancy, which is defined as the onset of labor in primates (e.g., humans) 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 newborns 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).

[0431] 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 outcome (including a clinical outcome) when administered to a subject, and thus, an "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 compared to the response obtained without 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 that will correspond to such an amount will vary according to various factors, such as a given dose, pharmaceutical formulation, route of administration, type of disease or condition, the subject (e.g., age, sex, weight) or identity of the host being treated, etc., but still can be readily determined by one of ordinary skill in the art by conventional methods known in the art. Dosage regimens can be adjusted to provide the optimal therapeutic response.

[0432] As used herein, the term "enhancer" refers to a type of regulatory element that is capable of increasing transcription efficiency and is independent of the distance or orientation of the enhancer relative to the transcription start site. Therefore, the enhancer can be placed upstream or downstream of the transcription start site or at a considerable distance from the promoter. The enhancer can also overlap with the promoter physically and functionally. Many polynucleotides comprising a promoter sequence (e.g., CMV promoter) also contain an enhancer sequence.

[0433] 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' cap formation, 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.

[0434] As used herein, the term "heterologous" refers to a combination of elements that do not occur naturally. For example, a heterologous transgene refers to a transgene that is not naturally expressed by the promoter to which it is operably linked.

[0435] As used herein, the terms "increase" and "decrease" refer to the regulation of a function, expression or activity that produces a greater or lesser amount, respectively, relative to a reference. For example, after the composition is administered as described herein, the amount of a marker of a measure (e.g., transgenic expression) as described herein may be increased or decreased 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 before administration. Typically, after administration, the metric is measured at the time when the effect has been achieved, for example, at least one week, one month, 3 months or 6 months after the start of the treatment regimen.

[0436] As used herein, the term "inner ear cell type" refers to a cell type 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 fiber cells, Scarpa ganglion neurons (vestibular ganglion neurons), endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner finger cells, inner column cells, outer column cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, spiral convex 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, fiber cells, cells of Reissner's membrane, and glial cells.

[0437] As used herein, "topical" or "local administration" refers to administration at a specific site of the body intended for local rather than systemic effect. 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., such as administration through the oval window, round window or horizontal semicircular canal into the perilymph or endolymph, such as intratympanic or transtympanic administration), and administration to the mucous membranes of a subject, where the administration is intended to produce a local rather than systemic effect.

[0438] As used herein, the term "operably connected" refers to a first molecule being linked to a second molecule, wherein the arrangement of the molecules causes the first molecule to affect the function of the second molecule. Two molecules may or may not be a part of a single adjacent 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, the promoter is operably connected to a transcribable polynucleotide molecule. In addition, if two parts of a transcriptional regulatory element are linked together so that the transcriptional activation functionality of one part is not adversely affected by the presence of another part, the two parts are operably connected to each other. Two transcriptional regulatory elements can be operably connected to each other by a joint nucleic acid (for example, inserting a non-coding nucleic acid), or can be operably connected to each other in the absence of an inserted nucleotide.

[0439] As used herein, the term "polynucleotide" refers to a polymer of nucleotides. Typically, polynucleotides are composed of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) naturally present in DNA or RNA connected by phosphodiester bonds. The term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not present in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When the application relates to polynucleotides, it should be understood that DNA, RNA are provided, and single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided in each case. As used herein, "polynucleotide sequence" may refer to polynucleotide material itself and / or biochemical characterization of sequence information of specific nucleic acids (i.e., a series of letters used as base abbreviations). Unless otherwise indicated, the polynucleotide sequence given herein is given in 5' to 3' directions.

[0440] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives transcription of a transgene operably linked to the promoter.

[0441] " Percentage (%) of sequence identity " with respect to reference polynucleotide or peptide sequence is defined as after comparing sequence and introducing room (if necessary) to realize maximum sequence identity percentage, the percentage of nucleic acid or amino acid identical with the nucleic acid in reference polynucleotide or peptide sequence in candidate sequence.Comparison for determining nucleic acid or amino acid sequence identity percentage can be realized in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2 or Megalign software.Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for maximum comparison on the total length of the sequence compared.For example, sequence comparison computer program BLAST can be used to generate sequence identity percentage value.As an illustration, given nucleic acid or amino acid sequence A for, with or for the sequence identity percentage of given nucleic acid or amino acid sequence B (it can be alternatively phrased as given nucleic acid or amino acid sequence A has for, with or for a certain sequence identity percentage of given nucleic acid or amino acid sequence B) is calculated as follows:

[0442] 100×(fraction X / Y)

[0443] wherein X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in the program's alignment of A and B, and wherein Y is the total number of nucleic acids in B. It will be understood that 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.

[0444] 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 in order to prevent, treat or control a specific disease or condition that affects or may affect the subject.

[0445] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of subjects without undue toxicity, irritation, allergic response and other problematic complications, and are commensurate with a reasonable benefit / risk ratio.

[0446] 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).

[0447] As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that at least partially controls the transcription of a polynucleotide. Transcriptional regulatory elements can include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help control gene transcription. Examples of transcriptional regulatory elements are described in, for example, Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0448] As used herein, the terms "subject" and "patient" refer to primates (e.g., humans). Subjects to be treated according to the methods described herein may be subjects who have 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 subjects at risk of developing these disorders (e.g., subjects 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, genetic mutations, or a family history of hearing loss, tinnitus, or vestibular dysfunction). Diagnosis may be performed by any method or technique known in the art. Those skilled in the art will appreciate that subjects to be treated according to the present disclosure may have been subjected to standard testing, or may have been identified as risky subjects without examination due to the presence of one or more risk factors associated with a disease or disorder.

[0449] As used herein, the terms "transduction" and "transduce" refer to a method of introducing a vector construct or a portion thereof into a cell. Where the vector construct is contained in 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 an inner ear cell type" is defined as the amount of AAV1 vector required to transduce 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%, or 100% of the cells of a given cell type). -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%,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% of a given cell type or of all inner ear cell types).

[0450] As used herein, "treatment" and "treating" with respect to a disease or disorder refer to methods for obtaining a beneficial or desired result (e.g., a clinical result). Beneficial or desired results may include, but are not limited to, alleviation or improvement of one or more symptoms or disorders; reduction in the extent of a disease or disorder; stabilization of the state of a disease, disorder, or illness (i.e., not worsening); prevention of the spread of a disease or disorder; delay or slowing of the progression of a disease or disorder; improvement or alleviation of a disease or disorder; and alleviation (whether partial or total), whether detectable or undetectable. "Improvement" or "alleviation" of a disease or disorder means that the extent and / or adverse clinical manifestations of the disease, disorder, or disorder are reduced and / or the time course of progression is slowed or prolonged compared to the extent or time course in the absence of treatment. "Treatment" may also mean prolonged survival compared to the expected survival if not receiving treatment. Those in need of treatment include those who already have a disease or disorder and those who are susceptible to a disease or disorder or those who intend to prevent a disease or disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0451] Figure 1A-1B Figure 1 is a series of fluorescent 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 unilateral injection was used to administer 1 μL of AAV1-CMV-GFP (virus titer 2.94×10 13Two weeks after injection, the inner ear was removed and the surface of the basilar membrane was prepared. A representative ear is shown. EGFP expression was observed in the apical, mid, and basal regions of the cochlea ( Figure 1A ; Upper left: apex of the cochlea; Lower right: mid-periphery of the cochlea; Upper right: basal period of the cochlea). High-magnification images show that EGFP is expressed in inner hair cells (IHCs), outer hair cells (OHCs) in the apical region, and spiral limbal cells ( Figure 1B ).

[0452] Figure 2A-2B A series of fluorescent images of the inner ear of 9-10 week old mice treated with AAV7m8 vector expressing EGFP under the control of CAG promoter. In 9-10 week old CBA / CaJ mice (n=8), unilateral injection was performed to administer 1 μL of AAV7m8-CAG-EGFP (virus titer 9.4×10 12 Two weeks after injection, the inner ear was removed and the surface of the basilar membrane was prepared. A representative ear is shown. EGFP expression was observed in the apical, mid, and basal regions of the cochlea ( Figure 2A ). High-magnification images show 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: the apex of the cochlea; middle: the mid-periphery of the cochlea; right: the base of the cochlea).

[0453] Figure 3A-3B is a series of fluorescent images of the inner ear of rhesus macaques treated with an 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 inject 30 μL of AAV2-CMV-GFP (virus titer 3.39 × 10 12 genomic copies / mL) were administered to the round window of the inner ear. Four weeks after injection, the inner ear was removed and the surface of the basilar membrane was prepared. A representative ear is shown. Immunohistochemistry of Myo7A was used to identify hair cells ( Figure 3A , right column), and GFP expression was used to monitor AAV1 infectivity ( Figure 3A , Figures in the left column). AAV2-CMV-GFP transduction of the rhesus macaque inner ear results in GFP expression across the basal-apical axis of the entire cochlea ( Figure 3A ; Upper left: apex of the cochlea, middle left: midperiphery of the cochlea; lower left: basal period of the cochlea). Cochlear GFP expression was observed in IHCs and some cells within the spiral limbus ( Figure 3A; Figures in the left column). Quantification of GFP expression in IHCs by frequency plot along the cochlea demonstrated that GFP was expressed in 40%-80% of IHCs ( Figure 3B ).

[0454] Figures 4A-4F 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 deliver 30 μL of AAV7m8-CAG-EGFP (virus titer 9.4 × 10 12 Two weeks after injection (n=1 animal) or four weeks after injection (n=1 animal), the inner ear was removed and embedded in paraffin for sectioning, fixation, immunostaining with anti-GFP antibody, and histological analysis. In one animal, EGFP expression was observed at the base of the cochlea two weeks after transduction of the inner ear with AAV7m8-CAG-EGFP ( Figure 4A , Upper panel: the dotted box indicates the basal periphery of the cochlea), as visualized by immunostaining with EGFP. EGFP is expressed in IHCs, OHCs, and fiber cells of the spiral ligament ( Figure 4B , Figure 4A In another animal, four weeks after injection, EGFP expression was observed across the basal-apical axis of the cochlea ( Figure 4C , the dotted box indicates the base of the cochlea and is Figure 4D EGFP is expressed in IHCs, OHCs, and fibrocytes of the spiral ligament ( Figure 4D , the organ of Corti is enlarged in the inset at the upper left of the figure). Figure 4E As shown, in the absence of the primary anti-GFP antibody, the secondary antibody did not stain the cochlea (the dotted box indicates the basal periphery of the cochlea). No staining was observed at the base ( Figure 4F , Figure 4E Magnification of the box in the middle; the organ of Corti is magnified in the upper left inset).

[0455] Figures 5A-5E 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 deliver 30 μL of AAV1-CAG-GFP (virus titer 9.9 × 10 12 Four weeks after injection, the inner ears were removed and embedded in paraffin for sectioning, fixation, immunostaining with anti-GFP antibody, and histological analysis. Figure 5AAs shown, staining with the secondary antibody alone in the absence of anti-GFP antibody did not stain the cochlea, 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 on the upper left represents the apical periphery of the cochlea, the small oval on the left represents the stria vascularis, and the small circle on the lower right represents the saccule), as visualized using immunostaining for GFP. Figure 5C , from Figure 5B ) and all layers of the balloon ( Figure 5E , from Figure 5B Immunostaining for GFP was detected in the right lower circle of the cochlea (enlargement of the lower right circle of the cochlea). Robust GFP expression was detected in the spiral ligament and marginal and intermediate cells of the stria vascularis. In this section, the basal cells of the stria vascularis showed no GFP expression, but GFP expression was seen in basal cells in other parts of the cochlea ( Figure 5D ).

[0456] Figures 6A-6C is a series of fluorescent images of the inner ear of rhesus macaques 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 inject 30 μL of AAV1-CMV-GFP (virus titer 2.01×10 13 genomic copies / mL) were administered to the round window of the inner ear. Four weeks after injection, the inner ear was removed and surface preparation of the basilar membrane, utricle, and crista was performed. One of the representative inner ears is shown. Immunohistochemistry for Myo7A was used to visualize hair cells, and GFP expression was used to monitor AAV1 infectivity ( Figures 6A-6C AAV1-CMV-GFP transduction of the inner ear of rhesus monkeys resulted in strong ubiquitous expression of GFP across the entire basal-apical axis of the cochlea ( Fig. 6A Cochlear GFP expression was observed in inner hair cells (IHCs), outer hair cells (OHCs), and supporting cells, as well as in the nonsensory spiral ligament and spiral limbus ( Figure 6B , Fig. 6A Vestibular structures such as the utricle and cristae also showed significant GFP expression in both sensory and non-sensory areas ( Figure 6C ).

[0457] Figures 7A-7F 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 injections were used to deliver 30 μL of AAV1-CAG-GFP (3.15 × 10 10 or 3.15 × 10 11genomic copies / ear were administered 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 anti-GFP antibody, and histological analysis. Fig. 7A As shown, the anti-GFP antibody did not stain the cochlea of ​​the ear that was not injected with AAV, thus confirming the specificity of the antibody. Figure 7B AAV1-CAG-GFP (3.15×10 10 Transduction of 100 genomic copies / ear) resulted in robust GFP expression throughout the cochlea, as visualized using immunostaining for GFP. Immunostaining for GFP was detected in hair cells, supporting cells, inner sulcus cells, outer sulcus cells, and cells of the spiral ligament. Figure 7C As shown, in these cell types, higher doses of AAV-CAG-GFP (3.15×10 11 genome copies / ear) showed stronger GFP expression. Figure 7D-7F Shown respectively with Figures 7A-7C The same images with nuclear staining removed (ie, these images show only GFP labeling). All scale bars are 100 μm. DETAILED DESCRIPTION

[0458] Described herein are compositions and methods for transducing developing primate (e.g., human) inner ear cells (e.g., 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). The invention features methods of transducing developing inner ear cells (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) in a primate (e.g., human) subject using a serotype 1 adeno-associated virus (AAV1) vector. The invention also features methods of using AAV1 vectors to express polynucleotides in specific inner ear cell types (e.g., outer hair cells, vestibular hair cells, or vestibular supporting cells) in primate (e.g., human) subjects, and AAV1 vectors containing promoters (e.g., promoters that induce gene expression in one or more inner ear cell types) and / or polynucleotides (e.g., polynucleotides corresponding to genes encoding therapeutic proteins, such as genes expressed in normal inner ear cells, or genes encoding proteins that regulate 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 primate (e.g., human) inner ear cells, and thus the compositions described herein can be administered to primate (e.g., human) subjects 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.

[0459] Inner ear cells

[0460] The inner ear is composed of many specialized cells. Both the cochlea and the vestibular system contain hair cells, which are the main sensory cells of the inner ear. The cochlear hair cells are composed of two main cell types: inner hair cells (IHCs) 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 vestibular cells and are involved in the sense of movement, which helps sense balance and spatial orientation. The spiral ganglion neurons innervate the cochlear hair cells and send axons into the central nervous system, while the 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, thereby maintaining the ionic composition of the endolymph and perilymph and regulating the synaptic formation of the ribbon synapse. Within the cochlea, supporting cells can be divided into five different types: 1) Hensen's cells, 2) Deiter's cells, 3) pillar cells; 4) inner digitiform cells; and 5) marginal 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, as have 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 therapeutic approach for treating hearing loss and vestibular dysfunction; however, given that a large number of cell types may need to be targeted to address various causes of hearing loss and vestibular dysfunction, viral vectors with broad tropism are needed to transduce many cell types of the primate (e.g., human) inner ear.

[0461] The present invention is based in part 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 pantropic transduction and strong expression of GFP in most or all cell types of 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 developing human inner ear cells (e.g., most or all inner ear cells in a human adult, adolescent, child, or full-term newborn). Thus, the compositions and methods described herein can be used to induce expression of polynucleotides in all cells of the inner ear of a developing primate (e.g., human), or they can be used to induce expression of polynucleotides in specific regions of the inner ear (e.g., cochlea or vestibular system) or cell types (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) based on the promoter contained in the AAV1 vector. Thus, the methods and compositions described herein can be administered to primate (e.g., human) subjects to treat conditions caused by genetic mutations or damage, degeneration, loss and / or dysfunction in all cell types of the inner ear, or to treat conditions caused by genetic mutations or damage, degeneration, loss and / or dysfunction in one or a portion of the cell types of the inner ear.

[0462] In some embodiments, an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid) is administered to a 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 fiber cells, Scarpa ganglion neurons (vestibular neurons), and vestibular dark cells. The invention relates to the expression of polynucleotides in the inner ear, such as endothelial cells of the cochlear capillaries, vestibular supporting cells, marginal cells, inner digitating cells, inner pillar cells, outer pillar cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, spiral convex 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, fiber cells, cells of Reissner's membrane, and glial cells. Pantropic transduction of inner ear cells by AAV1 vectors can be used to induce expression of polynucleotides 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 approaches in which it is desired to express a polynucleotide in many or most cell types of the inner ear, for example, to deliver a wild-type version of a gene that is mutated in all inner ear cell types or to produce large quantities of a secreted protein that produces a therapeutic effect at high concentrations.

[0463] 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., supporting cells, such as cochlear and / or vestibular supporting cells; OHCs; vestibular hair cells; or spiral ganglion neurons) or region (e.g., cochlear or vestibular system). A cell type-specific promoter may be included in an 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 in only one or a few cell types. This approach is beneficial because it can be used to avoid ectopic expression of a polynucleotide in cells where it is not normally expressed.

[0464] Expression of foreign polynucleotides in human cells

[0465] Mutations in various genes, such as myosin 7A (MYO7A), POU class 4 homeobox 3 (POU4F3), solute carrier family 17 member 8 (SLC17A8), gap junction protein β2 (GJB2), claudin 14 (CLDN14), coagulation factor C homologous protein (Cochlin) (COCH), protocadherin related 15 (PCDH15) and transmembrane 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 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 a 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). AAV1 vectors containing polynucleotides (e.g., AAV1 vectors containing wild-type AAV1 capsids) can be administered to a human subject (e.g., administered to the inner ear of a subject) to induce or increase 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 polynucleotides encoding proteins in human cells.

[0466] The AAV1 vectors described herein can be used to express polynucleotides 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. The accession numbers of the polynucleotides of Table 2 are provided in Table 3.

[0467] Table 2: Polynucleotides that can be expressed in one or more inner ear cell types

[0468]

[0469]

[0470]

[0471] Table 3: Accession numbers of the polynucleotides listed in Table 2

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481] Table 4: Amino acid sequences of Atoh1 variants

[0482]

[0483]

[0484] In some embodiments, polynucleotides are or encode components of gene editing systems. AAV1 vectors comprising components of gene editing systems operably connected to cell type-specific promoters (e.g., cell type-specific promoters listed in Table 5) can be used for cell type-specific gene editing. For example, components of gene editing systems can be used to introduce changes (e.g., insertions, deletions (e.g., knockouts), translocations, inversions, single point mutations or other mutations) in genes known to regulate inner ear cell function, such as those involved in sensorineural hearing loss or vestibular dysfunction. Exemplary gene editing systems include zinc finger nucleases (ZFNs), nucleases (TALENs) based on the sample effectors of transcription activators, and clustered regularly spaced short palindromic repeats (CRISPR) systems. Methods based on ZFNs, TALENs and CRISPRs are described in, for example, Gaj et al., Trends Biotechnol.31:397-405, 2013.

[0485] CRISPR refers to a group (or a group) of clustered regularly spaced short palindromic repeats (systems). The 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 repetitive sequences and spacer sequences. In naturally occurring CRISPR systems, the spacer is typically a sequence (e.g., a plasmid or phage sequence) foreign to bacteria. The CRISPR system has been modified for gene editing (e.g., changing, silencing and / or enhancing certain genes) in eukaryotic organisms. See, for example, Wiedenheft et al., Nature 482: 331, 2012. For example, such modifications to the system include introducing a plasmid containing a specially designed CRISPR and one or more appropriate Cas proteins into a eukaryotic cell. The CRISPR locus is transcribed into RNA and processed into small RNA by the Cas protein, the RNA comprising a repetitive sequence flanking the spacer. Depending on the spacer sequence, RNA acts as a guide to guide 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 examples, the CRISPR system includes the Cas9 protein, a nuclease that cuts on both strands of DNA. See, e.g., supra.

[0486] In some embodiments, in a CRISPR system used as described herein, e.g., according to one or more methods described herein, the spacer of the CRISPR is derived from a target gene sequence, e.g., from a gene known to regulate inner ear cell function, such as a gene involved in sensorineural hearing loss or vestibular dysfunction.

[0487] In some embodiments, the polynucleotide includes a guide RNA (gRNA) in a clustered regularly interspaced short palindromic repeat (CRISPR) system for gene editing. In some embodiments, the polynucleotide comprises or encodes a zinc finger nuclease (ZFN) or an mRNA encoding a ZFN, the zinc finger nuclease targeting (e.g., cleaving) a nucleic acid sequence (e.g., 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 TALEN or an mRNA encoding a TALEN, the TALEN targeting (e.g., cleaving) a nucleic acid sequence (e.g., DNA sequence) of a gene known to regulate inner ear cell function, such as a gene associated with sensorineural hearing loss or vestibular dysfunction.

[0488] For example, gRNA can be used in a CRISPR system to engineer changes in genes (e.g., genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction). In other examples, ZFN and / or TALEN can be used to engineer changes in genes (e.g., genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction). Exemplary changes include insertions, deletions (e.g., knockouts), translocations, inversions, single point mutations, or other mutations. Changes can be introduced into genes in cells, for example, in vitro, in vitro, or in vivo. In some embodiments, the changes reduce (e.g., knock down or knock out) the level and / or activity of genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction, for example, the changes are negative regulators of function. In another example, the changes correct defects (e.g., mutations that cause defects) in genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction.

[0489] In certain embodiments, the CRISPR system is used to edit (e.g., add or delete base pairs) a target 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 embodiments, the CRISPR system is used to introduce a premature stop codon, e.g., to reduce the expression of the target gene. In other embodiments, the CRISPR system is used to turn off the target gene in a reversible manner, e.g., similar to RNA interference. In some embodiments, the CRISPR system is used to guide Cas to the promoter of a target gene, e.g., 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.

[0490] In some embodiments, CRISPR systems can be generated to edit genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction, using techniques described, for example, in U.S. Pub. No. 20140068797; Cong, Science 339:819, 2013; Tsai, Nature Biotechnol., 32:569, 2014; and U.S. Pat. Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359.

[0491] In some embodiments, CRISPR interference (CRISPRi) technology can be used for transcriptional inhibition 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., a nuclease-free dCas9 or 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 transcriptional elongation. The complex can also block transcriptional initiation by interfering with transcription factor binding. The CRISPRi method is specific, has minimal off-target effects, and is reusable, for example, more than one gene can be repressed simultaneously (e.g., using multiple gRNAs). Similarly, the CRISPRi method allows reversible gene repression.

[0492] In some embodiments, CRISPR-mediated gene activation (CRISPRa) can be used for transcriptional activation of one or more genes described herein, such as genes known to regulate inner ear cell function, such as genes involved in sensorineural hearing loss or vestibular dysfunction. In CRISPRa technology, dCas9 fusion proteins recruit transcriptional activators. For example, dCas9 can be used to recruit polypeptides (e.g., activation domains), such as VP64 or p65 activation domains (p65D), and used with sgRNA (e.g., single sgRNA or multiple sgRNAs) to activate one or more genes, such as one or more endogenous genes. Multiple activating factors can be recruited by using multiple sgRNAs-this can improve activation efficiency. Multiple activation domains and single or multiple activation domains can be used. In addition to engineering dCas9 to recruit activating factors, sgRNAs can also be engineered to recruit activating factors. For example, RNA aptamers can be incorporated into sgRNAs to recruit proteins (e.g., activation domains), such as VP64. In some examples, a synergistic activation mediator (SAM) system can be used for transcriptional activation. In SAM, the MS2 aptamer is added to the sgRNA. MS2 recruits the MS2 coat protein (MCP) fused to p65AD and heat shock factor 1 (HSF1). CRISPRi and CRISPRa technologies are described in detail in, for example, Dominguez et al., Nat. Rev. Mol. Cell Biol. 17:5, 2016, which is incorporated herein by reference.

[0493] Polynucleotide encoding target protein

[0494] One platform that can be used to achieve therapeutically effective intracellular target protein concentrations in primate (e.g., human) cells is through the stable expression of nucleic acids encoding the target protein (e.g., by integration into the nuclear or mitochondrial genome of primate (e.g., human) cells, or by forming an additional concatemer in the nucleus of primate (e.g., human) cells). The nucleic acid is a polynucleotide encoding the primary amino acid sequence of the corresponding protein. In order to introduce exogenous genes into primate (e.g., human) cells, the gene can be incorporated into a vector, such as an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid). For introduction into inner ear cells, the AAV1 vector can be topically administered to the inner ear of a primate (e.g., human) subject.

[0495] The recognition and binding of primate (e.g., human) RNA polymerase to polynucleotides encoding target proteins is important for gene expression. Therefore, sequence elements that show high affinity for transcription factors that recruit RNA polymerase and promote the assembly of transcription complexes at transcription start sites can be included in the polynucleotide. Such sequence elements include, for example, promoters, whose sequences can be recognized and bound by specific transcription initiation factors and final RNA polymerases. Examples of promoters have been described in Smith et al., Mol. Sys. Biol., 3: 73, published online, and the disclosure thereof is incorporated herein by reference. The promoter used in the methods and compositions described herein can be a ubiquitous promoter (e.g., to induce or increase the expression of polynucleotides in all cells of the inner ear) or a cell type-specific promoter (e.g., to induce or increase the expression of polynucleotides in one or more inner ear cell types). Ubiquitous promoters include CAG promoter, cytomegalovirus (CMV) promoter, smCBA promoter (described in Haire et al., Invest. Opthalmol. Vis. Sci. 47: 3745-3753, 2006), dihydrofolate reductase (DHFR) promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter and EF1α promoter. Alternatively, promoters derived from viral genomes can also be used to stably express polynucleotides in primate (e.g., human) cells. Examples of functional viral promoters that can be used to express polynucleotides in primate (e.g., human) cells include adenovirus late promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, Epstein Barr virus (EBV) promoter and Rous sarcoma virus (RSV) promoter.

[0496] Cell type-specific promoters that can be included in the AAV1 vectors described herein to express nucleic acids encoding a protein of interest (eg, a polynucleotide listed in Table 2) in one or more inner ear cell types are provided in Table 5 below.

[0497] Table 5: Inner ear cell type specific promoters

[0498]

[0499] Once the polynucleotide encoding the target protein is incorporated into a primate (e.g., human) cell, the transcription of such polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the primate (e.g., human) cell to an external chemical reagent, such as an agent that regulates 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, such as by removing the repressor protein that has been bound to the promoter. Alternatively, chemical reagents can be used to enhance the affinity of the promoter to RNA polymerase and / or transcription factors, so that in the presence of chemical reagents, the transcription rate of the gene located downstream of the promoter increases. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanism include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA), and can be applied to primate (e.g., human) cells according to the established scheme to promote gene expression.

[0500] Other DNA sequence elements that may be included in the polynucleotide used in the compositions and methods described herein include enhancer sequences. Enhancers represent another type of regulatory element, which induces conformational changes in the polynucleotide containing the polynucleotide so that the DNA adopts a three-dimensional orientation that is conducive to the combination of transcription factors and RNA polymerases at the transcription start site. Therefore, the polynucleotides used in the compositions and methods described herein include those encoding target proteins, 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. The enhancers used in the compositions and methods described herein also include those enhancers derived from the genetic material of viruses that can infect eukaryotic cells. Examples include the SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer on the rear side of the replication origin (bp 100-270), and adenovirus enhancers. Other enhancer sequences that induce activation of eukaryotic gene transcription include CMV enhancers and RSV enhancers. The enhancer can be spliced ​​into the vector containing the polynucleotide encoding the target protein, for example, at a position 5' or 3' to the gene. In a preferred orientation, the enhancer is located 5' to the promoter, which is in turn located 5' to the polynucleotide encoding the target protein.

[0501] The AAV1 vectors described herein may include a woodchuck posttranscriptional regulatory element (WPRE). The 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 amount of total mRNA in the cell. Addition of the WPRE to the vector can result in significant improvements in the levels of transgene expression from several different promoters in vitro and in vivo.

[0502] In some embodiments, the AAV1 vectors described herein contain reporter-based sequences that can be used to verify the expression of nucleic acids contained in the AAV1 vectors, for example, in cells and tissues (e.g., in the cochlear and / or vestibular system, or in specific inner ear cell types, such as IHC, OHC, cochlear supporting cells, vestibular hair cells, and / or vestibular supporting cells). Reporter gene sequences that can be provided in transgenes 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 ubiquitous or cell type-specific promoters described herein), reporter gene sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescent or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunoassays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the signal-carrying vector is detected by assaying for β-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visualized by color or light production in a luminometer.

[0503] 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., AAV1 vectors mean polynucleotides encoding two different target proteins). In some embodiments, a bicistronic or multicistronic expression cassette is used to express two or more polynucleotides. In some embodiments, the multicistronic expression cassette comprises an internal ribosome entry site (IRES) between the two or more polynucleotides (e.g., an IRES between the polynucleotides encoding two different target proteins). In some embodiments, the multicistronic expression cassette comprises a foot-and-mouth disease virus 2A (FMDV2A) polynucleotide between the two or more polynucleotides (e.g., an FMDV 2A polynucleotide between each nucleic acid encoding a target protein).

[0504] In some embodiments, two or more AAV1 vectors described herein (e.g., 2, 3, 4 or more AAV1 vectors) can be used to express a single polynucleotide (e.g., a single polynucleotide listed in Table 2), such as a polynucleotide having a coding sequence of 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 otoferlin, the polynucleotide having a coding sequence of approximately 6 kb. In embodiments in which two or more AAV1 vectors are used to express a single polynucleotide, the coding sequence of the polynucleotide is distributed between the vectors so 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., a single polynucleotide listed in Table 2). A portion of the coding sequence of a polynucleotide (e.g., a polynucleotide having a coding sequence of 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 include the N-terminal portion of the coding sequence, and another AAV1 vector can include the C-terminal portion of the coding sequence). Exemplary dual vector systems include fragmented dual vectors, overlapping dual vectors, trans-splicing dual vectors, and dual-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.

[0505] In some embodiments, the AAV1 vectors described herein are polynucleotide sequences that enhance the translation rate of polynucleotides or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, IRES and polyadenylation signal sites to guide the efficient transcription of nucleic acids carried in the vector. Vectors suitable for the compositions and methods described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseoin.

[0506] AAV1 vectors for nucleic acid delivery

[0507] In some embodiments, polynucleotides (e.g., polynucleotides corresponding to genes expressed in healthy inner ear cells, such as 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 are wild-type AAV1 capsid proteins having the amino acid sequences of SEQ ID NOs: 1-3, as shown below.

[0508] In some embodiments, the wild-type AAV1 capsid protein VP1 has the amino acid sequence of SEQ ID NO:1.

[0509] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL(SEQ ID NO:1)

[0510] In some embodiments, the wild-type AAV1 capsid protein VP2 has the amino acid sequence of SEQ ID NO:2.

[0511] TAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL(SEQ ID NO:2)

[0512] In some embodiments, the wild-type AAV1 capsid protein VP3 has the amino acid sequence of SEQ ID NO:3.

[0513] MASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL(SEQ ID NO:3)

[0514] AAV1 vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs comprising 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 promote integration and expression of heterologous genes. The viral sequences may include those sequences of AAV that are required (cis) for DNA replication and packaging (e.g., functional ITRs) into virions. In typical applications, the heterologous transgene encodes a protein expressed in healthy inner ear cells that promotes inner ear cell development, function, cell fate determination, regeneration, survival, proliferation and / or maintenance, or a wild-type form of a hair cell protein that is mutated in a subject with inherited or genetic hearing loss, deafness, and / or vestibular dysfunction (e.g., dizziness, vertigo, or imbalance). Such AAV1 vectors may also contain a marker or reporter gene. Available AAV1 vectors have one or more AAV WT genes that are completely or partially deleted but still retain functional flanking ITR sequences. The AAV ITRs contained in the AAV1 vectors described herein may have any serotype suitable for a particular application (e.g., ITRs may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs). For use in the methods and compositions described herein, the ITRs may be AAV2 ITRs. Methods for using AAV vectors are described in, for example, Tal et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000), each of which is incorporated herein by reference for disclosures relating to AAV vectors for gene delivery.

[0515] The promoters and polynucleotides described herein can be incorporated into AAV1 virions to facilitate the introduction of the promoters and / or polynucleotides into primate (e.g., human) inner ear cells. The capsid protein of AAV1 constitutes the external non-nucleic acid portion of the virion and is encoded by the AAV1 cap gene. The cap gene encodes three viral coat proteins VP1, VP2, and VP3 required for virion assembly. The construction of AAV virions has been described in, for example, 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; and Rabinowitz et al., J. Virol. 76: 791 (2002) and Bowles et al., J. Virol. 77: 423 (2003), each of which is incorporated herein by reference for disclosures relating to AAV vectors for gene delivery. The capsid protein of the AAV1 vector used in the compositions and methods described herein may have the amino acid sequence described in U.S. Pat. No. 6,759,237 (and be encoded by the polynucleotide sequence described therein, which is incorporated herein by reference).

[0516] AAV virions that can be used in conjunction with the compositions and methods described herein include those 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 are described in, for example, 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), each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

[0517] Also useful in combination with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors (e.g., AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV8) of a given serotype pseudotyped with a capsid gene derived from AAV1 (e.g., a wild-type AAV1 capsid). Techniques for the construction and use of pseudotyped rAAV virions are known in the art and are described in, for example, 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).

[0518] Pharmaceutical composition

[0519] The AAV1 vectors described herein (e.g., AAV1 vectors comprising wild-type AAV1 capsids) can be incorporated into a vehicle for administration to a patient, such as a primate (e.g., human) patient with sensorineural hearing loss, deafness, auditory neuropathy, tinnitus, and / or vestibular dysfunction. Pharmaceutical compositions containing vectors (e.g., AAV1 vectors) can be prepared using methods known in the art. For example, such compositions can use, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington: The Science and Practice of Pharmacology 22nd Edition, Allen, L. Ed. (2013); incorporated herein by reference) and in a desired form, such as in the form of a lyophilized formulation or an aqueous solution.

[0520] A mixture of AAV1 vectors can be prepared in water appropriately mixed with one or more excipients, carriers or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oil. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth. Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions and sterile powders for 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 preparation may be sterile and may be fluid to the extent that there is easy injectability. The preparation may be stable under manufacturing and storage conditions and may be preserved under conditions that prevent the contamination of microorganisms (such as bacteria and fungi). The carrier may be a solvent or dispersion medium containing the following: for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and / or vegetable oils. Appropriate 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 a surfactant. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by the use in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0521] For example, if necessary, the solution comprising the pharmaceutical composition described herein can be appropriately buffered, and the liquid diluent can be first made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous medium that can be used will be known to those skilled in the art according to the present disclosure. For example, a dose can be dissolved in 1ml isotonic NaCl solution and added to 1000ml subcutaneous infusion fluid or injected at the infusion site of suggestion. According to the disease of the treated subject, a certain dosage variation will necessarily occur. In order to be applied topically to the inner ear, the composition can be formulated to include synthetic perilymph. Exemplary synthetic perilymph includes 20-200mM NaCl, 1-5mM KCl, 0.1-10mM CaCl2, 1-10mM glucose and 2-50mM HEPE, with a pH between about 6 and 9 and an osmotic degree of about 300mOsm / kg. The person responsible for administration will determine the appropriate dose for individual subjects in any case. Moreover, for primate (eg, human) administration, preparations can meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biologics standard.

[0522] Treatment

[0523] The AAV1 vectors described herein (e.g., AAV1 vectors containing wild-type AAV1 capsids) can be administered to subjects suffering from sensorineural hearing loss, auditory neuropathy, deafness, tinnitus, and / or vestibular dysfunction by a variety of routes, such as topical administration to the inner ear or middle ear (e.g., administration into the perilymph or endolymph, such as through the oval window, endolymphatic sac, round window, or semicircular canals, e.g., transtympanic or intratympanic administration), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, infusion, lavage, and oral administration. In any given case, the most appropriate route of administration will depend on the specific composition being administered, the patient, the method of drug formulation, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. The composition can be administered once or more than once (e.g., once a year, twice a year, three times a year, once every two months, or once a month).

[0524] A subject that can be treated as described herein is a subject having or at risk of developing sensorineural hearing loss, deafness, auditory neuropathy, tinnitus, and / or vestibular dysfunction (e.g., a subject having or at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods described herein can be used to treat a subject having or at risk of developing inner ear cell damage (e.g., cochlear hair cells, vestibular hair cells, cochlear supporting cells, vestibular supporting cells, and / or spiral ganglion neurons, such as damage associated with acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging), a subject having or at risk of developing sensorineural hearing loss, deafness, tinnitus, or auditory neuropathy, a subject having or at risk of developing vestibular dysfunction (e.g., dizziness, vertigo, or imbalance), a subject having tinnitus (e.g., tinnitus alone, or tinnitus associated with sensorineural hearing loss or vestibular dysfunction), a subject having a genetic mutation associated with hearing loss and / or vestibular dysfunction, or a subject with 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 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 may include a step of screening the subject for one or more mutations in genes known to be associated with hearing loss or vestibular dysfunction before treatment or administration with the compositions described herein. Subjects may be screened for genetic mutations using standard methods known to those skilled in the art (e.g., genetic testing). The methods described herein may also include a step of assessing the hearing and / or vestibular function of the subject before treatment or administration with the compositions described herein. Hearing may be assessed using standard tests such as audiometry, auditory brainstem response (ABR), electrocochlear oscopy (ECOG) and otoacoustic emissions. Vestibular function can be assessed using standard tests, such as eye movement testing (e.g., electronystagmography (ENG) or videonystagmography (VNG)), posturography, swivel chair testing, ECOG, vestibular evoked myogenic potentials (VEMPs), and specialist 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 hearing and / or vestibular function in a subject after treatment or administration with a composition described herein.The compositions and methods described herein can also be administered as a preventive 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 who carry a genetic mutation associated with hearing loss or vestibular dysfunction but have not yet manifested hearing loss 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., otitis interna or vestibular neuritis), or Meniere's disease.

[0525] The compositions and methods described herein can be used to promote or induce inner ear cell regeneration (e.g., regeneration of cochlear hair cells, vestibular hair cells, or spiral ganglion neurons) in primate (e.g., human) subjects and / or increase the number of hair cells (e.g., IHCs, OHCs, and / or vestibular hair cells) and / or SGNs in primate (e.g., human) subjects. 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 inducing supporting cells to differentiate into hair cells (e.g., to induce cochlear supporting cells to differentiate into IHCs and / or OHCs, and / or to induce vestibular supporting cells to differentiate into vestibular hair cells). Subjects who may benefit from compositions that promote or induce 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 differentiation of supporting cells (e.g., cochlear and / or vestibular supporting cells) into hair cells include subjects with hearing loss or vestibular dysfunction due to 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), such as 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), as well as subjects with abnormal inner ear cells (e.g., inner ear cells that do not function normally compared to normal healthy inner ear cells) or with a reduction in the 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 a subject at risk for damage, degeneration, or loss of inner ear cells (e.g., loss of inner ear cells due to age, exposure to loud noise, disease or infection, head trauma, or ototoxic drugs)).

[0526] 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 are about to be treated with ototoxic drugs. Ototoxic drugs are toxic to the cells of 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 found to have ototoxicity include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin and amikacin), puromycin, antineoplastic agents (e.g., platinum-containing chemotherapeutics, such as cisplatin, carboplatin and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, particularly at high doses) and quinine. In some embodiments, the methods described herein prevent or reduce inner ear cell damage or death (e.g., IHC, OHC, SGN, cochlear supporting cell, vestibular supporting cell, and / or vestibular hair cell damage or death) associated with acoustic trauma, disease or infection, head trauma, or aging.

[0527] 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., synaptic connections between cochlear hair cells (IHCs and / or OHCs) and SGNs or 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 (IHCs and / or OHCs) or vestibular hair cells) by neurons in the inner ear (e.g., SGNs or vestibular ganglion neurons).

[0528] Treatment may include administering a composition comprising an AAV1 vector described herein (e.g., an AAV1 vector comprising a wild-type AAV1 capsid) in various unit doses. Each unit dose will typically contain a predetermined amount of the therapeutic composition. The amount to be administered, as well as the specific route of administration and formulation, are within the skill of those skilled in the clinical art. The unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. Administration may be performed using a syringe pump to control the infusion rate so as to minimize damage to the inner ear (e.g., cochlea). The AAV1 vector may have, for example, a volume of 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 administered in a volume of 1×10 7 VG / ear up 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 VG / ear) was administered to the subjects.

[0529] In some embodiments, a composition described herein is 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) developing inner ear cell types selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fiber cells, Scarpa ganglion neurons (vestibular ganglion neurons), endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitating 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 convex 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, fiber cells, cells of Reissner's membrane, and glial cells. In some embodiments, a composition described herein is administered in an amount sufficient to transduce all or the most developed inner ear cell types.

[0530] The compositions described herein are administered in an amount sufficient to improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), reduce tinnitus, increase expression of a polynucleotide contained in an AAV1 vector, increase function of a protein encoded by a polynucleotide contained in an 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., 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, e.g., increase the proliferation of supporting cells), increase the differentiation of supporting cells into hair cells (e.g., cause the differentiation of cochlear supporting cells into IHCs and / or OHCs, and / or cause the differentiation of vestibular supporting cells into vestibular hair cells), prevent or reduce inner ear cell damage or death (e.g., associated with acoustic trauma, head trauma In some embodiments, the invention relates to an intramural synapse, a cochlear supporting cell, a vestibular supporting cell, and / or a vestibular hair cell, which is damaged or dead, and / or is caused by injury, ototoxic drugs, disease or infection, or aging, promoting or increasing inner ear cell development, promoting or increasing 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 damage or 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), improving inner ear cell function, preserving ribbon synapses, promoting or increasing ribbon synapse formation, maintaining connections (e.g., synaptic connections) between hair cells and neurons (e.g., SGNs and / or vestibular ganglion neurons), or increasing or restoring connections (e.g., synaptic connections) between hair cells and neurons (e.g., SGNs and / or vestibular ganglion neurons). Hearing can be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochlear gamma (ECOG), and otoacoustic emissions) and can improve 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 audiometric results obtained before treatment. Vestibular function can be assessed using standard tests for balance and vertigo (e.g., eye movement tests (e.g., ENG or VNG), posturography, swivel chair tests, ECOG, VEMP, and specialist clinical balance tests) and can improve 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 measurements obtained before 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 subjects who carry a genetic mutation associated with hearing loss or vestibular dysfunction, have a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), or have been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic medications, head trauma, disease or infection, or acoustic trauma), but do not exhibit hearing loss or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), or in subjects who exhibit mild to moderate hearing loss or vestibular dysfunction). Expression of the protein encoded by the polynucleotide can be assessed using immunohistochemistry, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detecting protein or mRNA, 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 expression prior to 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 assessed based on an audiometric test or a vestibular function test, 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, or, hair cell or SGN regeneration, or the function of the protein encoded by the polynucleotide before administration of the composition described herein. Inner ear cell damage 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 damage 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 dosage 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 compositions. Depending on the results of the evaluation, the patient may receive additional treatment.

[0531] Pill Box

[0532] The compositions described herein can be provided in a kit for treating sensorineural hearing loss or vestibular dysfunction. The composition can include an AAV1 vector containing a promoter and / or a nucleic acid sequence of a polynucleotide and can be provided in a 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) developing inner ear cell types selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fiber cells, Scarpa ganglion The kit may also include a package insert that instructs a user of the kit (such as a physician) to perform the methods described herein. The kit may optionally include a syringe or other device for administering the composition.

[0533] Example

[0534] The following examples are put forth in order to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared, and evaluated, and are intended solely to illustrate the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0535] 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. Unlike AAV1 tropism in mice, which exhibits limited transduction of a few cell types of the inner ear, AAV1-mediated transgene delivery to the inner ear of non-human primates exhibits broad transduction of multiple cell types. This is in contrast to serotypes such as AAV7m8, which exhibit broad transduction of multiple inner ear cell types in mice, but show more restricted tropism in the inner ear of non-human primates.

[0536] In all of the examples described below, the AAV1 vector contains a capsid protein having the amino acid sequence of SEQ ID NOs: 1-3.

[0537] Example 1. In vivo administration of a composition containing an AAV1 viral vector carrying a transgene to the cochlea of ​​mice

[0538] To determine the transduction efficacy of AAV1 viral vectors in the mouse inner ear, a composition containing an AAV1 vector carrying an EGFP transgene under the control of a 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. The virus titer was 2.94×10 13 Two weeks after injection, the inner ear was surgically removed and the surface of the basilar membrane was prepared. To evaluate AAV-1-mediated inner ear transduction, EGFP expression was directly visualized using fluorescence microscopy. EGFP expression was observed throughout the basal-apical axis of the cochlea, but varied between cell types along the length of the frequency map. ( Figure 1A ; Upper left: apical part of the cochlea; Lower left: mid-periphery of the cochlea; Upper right: basal part of the cochlea). Inner hair cells (IHCs) expressed EGFP across the basal-apical axis of the cochlea, but outer hair cells (OHCs) showed GFP expression primarily in the apical region of the cochlea. EGFP expression was also observed in some cells of the spiral limbus ( Figure 1B ).

[0539] Example 2. In vivo administration of a composition containing an AAV7m8 viral vector carrying a transgene to the cochlea of ​​mice

[0540] To determine the transduction efficacy of AAV7m8 viral vectors in the mouse inner ear, a composition containing an AAV7m8 vector carrying an 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. The virus titer was 9.4×10 12 Two weeks after injection, the inner ear was surgically removed and the surface of the basilar membrane was prepared. EGFP expression was observed throughout the cochlea ( Figure 2A ). High-magnification images show 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: the apex of the cochlea; middle: the mid-periphery of the cochlea; right: the base of the cochlea).

[0541] Example 3. In vivo administration of a composition containing an AAV2 vector carrying a transgene to the inner ear of an adult non-human primate

[0542] To determine the transduction efficacy of AAV2 viral vectors in the inner ear of adult non-human primates, a composition containing an AAV2 vector carrying a GFP transgene under the control of a CMV promoter was delivered unilaterally (n=1 animal) and bilaterally (n=2 animals) into the inner ear of rhesus monkeys by round window membrane injection. The virus titer was 3.39×10 12 Four weeks after injection, the inner ear was surgically removed and the basilar membrane surface was prepared. Immunolabeling of Myo7A was used to identify the hair cells of the inner ear ( Figure 3A , right column), and AAV2-mediated transduction of inner ear cells was measured by direct GFP fluorescence ( Figure 3A , Figures in the left column). GFP expression was observed across the basal-apical axis of the cochlea, particularly in IHCs and some cells of the spiral limbus ( Figure 3A , graphs in the left column). To determine the transduction efficacy of the AAV2-CMV-GFP vector in IHCs of the inner ear of rhesus monkeys, frequency plots along the cochlea were quantified for GFP expression in IHCs, demonstrating GFP expression in 40%-80% of cells ( Figure 3B )

[0543] Example 4. In vivo administration of a composition containing an AAV7m8 vector carrying a transgene to the inner ear of an adult non-human primate

[0544] To determine the transduction efficacy of AAV7m8 viral vectors in the inner ear of adult nonhuman primates, a composition containing an AAV7m8 vector carrying an EGFP transgene under the control of a CAG promoter was delivered bilaterally into the inner ear of cynomolgus monkeys by round window membrane injection. The virus titer was 9.4 × 10 12 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 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 base of the cochlea ( Figure 4A , Upper panel: the dotted box indicates the base of the cochlea). EGFP was observed in IHCs, OHCs, and fiber cells of the spiral ligament ( Figure 4B , Figure 4A In another animal, four weeks after injection, EGFP expression was observed across the basal-apical axis of the cochlea ( Figure 4C , the dotted box indicates the base of the cochlea and is Figure 4DEGFP is 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 a negative control, immunostaining with secondary antibody in the absence of anti-GFP primary antibody showed no staining within the cochlea ( Figure 4E -F).

[0545] Example 5. In vivo administration of a composition containing an AAV1 viral vector carrying a transgene to the inner ear of an adult non-human primate

[0546] To determine the transduction efficacy of AAV1 viral vectors in the inner ear of adult nonhuman primates, a composition containing an AAV1 vector carrying a GFP transgene under the control of a CAG promoter (AAV1-CAG-GFP) was delivered bilaterally into the inner ear of cynomolgus monkeys by round window membrane injection. The virus titer was 9.9 × 10 12 GC / mL injection (30 μL). Four weeks after injection, the inner ear was surgically excised and embedded in paraffin for sectioning and histological analysis. To evaluate AAV1-mediated inner ear transduction, paraffin sections were immunolabeled with rabbit monoclonal anti-GFP antibody (Abcam EPR14104). Figure 5B and 5C ) and balloon ( Figure 5B and 5E GFP expression was observed in multiple inner ear structures of the cochlea. Robust GFP expression was detected in the spiral ligament and in marginal and intermediate cells of the stria vascularis. In the slice shown in the image, the basal cells of the stria vascularis showed no GFP expression, but GFP expression was seen in basal cells in other parts of the cochlea ( Figure 5D ). Staining with secondary antibody alone in the absence of anti-GFP antibody was used as a negative control to confirm the specificity of the antibody ( Figure 5A ).

[0547] To determine whether the widespread transduction of AAV1 vectors 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 AAV1 vectors carrying the GFP transgene under the control of the CMV promoter (AAV1-CMV-GFP). 2.01×10 13GC / mL of virus titer, 30 μL of the composition was delivered at an injection rate of 15 μL / min. The tropism of the AAV1 vector was determined 4 weeks after injection by surgical resection of the inner ear and surface preparation of the basilar membrane, utricle, and cristae. AAV1-mediated GFP expression was directly monitored by GFP fluorescence, and hair cells of the inner ear were identified by immunolabeling with antibodies against Myo7a. Fluorescence imaging revealed robust GFP expression throughout the basal-apical axis of the cochlea ( Fig. 6A ), the GFP is expressed in the inner hair cell (IHC) and outer hair cell (OHC) layers ( Figure 6B GFP expression was also observed in supporting cells and nonsensory spiral ligaments and spiral limbus ( Figure 6B In addition, the vestibular structures of the inner ear showed strong GFP expression in sensory and non-sensory cells of the utricle and cristae ( Figure 6C ).

[0548] Thus, as shown in Figures 5 and 6, AAV1 exhibits robust pantropic transduction in the inner ear of non-human primates. This broad tropism in sensory and non-sensory cells of the cochlea and vestibule can be exploited for therapeutic benefit. Table 6 below provides a summary of the tropism observed in the inner ear of non-human primates using different AAV capsids.

[0549] Table 6: Tropism of different capsids in the inner ear of non-human primates

[0550]

[0551] Strong = strong expression; Medium = medium expression; Weak = weak expression; Little / None = little or no expression

[0552] Example 6. In vivo administration of compositions containing AAV1 viral vectors carrying a transgene to the inner ear of adult non-human primates using low and high viral titers

[0553] To determine the effect of viral titer on the transduction efficacy of AAV1 viral vectors in the inner ear of adult nonhuman primates, a composition containing an AAV1 vector carrying a GFP transgene under the control of the CAG promoter (AAV1-CAG-GFP) was delivered bilaterally into the inner ear of cynomolgus monkeys by round window membrane injection. 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 11GC / ear; "AAV1 high"). Four weeks after injection, the inner ear was surgically excised 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). Figures 7A-7C Shown in the ear not injected with AAV ( Fig. 7A ), in the ear with low AAV injection ( Figure 7B ) and in the AAV-high injected ear ( Figure 7C ) in the GFP marker. FIG. 7D to FIG. 7F In the case of no nuclear staining (i.e., only GFP staining), the Figures 7A-7C The same image. The anti-GFP antibody did not label the cochlea of ​​the ear that was not injected with AAV, confirming the specificity of the antibody ( Fig. 7A and 7D AAV1-low injected ears showed robust GFP expression from the base to the apex of the cochlea in multiple cell types, including hair cells, supporting cells, inner sulcus cells, outer sulcus cells, and cells in the spiral ligament ( Figure 7B and 7E Increasing viral titers resulted in 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 pantropic transduction in the inner ear of non-human primates, which can be enhanced by increasing viral titers.

[0554] Example 7. Expression of polynucleotides in outer hair cells of the inner ear of a human subject

[0555] According to the methods and compositions disclosed herein, a person skilled in the art may administer a composition containing an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid) to a human subject to induce polynucleotide expression in OHC. To this end, a person skilled in the art may locally administer a composition containing an AAV1 vector to the inner ear of a human subject (e.g., to the endolymph or perilymph, such as to or through the round window, oval window, or horizontal semicircular canal, or by transtympanic or intratympanic injection), wherein the AAV1 vector contains a polynucleotide (e.g., a polynucleotide encoding Strc, Chrna9, Chrna10, Ocm, Tmc1, Myo7a, or Ush1c) operably linked to an OHC-specific promoter (e.g., SLC26A5 promoter, OCM promoter, STRC promoter, or ATP2B2 promoter). In order to induce expression of a polynucleotide in OHC, for example, 1x10 7 VG / ear up to 2x10 14VG / 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 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, 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 The AAV1 vector was administered at a dose of 10 VG / ear).

[0556] After the composition is applied to a human subject, skilled practitioners in the art can monitor gene expression and / or the expression of proteins encoded by polynucleotides by a variety of methods, as well as the improvement of patients in response to therapy. For example, after applying the composition, a physician can indirectly assess gene expression and / or protein production and monitor the hearing of a patient by performing standard tests such as audiometry, ABR, electrocochlear osmography (ECOG) and distortion products of 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 applying the composition, it is found that after applying the composition, the patient shows an improvement in hearing in one or more tests (for example, as indicated by the improved OHC function of the DPOAE measurement value), which indicates that the patient has a good response to treatment. Subsequent doses can be determined and applied as needed.

[0557] Example 8. Treatment of Vestibular Dysfunction in Human Subjects by Administration of AAV1 Vectors

[0558] According to the methods disclosed herein, one skilled 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 reflexes). To this end, a physician skilled in the art can administer to a human patient a composition containing an AAV1 vector (e.g., an AAV1 vector containing a wild-type AAV1 capsid) containing a polynucleotide encoding a transgene (e.g., a polynucleotide encoding Atoh1, Gfi1, Sox11, Ntf3, Bdnf, Whirlin, Sox11, Tmtc4 or Pou4f3) operably linked to a vestibular cell type-specific promoter (e.g., a utricle 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 MYOSIN 15 promoter, a GFI1 promoter, a POU4F3 promoter or a MYOSIN 7A promoter). Compositions containing AAV vectors can be administered to a patient, for example, by topical administration to the inner ear (e.g., endolymph or perilymph), such as by administration to or through the round window, oval window, or horizontal semicircular canal, or by administration to the semicircular canal to treat vestibular dysfunction. For the treatment of vestibular dysfunction, for example, 1x10 7 VG / ear up 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 The AAV1 vector was administered at a dose of 10 VG / ear).

[0559] After the composition is applied to the patient, those skilled in the art can monitor the patient's improvement in response to treatment by a variety of methods. For example, a physician can monitor the patient's vestibular function by performing standard tests (such as electronystagmography, video nystagmography, rotation test, vestibular evoked myogenic potential or computer dynamic posture map). Compared with the test results before the application of the composition, it is found that after the application of the composition, the patient shows improved balance, gait, posture and / or vestibular-ocular reflexes in one or more tests, which indicates that the patient has a good response to treatment. Subsequent dosages can be determined and applied as needed.

[0560] Other Implementations

[0561] Various modifications and alterations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described manner for carrying out the invention that are apparent to those skilled in the art are intended to be within the scope of the invention. Other embodiments are in the claims.

Claims

1. Use of a serotype 1 adeno-associated virus (AAV1) vector in the preparation of a medicament for transducing developing inner ear cells in a human subject, wherein the AAV1 vector comprises VP1, VP2 and VP3 capsid proteins, wherein the sequence of the VP1 capsid protein is SEQ ID NO: 1, the sequence of the VP2 capsid protein is SEQ ID NO: 2, and the sequence of the VP3 capsid protein is SEQ ID NO:

3. NO:3, and wherein the cells are three or more types of inner ear cells selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate 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 convex 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, fiber cells, cells of Reissner's membrane and glial cells.

2. The use of claim 1, wherein the cells are five or more cell types selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate cells, inner column cells, outer column cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, spiral convex cells, root cells, interdental cells, basal cells of stria vascularis, intermediate cells of stria vascularis, marginal cells of stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fiber cells, cells of Reissner's membrane and glial cells.

3. The use of claim 2, wherein the cells are ten or more cell types selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate cells, inner pillar cells, outer pillar cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, spiral convex cells, root cells, interdental cells, basal cells of stria vascularis, intermediate cells of stria vascularis, marginal cells of stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fiber cells, cells of Reissner's membrane and glial cells.

4. The use of claim 3, wherein the cells are fifteen or more cell types selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate cells, inner column cells, outer column cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, spiral convex cells, root cells, interdental cells, basal cells of stria vascularis, intermediate cells of stria vascularis, marginal cells of stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fiber cells, cells of Reissner's membrane and glial cells.

5. The use of claim 4, wherein the cells are twenty or more cell types selected from the group consisting of outer hair cells, vestibular hair cells, vestibular dark cells, vestibular fiber cells, Scarpa ganglion neurons, endothelial cells of vestibular capillaries, vestibular supporting cells, marginal cells, inner digitate cells, inner column cells, outer column cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, spiral convex cells, root cells, interdental cells, basal cells of stria vascularis, intermediate cells of stria vascularis, marginal cells of stria vascularis, spiral ganglion neurons, endothelial cells of cochlear capillaries, fiber cells, cells of Reissner's membrane and glial cells.

6. The use of claim 1, wherein the AAV1 vector transduces at least 20% of cells of a given cell type or all inner ear cell types.

7. The use of claim 1, wherein the medicament is formulated for topical administration to the middle ear or inner ear of the subject.

8. The use of claim 7, wherein the medicament is formulated for transtympanic or intratympanic administration.

9. The use as claimed in claim 1, wherein the AAV1 vector comprises a promoter selected from the list of promoters in Table 5.

10. The use according to claim 1, wherein the AAV1 vector comprises a ubiquitous promoter.

11. The use as claimed in claim 1, wherein the AAV1 vector comprises a polynucleotide selected from the list in Table 2.

12. The use of claim 11, wherein the AAV1 vector comprises a polynucleotide encoding solute transporter family 26 member 4 (Slc26a4), gap junction protein β2 (Gjb2) or gap junction protein β6 (Gjb6), and wherein the cells are three or more inner ear cell types selected from the group consisting of: vestibular dark cells, marginal cells, inner digitate 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 convex cells, root cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells and fiber cells.

13. The use of claim 12, wherein the AAV1 vector comprises a polynucleotide encoding Slc26a4, and wherein the three inner ear cell types are spiral lobes cells, root cells and vestibular dark cells.

14. The use of claim 12, wherein the AAV1 vector comprises a polynucleotide encoding Gjb2 or Gjb6, and wherein the cells are three or more cell types selected from the group consisting of marginal cells, inner interdigitating cells, inner pillar cells, outer pillar cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells and fiber cells.

15. The use of claim 14, wherein the AAV1 vector comprises a polynucleotide encoding Gjb2 or Gjb6, and wherein the cells are five or more cell types selected from the group consisting of marginal cells, inner interdigitating cells, inner pillar cells, outer pillar cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells and fiber cells.

16. The use of claim 15, wherein the AAV1 vector comprises a polynucleotide encoding Gjb2 or Gjb6, and wherein the cells are ten or more cell types selected from the group consisting of marginal cells, inner interdigitating cells, inner pillar cells, outer pillar cells, first row Deiter cells, second row Deiter cells, third row Deiter cells, Hensen cells, Claudius cells, interdental cells, stria vascularis basal cells, stria vascularis intermediate cells, stria vascularis marginal cells and fiber cells.

17. The use according to any one of claims 1 to 16, wherein the AAV1 vector comprises AAV2 inverted terminal repeats (ITRs).

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