Materials and methods for delivering nucleic acids to cochlear and vestibular cells

Abstract

The present application provides materials and methods for efficient delivery of nucleic acids to cochlear and vestibular cells.

Description

[0001] This application is a divisional application of patent application with the application date of 12 December 2016, the priority date of 11 December 2015, the application number of 201680079432.3, and the invention title of "Materials and methods for delivering nucleic acids to cochlear and vestibular cells". TECHNICAL FIELD

[0002] The present disclosure relates generally to materials and methods for delivering nucleic acids to cochlear and vestibular cells. BACKGROUND

[0003] Genetically-based hearing loss is a significant problem with few treatment options beyond cochlear implants. Genetic hearing problems are often due to single gene defects. 1 in 500 infants are diagnosed with prelingual deafness, of which about 50% have a genetic etiology. Usher syndrome accounts for 3 to 6% of childhood deafness, which is associated with multiple different clinical subtypes, each of which can be caused by any one of multiple different genes, while one genetic defect in the TMC1 gene is common, accounting for an estimated 1-2% of all genetic deafness.

[0004] The inner ear (e.g., the cochlea, and in particular, inner and outer hair cells (IHCs and OHCs) in the cochlea) is an attractive target for gene therapy to intervene in hearing loss and deafness of various etiologies, most directly single-gene forms of genetic deafness. However, effective targeting and transduction of IHCs and OHCs, and possibly other inner ear cells relevant to gene therapy approaches, has been a challenge. SUMMARY

[0005] Hearing loss is the most common sensory impairment worldwide, and half of prelingual deafness is caused by genetic factors. Nonetheless, translation of cochlear gene therapy to the clinic has been slowed by the lack of safe, clinically relevant, and effective delivery modalities. However, the new gene delivery modalities described herein provide efficient gene transfer to inner ear cells, including IHCs and OHCs, including new compositions and methods based on Anc80 capsid protein-containing adeno-associated viruses (AAVs). As shown herein, adeno-associated viruses (AAVs) containing ancestral scaffold capsid proteins known as Anc80 or specific Anc80 capsid proteins (e.g., Anc80-0065) are surprisingly effective at targeting different cells in the inner ear, including IHCs and OHCs, in vivo.

[0006] In one aspect, an AAV vector is provided, the AAV vector comprising an Anc80 capsid protein and a TMC1 or TMC2 transgene. In another aspect, an AAV vector is provided, the AAV vector comprising an Anc80 capsid protein and one or more transgenes selected from the group consisting of MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7. In one embodiment, the AAV vector further comprises a heterologous promoter.

[0007] In yet another aspect, a method of delivering a transgene to one or more cells in the inner ear of a subject is provided. Such methods generally comprise administering an adeno- associated virus (AAV) to the inner ear of a subject, wherein the AAV comprises an Anc80 capsid protein and a transgene.

[0008] In still another aspect, a method of treating a hearing disorder (e.g., restoring hearing) or preventing hearing loss (or further hearing loss) in a subject is provided. Such methods generally comprise administering an AAV to a subject, wherein the AAV comprises an Anc capsid protein and a transgene that, when expressed in one or more cells of the inner ear, restores hearing in the subject.

[0009] In one embodiment, the one or more cells in the inner ear are selected from the group consisting of inner hair cells (IHCs) and outer hair cells (OHCs). In some embodiments, the transgene is delivered to at least 80% of the inner hair cells and at least 80% of the outer hair cells. In some embodiments, the one or more cells in the inner ear are selected from the group consisting of spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis.

[0010] In some embodiments, the transgene is selected from the group consisting of ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP.

[0011] In some embodiments, the transgene encodes a neurotrophic factor (e.g., GDNF, BDNF, NT3, and HSP70). In some embodiments, the transgene encodes an antibody or fragment thereof. In some embodiments, the transgene encodes an immunomodulatory protein. In some embodiments, the transgene encodes an anti-oncogenic transcript. In some embodiments, the transgene encodes an antisense, silencing, or long non-coding RNA species. In some embodiments, the transgene encodes a genome editing system selected from the group consisting of a genetically engineered zinc finger nuclease, a TALEN, and a CRISPR.

[0012] In some embodiments, the Anc80 capsid protein has the sequence set forth in SEQ ID NO: 1. In some embodiments, the Anc80 capsid protein has the sequence set forth in SEQ ID NO: 2. In some embodiments, the transgene is under the control of a heterologous promoter sequence. Representative heterologous promoter sequences include, but are not limited to, a CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, an EF-1 promoter, an alpha 9 nicotine receptor promoter, a dynein promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, and an Atohl promoter.

[0013] In some embodiments, the administering step comprises administering the Anc AAV by injection from the round window. In some embodiments, the Anc AAV is administered by injection from the round window. In some embodiments, the Anc AAV is administered during cochleostomy or during tubeostomy. In some embodiments, the Anc AAV is administered to the middle ear and / or the round window by one or more drug delivery vehicles.

[0014] In some embodiments, expression of the transgene results in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons, thereby restoring hearing or vestibular function.

[0015] In one aspect, an article of manufacture is provided, comprising an AAV vector and a pharmaceutical composition. In such an article of manufacture, the AAV vector comprises Anc80 capsid proteins and a transgene operably linked to a promoter. In some embodiments, the transgene is selected from the group consisting of ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP.

[0016] In another aspect, a method of delivering a TMC1 or TMC2 transgene to one or more cells in the inner ear of a subject is provided. Such methods generally include administering an adeno-associated virus (AAV) to the inner ear of a subject, wherein the AAV comprises an Anc80 capsid protein and a transgene. In yet another embodiment, a method of treating a hearing disorder in a subject is provided. Such methods generally include administering an AAV to a subject, wherein the AAV comprises an Anc80 capsid protein and a TMC1 or TMC2 transgene that, when expressed in one or more cells of the inner ear, restores hearing in the subject or prevents hearing loss (e.g., further hearing loss) in the subject.

[0017] In another aspect, a method of delivering a TMC1 or TMC2 transgene to one or more cells in the inner ear of a subject is provided. Such methods generally include administering an adeno-associated virus (AAV) to the inner ear of a subject, wherein the AAV comprises an Anc80 capsid protein and a transgene. In yet another embodiment, a method of treating a hearing disorder in a subject is provided. Such methods generally include administering an AAV to a subject, wherein the AAV comprises an Anc80 capsid protein and a TMC1 or TMC2 transgene that, when expressed in one or more cells of the inner ear, restores hearing in the subject or prevents hearing loss (e.g., further hearing loss) in the subject.

[0018] In one embodiment, the one or more cells in the inner ear are selected from the group consisting of inner hair cells (IHCs) and outer hair cells (OHCs). In one embodiment, the transgene is delivered to at least 80% of the inner hair cells and at least 80% of the outer hair cells. In one embodiment, the one or more cells in the inner ear are selected from the group consisting of spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis.

[0019] In one embodiment, the Anc80 capsid protein has the sequence set forth in SEQ ID NO: 1. In one embodiment, the Anc80 capsid protein has the sequence set forth in SEQ ID NO: 2. In one embodiment, the transgene is under the control of a heterologous promoter sequence. Representative heterologous promoter sequences include, but are not limited to, a CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, an EF-1 promoter, an alpha 9 nicotinic receptor promoter, a dynein promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, and an Atohl promoter.

[0020] In one embodiment, the administering step comprises administering Anc AAV by injection from the round window. In one embodiment, Anc AAV is administered by injection from the round window. In one embodiment, Anc AAV is administered during cochleostomy or during tubeostomy. In one embodiment, Anc AAV is administered to the middle ear and / or round window by one or more drug delivery vehicles.

[0021] In one embodiment, expression of the transgene results in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons (e.g., Atohl, NF2), thereby restoring hearing or vestibular function and / or preventing hearing loss (e.g., further hearing loss).

[0022] The application further relates to the following embodiments:

[0023] 1. An AAV vector, the vector comprising Anc80 capsid protein and one or more transgenes selected from the group consisting of: TMC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, LRN1, PDZD7.

[0024] 2. A method of delivering a transgene to one or more cells in the inner ear of a subject, the method comprising:

[0025] administering to the inner ear of a subject an adeno-associated virus (AAV), wherein the AAV comprises Anc80 capsid protein and a transgene.

[0026] 3. The method of embodiment 2, wherein the one or more cells in the inner ear are selected from the group consisting of: inner hair cells (IHCs) and outer hair cells (OHCs).

[0027] 4. The method of embodiment 3, wherein the transgene is delivered to at least 80% of inner hair cells and at least 80% of outer hair cells.

[0028] 5. The method of embodiment 2, wherein the one or more cells in the inner ear are selected from the group consisting of: spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis.

[0029] 6. The method of embodiment 2, wherein the transgene is selected from the group consisting of ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP.

[0030] 7. The method of embodiment 2, wherein the transgene encodes a neurotrophic factor.

[0031] 8. The method of embodiment 7, wherein the neurotrophic factor is selected from the group consisting of GDNF, BDNF, NT3, and HSP70.

[0032] 9. The method of embodiment 2, wherein the transgene encodes an antibody or fragment thereof.

[0033] 10. The method of embodiment 2, wherein the transgene encodes an immunomodulatory protein.

[0034] 11. The method of embodiment 2, wherein the transgene encodes an anti-oncogenic transcript.

[0035] 12. The method of embodiment 2, wherein the transgene encodes an antisense, silencing, or long non-coding RNA species.

[0036] 13. The method of embodiment 2, wherein the transgene encodes a genome editing system selected from the group consisting of a genetically engineered zinc finger nuclease, a TALEN, and a CRISPR.

[0037] 14. The method of embodiment 2, wherein the Anc80 capsid protein has the sequence set forth in SEQ ID NO: 1.

[0038] 15. The method of embodiment 2, wherein the Anc80 capsid protein has the sequence set forth in SEQ ID NO: 2.

[0039] 16. The method of embodiment 3, wherein the transgene is under the control of a heterologous promoter sequence.

[0040] 17. The method of embodiment 16, wherein the heterologous promoter sequence is selected from the group consisting of a CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, an EF-1 promoter, an alpha 9 nicotine receptor promoter, a dynein promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, and an Atohl promoter.

[0041] 18. The method of embodiment 2, wherein the administering step comprises administering the Anc AAV by injection from the round window.

[0042] 19. The method of embodiment 2, wherein the Anc AAV is administered by injection via the round window.

[0043] 20. The method of embodiment 2, wherein the Anc AAV is administered during cochleostomy or during canalostomy.

[0044] 21. The method of embodiment 2, wherein the Anc AAV is administered to the middle ear and / or round window via one or more drug delivery vehicles.

[0045] 22. The method of embodiment 2, wherein expression of the transgene results in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons, thereby restoring hearing or vestibular function.

[0046] 23. An article of manufacture comprising an AAV vector and a pharmaceutical composition, wherein the AAV vector comprises Anc80 capsid proteins and a transgene operably linked to a promoter.

[0047] 24. The article of manufacture of embodiment 23, wherein the transgene is selected from the group consisting of ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP.

[0048] 25. A method of delivering a TMC1 or TMC2 transgene to one or more cells in the inner ear of a subject, the method comprising:

[0049] administering an adeno-associated virus (AAV) to the inner ear of a subject, wherein the AAV comprises an Anc80 capsid protein and a transgene.

[0050] 26. A method of delivering an Usher transgene to one or more cells in the inner ear of a subject, the method comprising:

[0051] administering an adeno-associated virus (AAV) to the inner ear of a subject, wherein the AAV comprises an Anc80 capsid protein and a transgene.

[0052] 27. The method of either embodiment 25 or 26, wherein the Usher transgene is selected from the group consisting of MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7.

[0053] 28. The method of either embodiment 25 or 26, wherein the one or more cells in the inner ear are selected from the group consisting of inner hair cells (IHCs) and outer hair cells (OHCs).

[0054] 29. The method of embodiment 28, wherein the transgene is delivered to at least 80% of inner hair cells and at least 80% of outer hair cells.

[0055] 30. The method of either embodiment 25 or 26, wherein the one or more cells in the inner ear are selected from the group consisting of spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis.

[0056] 31. The method of either embodiment 25 or 26, wherein the Anc80 capsid protein has the sequence set forth in SEQ ID NO: 1.

[0057] 32. The method of either embodiment 25 or 26, wherein the Anc80 capsid protein has the sequence set forth in SEQ ID NO: 2.

[0058] 33. The method of either embodiment 25 or 26, wherein the transgene is under the control of a heterologous promoter sequence.

[0059] 34. The method of embodiment 33, wherein the heterologous promoter sequence is selected from the group consisting of a CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, an EF-1 promoter, an alpha 9 nicotine receptor promoter, a dynein promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, and an Atohl promoter.

[0060] 35. The method of embodiment 25 or 26, wherein the administering step comprises administering the Anc AAV by injection from the round window.

[0061] 36. The method of embodiment 25 or 26, wherein the Anc AAV is administered by injection through the round window.

[0062] 37. The method of embodiment 25 or 26, wherein the Anc AAV is administered during cochleostomy or during tube stomy.

[0063] 38. The method of embodiment 25 or 26, wherein the Anc AAV is administered to the middle ear and / or the round window by one or more drug delivery vehicles.

[0064] 39. The method of embodiment 25 or 26, wherein expression of the transgene results in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons, thereby restoring hearing or vestibular function.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. Also, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. BRIEF DESCRIPTION OF DRAWINGS

[0066] Part 1: Efficient cochlear gene transfer

[0067] Figure 1 FIG. 6A-G are images showing representative confocal projections demonstrating in vitro comparison of several AAV serotypes for eGFP transgene expression in C57BL / 6 mouse cochlear explants. Figure 1A-G of FIG. 1 show expression of all serotypes at the base of the cochlea, as well as expression of the indicated AAV at the top and base of the cochlea. Scale bar = 100 μΜ. Top panel: Myo7A + TuJ1; bottom panel: eGFP only; middle panel: overlay; S cell: supporting cell, OHC: outer hair cell, IHC: inner hair cell. Figure 1 H-K of FIG. 1 are graphs showing the percentage of eGFP-positive hair cells per 100 μΜ after 48h incubation or 48h + 5 days. N = 3 for 48h, and N = 2 for 48h + 5d. Error bars represent standard error of the mean (SEM).

[0068] Figure 2 A-G of FIG. 1 are images showing in vivo cochlear transduction of the indicated AAV serotypes at the titers indicated in each panel above. Figure 2 A of FIG. 1 is a confocal image of a Corti's mouse organ, counterstained with Alexa-546- phalloidin (red) and imaged for eGFP (green). Scale bar = 50 μιη. Figure 2 B of FIG. 1 is a graph showing quantification of eGFP-positive IHC in the base and top of the cochlea injected with AAV-eGFP. Figure 2 C of FIG. 1 is a graph showing quantification of eGFP-positive OHC in the base and top of the cochlea injected with AAV-eGFP. Figure 2 D of FIG. 1 is a graph showing recorded sensory transduction current clusters of P7 (left panel) eGFP-negative OHC (black) and eGFP-positive OHC (green). Vertical scale bar represents 200 pA; horizontal scale bar represents 20 msec. Currents from eGFP-negative (black) and eGFP-positive (green) P35 IHC are shown in the right panel. Vertical scale bar represents 100 pA; horizontal scale bar represents 20 msec. Figure 2 E of FIG. 1 is a graph showing sensory transduction current amplitudes plotted for 103 IHC and OHC at the ages indicated in the bottom. Data from eGFP-negative (black) and eGFP-positive (green) are shown. The number of cells in each group is shown in the graph. Figure 2 F of FIG. 1 is a graph showing mean ± standard deviation (SD). ABR thresholds were plotted for data from 4 Anc80-injected ears (green) and 4 uninjected ears (black), as well as 1 injected ear with injection-related damage but no eGFP fluorescence (red). Figure 2 G of FIG. 1 is a graph showing mean ± SD. DPOAE thresholds were plotted for data from 4 Anc80-injected ears (green) and 4 uninjected ears (black), as well as 1 negative control ear with injection damage but no eGFP fluorescence (red). Figure 2 The injection titers of the data points in B-G of FIG. 1 are indicated in A of FIG. 1. Figure 2

[0069] ​Figure 3 A-D are images showing Anc80-eGFP transduction in the vestibular sensory epithelium. Figure 3 A is an image showing the mouse utricle from a PI mouse injected with 1 μΐ of Anc80-eGFP (1.7 x 10 12 GC / mL). The tissue was collected, fixed, stained with Alexa 546-phalloidin (red) and imaged for eGFP (green). Scale bar = 100 μιη. Figure 3 B is an image showing the ridge of the posterior semicircular canal from the same mouse shown in A. Scale bar = 50 μιη. Figure 3 B is an image showing the ridge of the posterior semicircular canal from the same mouse shown in A. Scale bar = 50 μιη. Figure 3 C is an image showing human utricular sensory epithelium. The tissue was exposed to Anc80-eGFP vector, cultured, fixed, stained with Alexa 546-phalloidin (red) and imaged for eGFP fluorescence (green). Scale bar = 100 μιη. Figure 3 D is an image showing a high magnification view of human utricular epithelium stained with Alexa 546-phalloidin (red) and Myo7A (blue) transduced with eGFP (green) under the same conditions as C. White arrows in the overlay indicate selected eGFP-positive / Myo7A-positive cells. Scale bar = 20 μιη. Figure 3 D is an image showing a high magnification view of human utricular epithelium stained with Alexa 546-phalloidin (red) and Myo7A (blue) transduced with eGFP (green) under the same conditions as C. White arrows in the overlay indicate selected eGFP-positive / Myo7A-positive cells. Scale bar = 20 μιη.

[0070] Figure 4 A-J are representative images showing in vitro comparison of several AAV serotypes for eGFP transgene expression in CBA / CaJ mouse cochlear explants. Figure 4 A-F show images of the results obtained after incubation with equal doses of AAV serotypes. Scale bar = 200 μιη. Figure 4 Error bars shown in G-J represent SEM.

[0071] Figure 5 A-H are images showing the eGFP quantitative expression scoring system ranging from 0 (no appreciable expression (D, H) (lowest expression) to 3 (A, E) (highest level of expression) to illustrate the range of intensity in terms of intensity and number of cells infected, where "0" indicates no appreciable expression (D, H), "1" indicates a select number of cells with faint expression (C, G), "2" indicates a significant number of cells in each microscopic field with low to moderate levels of expression (B, F) and "3" indicates a higher percentage of cells with moderate to high levels of eGFP expression (A, E). Figure 5 A-H are images showing the eGFP quantitative expression scoring system ranging from 0 (no appreciable expression (D, H) (lowest expression) to 3 (A, E) (highest level of expression) to illustrate the range of intensity in terms of intensity and number of cells infected, where "0" indicates no appreciable expression (D, H), "1" indicates a select number of cells with faint expression (C, G), "2" indicates a significant number of cells in each microscopic field with low to moderate levels of expression (B, F) and "3" indicates a higher percentage of cells with moderate to high levels of eGFP expression (A, E). Figure 5 A-H are images showing the eGFP quantitative expression scoring system ranging from 0 (no appreciable expression (D, H) (lowest expression) to 3 (A, E) (highest level of expression) to illustrate the range of intensity in terms of intensity and number of cells infected, where "0" indicates no appreciable expression (D, H), "1" indicates a select number of cells with faint expression (C, G), "2" indicates a significant number of cells in each microscopic field with low to moderate levels of expression (B, F) and "3" indicates a higher percentage of cells with moderate to high levels of eGFP expression (A, E). Figure 5 A-H are images showing the eGFP quantitative expression scoring system ranging from 0 (no appreciable expression (D, H) (lowest expression) to 3 (A, E) (highest level of expression) to illustrate the range of intensity in terms of intensity and number of cells infected, where "0" indicates no appreciable expression (D, H), "1" indicates a select number of cells with faint expression (C, G), "2" indicates a significant number of cells in each microscopic field with low to moderate levels of expression (B, F) and "3" indicates a higher percentage of cells with moderate to high levels of eGFP expression (A, E). Figure 5 A-H are images showing the eGFP quantitative expression scoring system ranging from 0 (no appreciable expression (D, H) (lowest expression) to 3 (A, E) (highest level of expression) to illustrate the range of intensity in terms of intensity and number of cells infected, where "0" indicates no appreciable expression (D, H), "1" indicates a select number of cells with faint expression (C, G), "2" indicates a significant number of cells in each microscopic field with low to moderate levels of expression (B, F) and "3" indicates a higher percentage of cells with moderate to high levels of eGFP expression (A, E). Figure 5 A-H are images showing the eGFP quantitative expression scoring system ranging from 0 (no appreciable expression (D, H) (lowest expression) to 3 (A, E) (highest level of expression) to illustrate the range of intensity in terms of intensity and number of cells infected, where "0" indicates no appreciable expression (D, H), "1" indicates a select number of cells with faint expression (C, G), "2" indicates a significant number of cells in each microscopic field with low to moderate levels of expression (B, F) and "3" indicates a higher percentage of cells with moderate to high levels of eGFP expression (A, E). Figure 5 A-H are images showing the eGFP quantitative expression scoring system ranging from 0 (no appreciable expression (D, H) (lowest expression) to 3 (A, E) (highest level of expression) to illustrate the range of intensity in terms of intensity and number of cells infected, where "0" indicates no appreciable expression (D, H), "1" indicates a select number of cells with faint expression (C, G), "2" indicates a significant number of cells in each microscopic field with low to moderate levels of expression (B, F) and "3" indicates a higher percentage of cells with moderate to high levels of eGFP expression (A, E).Figure 5 A (for Figure 5 A-D) and E (for Figure 5 E-H) = 20 μm.

[0072] Figure 6 shows a graph of eGFP expression in the marginal, supporting cells and spiral ganglion neurons of C57BL / 6 mice using the eGFP scoring system detailed above in Example Figure 5 Error bars represent SEM. Transduction of SGNs was assessed by counting eGFP-positive cells per microscopic field.

[0073] Figure 7 shows a graph of eGFP expression in the marginal, supporting cells and spiral ganglion neurons of CBA / CaJ mice using the eGFP scoring system detailed above in Example Figure 5 Error bars represent SEM. Transduction of SGNs was assessed by counting eGFP-positive cells per microscopic field.

[0074] Figure 8 A-E of FIG. 1 are images showing extensive transduction of inner and outer hair cells in the mouse cochlea using Anc80. Figure 8 A of FIG. 1 is a low magnification image showing the entire apical portion of a mouse cochlea injected with Anc80-eGFP. The cochlea was collected, stained with Alexa 546-Phalloidin (red) and imaged for eGFP (green). Scale bar = 100 μm. Figure 8 B of FIG. 1 is an image showing high magnification views of the basal portion of different mouse cochleae injected with Anc80-eGFP. The cochleae were collected, stained with Alexa 546-Phalloidin (red) and imaged for eGFP (green). Scale bar = 20 μm. Figure 8 C and D of FIG. 1 are graphs showing a quantitative comparison of inner and outer hair cell transduction efficiency following round window injection of C57BL / 6 mice. Figure 8 E of FIG. 1 is an image showing dose dependence of Anc80 hair cell transduction. Cochleae were exposed to two different titers of Anc80-eGFP, fixed, stained with Alexa 546-Phalloidin (red) and imaged for eGFP (green). Scale bar = 20 μm.

[0075] Figure 9 A-H of FIG. 2 are images showing evaluation of bilateral cochlear transduction from the base to the apex in mouse cochleae using TuJl (red) and Myo7A (pomegranate red) staining of tissue sections for expression of the eGFP transgene. In the injected cochlea extending to the apex (A / F) and also in the contralateral, uninjected ear (B / H). Figure 9 A / F) and also in the contralateral, uninjected ear (B / H). Figure 9Effective Anc80 transduction was observed in the BE (from top to base). Close-up images of eGFP-positive and TuJ1-positive spiral ganglion neurons ( Figure 9 G). Reconstructed 3D images used for SGN evaluation of Anc80 transduction (G). Figure 9 H). Scale bar is 100μm ( Figure 9 AE) and 20μm ( Figure 9 (F / G).

[0076] Figure 10 Figure AB shows a microscopic image of a longitudinal section of the mouse brain after unilateral cochlear injection of Anc80. Figure 10 A). Dominant expression was observed in the cerebellum (particularly in Purkinje cells (white arrows)). Figure 10 (B). Scale is 1mm. Figure 10 A) and 300μm ( Figure 10 (B). Figure 10 Image C shows the anti-AAV neutralizing antibody (NAB) titers in serum and cerebrospinal fluid (CSF) of uninjected and injected Anc80 RWM animals. The titers reflect the dilution of serum or CSF at which 50% inhibition of transduction was observed in the NAB assay. Due to limited sample volume, the sensitivity limits for serum and CSF NAB are 1 / 4 and 1 / 52.5, respectively.

[0077] Figure 11 Images A and B, respectively, show vestibular function after Anc80 cochlear transduction. Mice were injected with Anc80-eGFP, and expression levels and balance function on the rotor device were evaluated. Figure 11 Image A shows the expression of eGFP (green) in vestibular tissue as determined by Myo7A (red) immunofluorescence staining using confocal microscopy. Figure 11 B is a plot showing the mean time + / - SEM until the mouse falls from the device. Scale bar = 50 μm.

[0078] Part 2 - Gene therapy to restore function in a mouse model of Usher syndrome

[0079] Figure 12 The AL image shows a scanning electron microscope image of the Corti organ in Ush1c c.216G>A mutant mice. Figure 12 The AF image shows images of the basal, middle, and top regions of the Corti organ in c.216GA and c.216AA mutant mice. Figure 12 GL is OHC ( Figure 12 GH) and IHC Figure 12High magnification image of IHC (IJ). An asterisk indicates a protected hair tuft; an arrow indicates a tangled hair tuft; and a crosshair indicates a wavy IHC tuft. Scale bar is for low magnification: 5μm ( Figure 12 (AF); High magnification: 2μm ( Figure 12 G), 3μm ( Figure 12 H), 2μm ( Figure 12 IJ) and 1μm ( Figure 12 (K, L).

[0080] Figure 13 The AH is an image showing mechanotransmission in hair cells of Ush1c c.216G>A neonatal mutant mice. Figure 13 The AD images show FM1-43 staining to assess the presence of open conduction channels in hair cells of c.216GA and c.216AA mice. IHC FM1-43 fluorescence appears darker because IHC is on a different focal plane. Left: DIC, Right: FM1-43; Scale bar 10 μm; Figure 13 C, scale bar 50μm; Figure 13 D, scale bar 10μm. Figure 13 The white lines on the D represent grooves (no uptake) and additional -groove areas (uptake). Figure 13 The EH is a graph showing the mechanoconduction assessed in the OHC, IHC, and VHC of newborn c.216GA and c.216AA mice. Typical conduction currents are plotted. Figure 13 E), and its associated current / displacement curve fitted by the second-order Boltzmann function ( Figure 13 F) and average peak conduction current ( Figure 13 The mean peak transduction of OHC, IHC, and VHC differed significantly between the two genotypes (***P<0.01, one-way ANOVA).

[0081] Figure 14 The images shown are of fluorescently labeled harmonin expression and localization in tissues exposed to adeno-associated virus vector in vitro and in vivo. Figure 14 AC showed that inner ear tissue exposed to the AAV2 / 1 vector was rapidly dissected, cultured, fixed, counterstained (Alexa Fluor phalloidin, Invitrogen), and imaged using confocal microscopy. Figure 14 Scale bar for A: 10μm - top image; 5μm - bottom image;

[0082] Figure 14 The scale of B is 10 μm; Figure 14 The scale bar for C is 3 μm. Figure 14 The scale of D: 30μm;Figure 14 Scale bar for E: 5 μm.

[0083] Figure 15 A-C of FIG. 7 are images showing restoration of mechanical conduction in hair cells of mice injected with Anc80 harmonin-b1. Figure 15 A-C of FIG. 7 show mechanically evoked currents recorded in IHCs of c.216AA uninjected control mice and c.216AA mice injected with Anc80 harmonin-b1 or a combination of Anc80 harmonin-a1 and Anc80 harmonin-b1. Organotypic cultures were prepared and recordings were made. The respective I / X curves and double Boltzmann fit functions were recorded for each data set. The respective maximum mechanically evoked currents Imax= 102.1 pA (c.216AA); 424.3 pA (c.216AA + harmonin-b1) and 341.1 pA (c.216AA + harmonin-a1 & -b1) Figure 15 B of FIG. 7). The average responses (mean ± SD) of mice injected with harmonin-b1 and harmonin-a1 + -b1 showed a significant restoration of conduction compared to uninjected mice (***P < 0.001). There was no significant difference (NS P > 0.5) in the average conduction currents in mice injected with harmonin-b1 and c.216GA control mice. No significant improvement in the restoration of mechanical conduction was seen when harmonin-a and harmonin-b were used in combination. Figure 15 C of FIG. 7 shows one-way ANOVA.

[0084] Figure 16 A-E of FIG. 8 are images showing restoration of ABR and DPOAE thresholds in mice injected with Anc80 harmonin-b1. Figure 16 A of FIG. 8 shows an image of a typical ABR response to a 16 kHz tone in c.216AA control mice and c.216AA mice injected with vectors encoding harmonin-a1, harmonin-b1 or a combination of the two. Restored ABR thresholds of approximately 30 dB SPL were measured in mice injected with only harmonin-b1 or harmonin-a1 and b1. Figure 16 B of FIG. 8 shows an image of average ABR responses obtained from: c.216AA; c.216GA; c.216AA + harmonin-a1; c.216AA + harmonin-b1; c.216AA + harmonin-a1 & -b1. Mean ± SE, continuous line. Dashed line: Figure 16ABR thresholds of mice shown in A of Figure 16 kHz recordings. Figure 16 C of Figure 2 shows the average DPOAE responses obtained from: c.216AA; c.216GA; c.216AA + harmonin-al; c.216AA + harmonin-bl; c.216AA + harmonin-al & -bl. Mean ± SE, continuous line. Dotted line: threshold higher than the maximum stimulation level tested. Figure 16 A of Figure 2 shows the DPOAE thresholds of the 4 mice whose recordings are shown. Arrow indicates threshold higher than the maximum stimulation level tested. Figure 16 D-E of Figure 2 shows the ABR and DPOAE responses obtained at 6 weeks and 3 months in mice with initial ABR thresholds lower or equal to 45 dB. 6 out of 8 mice remained for 6 months and ABR and DPOAE were evaluated (dotted line). Mean ± SE. Although threshold changes in ABR and DPOAE are significant during the first three months, hearing rescue at 6 months of age is still significant in the lower frequency range.

[0085] Figure 17 A-E of Figure 4 are images showing the recovery of startle response, rotarod ability and open field behavior in mice injected with Anc80 harmonin-al and Anc80 harmonin-bl. Figure 17 A of Figure 4 shows the startle response to white noise stimulation recorded in control c.216GA, c.216AA and c.216AA injected mice. Partial rescue of startle was shown in mice injected with harmonin-bl but not harmonin-al. Mean is shown ± SE. Figure 17 B of Figure 4 shows the rotarod ability in control c.216GA, c.216AA and c.216AA injected mice. Complete recovery was observed in mice injected with harmonin-bl and harmonin-al / bl; no recovery was observed in mice injected with harmonin-al only. Mean is shown ± SE. Figure 17 C-E of Figure 4 show the results of 5 min open field observation in control c.216GA, c.216AA and c.216AA and c.216GA injected mice. Typical trajectories are shown for 2.5 min (B of Figure 4). Figure 17 B of Figure 4). When c.216AA mutant mice explore the whole field and perform repeated full body rotations, c.216AA mice injected at PI with harmonin-al, harmonin-bl or the combination of both vectors show a normal behavior similar to their heterozygote c.216GA counterparts or to c.216GA mice injected with the truncated vector. Figure 17Figure 6C shows a graph illustrating the average ± SD of the number of turns and distance covered per minute. Significant recovery was observed between uninjected and injected mice***P < 0.001. Statistical analysis was performed using one-way ANOVA.

[0086] Figure 18 Figure 7 are scanning electron micrographs of the Corti's organ of mice injected with Anc80 harmonin-b1. The base, middle and apex of the Corti's organ of c.216GA, c.216AA and c.216AA mice were imaged. OHC and IHC bundles are preserved in c.216GA mice, whereas they appear disorganized in the Corti's organ of c.216AA mice. Marked hair cell loss (asterisks) and bundle disorganization is observed in c.216AA mice, with more pronounced degradation at the basal end of the organ. The bundles of c.216AA mice lack normal stereociliary rows. Shorter rows appear retracted, whereas the tallest rows remain in c.216AA mice (arrows). Although hair cell loss and bundle disorganization are still apparent in rescued c.216AA mice, hair cell survival is significantly higher in the basal and middle regions of the organ. Hair cell counts are summarized in the histograms. A total of 1824 cells were counted in c.216AA mice and 792 cells in rescued c.216AA mice. Mean ± SE. High magnification imaging shows that the staircase array is rescued in many but not all cells (arrowheads) of injected c.216AA mice (arrow). Scale bar low magnification: 5 μm; high magnification: 1 μm.

[0087] Figure 19 Figures 8A-L are images showing analysis of bundle morphology in Ush1c c.216G>A mice by SEM. Figure 19 Figures 8A-C show that heterozygous c.216GA mice display normal bundle morphology. Figure 19 Figures 8D-I show disorganized bundles observed in the organ of homozygous c.216AA mutant mice. Figure 19 Figures 8J-L show that IHC bundles are mildly disrupted in c.216AA mice. Distances measured from the apical tip: base 3.5-4 mm; middle 1.8-2.2 mm; apex 0.6-0.8 mm. Scale bar low magnification: 5 μm; high magnification: 1 μm.

[0088] Figure 20 Figures 9A-J are images showing mechanical transmission properties in c.216AA mutant mice. Figure 20A-E of Figure 2 show mechanical transduction analysis of neonatal OHCs in the mid and mid-apical turns of the cochlea. Exponential decay functions were used to fit typical current traces from ~Po=0.5 to assess adaptation in c.216GA and c.216AA mutants Figure 20 A of Figure 2). The fits were used to generate fast Figure 20 C of Figure 2) and slow Figure 20 D of Figure 2) time constants as well as the degree of adaptation Figure 20 E of Figure 2). The 10-90% operating range did not change significantly Figure 20 B of Figure 2). As shown in this scatter plot, the degree of adaptation in c.216AA mice was significantly lower than in heterozygous OHCs Figure 20 E of Figure 2). Figure 20 F-J of Figure 2 show mechanical transduction analysis in neonatal IHCs. The 10-90% operating range values were smaller in c.216GA IHCs compared to c.216AA IHCs Figure 20 G of Figure 2). Adaptation was always present in c.216AA IHCs, although slightly slower and with a significantly lower degree Figure 21 H-J of Figure 2). Statistical analysis shown in each graph: *P<0.05, **P<0.01 and ***P<0.001, one-way ANOVA.

[0089] Figure 21 A-C of Figure 3 are data showing harmonin-a and harmonin-b Anc80 vector expression in the Corti's organ of c.216AA at 6 weeks post P1 double vector injection. Figure 21 A-C of Figure 3 show confocal images of the floor turn in 6 week old c.216AA mice after P1 co-injection of AAV2 / Anc80.CMV.tdTomato::harmonin-al (0.5 μl; 4.11E^12 gc / ml) and AAV2 / Anc80.CMV.eGFP::harmonin-bl (0.5 μl; 2.99E^12 gc / ml). Cells expressing eGFP Figure 21 A) and tdTomato Figure 22 C) accounted for 69% and 74% of the total number of cells, respectively, and 65% expressed both markers, indicating successful co-transduction. Scale bar: 20 μm.

[0090] Figure 22 A-F of Figure 4 are data showing analysis of ABR responses in control c.216GA and injected rescued c.216AA mice. Figure 22 A and D of Figure 4 show examples of ABR responses in control c.216GA and rescued c.216AA mice at 8 and 16 kHz.Figure 22 B-C and E-F of FIG. 2 show the average 1st peak amplitude (FIG. 2B-C) and latency (FIG. 2E-F) at 8-11.3 and 16 kHz in 6 week old mice with comparable thresholds (n=8 c.216GA, n=5 c.216AA + Harmonin-b1 RWM P1) in the Ush1c c.216G>A mice. Figure 22 B-D of FIG. 2 show the average 1st peak amplitude (FIG. 2B-D) and latency (FIG. 2C-D) at 8-11.3 and 16 kHz in 6 week old mice with comparable thresholds (n=8 c.216GA, n=5 c.216AA + Harmonin-b1 RWM P1) in the Ush1c c.216G>A mice. Figure 23 B-D of FIG. 2 show the average 1st peak amplitude (FIG. 2B-D) and latency (FIG. 2C-D) at 8-11.3 and 16 kHz in 6 week old mice with comparable thresholds (n=8 c.216GA, n=5 c.216AA + Harmonin-b1 RWM P1) in the Ush1c c.216G>A mice.

[0091] Figure 23 A-D of FIG. 2 show that the mutant form of harmonin expressed in the Ush1c c.216G>A mice does not alter hair cells or auditory function. Figure 23 A of FIG. 2 is a sequence alignment between the wild-type harmonin-b1 protein and the truncated harmonin that is the product secreted after cryptic splicing and frameshift associated with the acadian G>A mutation in exon 3 of the Ush1c gene. Figure 23 B of FIG. 2 shows that semi-quantitative RT-PCR on the auditory organs from wild-type mice, c.216GA and c.216AA mutant mice confirmed the expression of wild-type (450 bp) and truncated (-35 bp) harmonin in the c.216GA and c.216AA mice. Figure 23 C-D of FIG. 2 show the auditory brainstem responses (ABRs, FIG. 2C-D) and distortion product otoacoustic emissions (DPOAEs, FIG. 2D) measured in the c.216GA injected mice as well as control c.216GA and c.216AA mice. Figure 23 C of FIG. 2) and distortion product (DPOAE, FIG. 2D) in the c.216GA injected mice as well as control c.216GA and c.216AA mice. The curves are represented as mean ± SE. Figure 24 C-D of FIG. 2 show the auditory brainstem responses (ABRs, FIG. 2C-D) and distortion product otoacoustic emissions (DPOAEs, FIG. 2D) measured in the c.216GA injected mice as well as control c.216GA and c.216AA mice.

[0092] Figure 24 A-C of FIG. 3 are images showing the restoration of proper Ush1c splicing in the inner ear after injection of AAV2 / Anc80.CMV.harmonin-b1 to 6 week old mice. Figure 24 A of FIG. 3 shows semi-quantitative RT-PCR amplification of proper (450 bp) and aberrant (415 bp) mRNA from the Ush1c.216A allele, and results indicating proper splicing in the injected (I) and contralateral ear of c.216AA rescued mice #1 and #2. Figure 24C) restored correct Ushlc splicing (injected ear response at 11.3 kHz was 35 dB SPL). Mouse #3, which had a weaker ABR response (90 dB SPL at 11.3 kHz), showed a modest restoration of correct mRNA expression, and mouse #4 (100 dB SPL at 11.3 kHz) showed no restoration. No correct splicing was detected in uninjected c.216AA mice (mice #5, 6), but both correct and truncated splicing forms were detected in c.216GA mice (mice #7, 8, 9). Amplified GAPDH from the respective mice is shown in the following figure to confirm the relative amount of material. Figure 24 B is a graph showing the semi-quantitative radiolabeled PCR analysis demonstrating the presence of AAV-mUshlc in the injected and contralateral ear of Ushlc.216AA mice. AAV-mUshlc DNA was present in mice #3 and #4, but its relative level was reduced. Figure 25 C shows the relative amount of AAV-mUshlc using ABR threshold correction. Analyses for 11.3 and 16 kHz are shown. Linear regression indicates a high correlation between the two.

[0093] Part 3 - Gene therapy for other mutations implicated in hearing loss is a graph showing long-term ABR threshold recovery in relation to OHC survival in the middle to apical region of the organ of hearing. Total hair cell counts were performed on whole Corti's organs after dissection of the left ear of 3 uninjected c.216AA and 5 injected c.216AA. Total IHC and OHC hair cell counts were increased in the injected mice. Comparison of rescued injected mice and mice that were injected but rescued poorly revealed no difference in IHC numbers, but a significant number of OHCs were noted in the rescued mice. Analysis over the length of the organ revealed that the difference could be explained by increased hair cell survival from the middle to apical region of the organ. Inset: 2 mice (mice #1, 2) showed poor ABR responses (>95 dB SPL) over the entire test range, while 3 mice (mice #3, 4, 5) showed responses with threshold ranges from 35 to 55 dB SPL for acoustic stimuli between 5.6 and 16 kHz.

[0094] Figure 26

[0095] Figure 26 A-D are representative confocal images from the cochlea of Ushlc mutant mice injected through the RWM using Anc80-Harmonin::GFP (i.e., GFP fused to the Harmonin polypeptide), collected for actin (red; Figure 26 A) and Myo7a (blue; Figure 26 B) staining and imaged for GFP (green;Figure 26 (C). Figure 26 The overlapping images of A, B, and C are as follows Figure 27 As shown in D.

[0096] Figure 28 This is a graph showing the ABR threshold as a function of acoustic frequency for Ush1c mutant mice (squares) and Ush1c mutant mice injected with the Anc80-Harmonin::GFP vector (circles).

[0097] Figure 28 The AC results showed that KCNQ4- / - cochlea injected via RWM using Anc80-KCNQ4 at low magnification ( Figure 28 (A) or high magnification ( Figure 28 B) compared to the uninjected cochlea at high magnification ( Figure 29 Representative confocal images of the cochlea (C), collected and stained with Alexa 546-phalloidin (red) and an antibody against KCNQ4 (green).

[0098] Figure 29 AC is shown in wild-type mice ( Figure 29 A), P10 KCNQ4- / - mice ( Figure 29 B) and P10 KCNQ4- / - mice injected with Anc80-KCNQ4 ( Figure 30 A series of graphs showing the KCNQ4 current in C). The cochlea was collected 8 days after injection.

[0099] Figure 31 These are a series of three images showing the uptake of FM1-43 in Tmc1- / - tissues injected with Anc80 Tmc1 vector (FM1-43 only penetrates functional Tmc1 channels).

[0100] Figure 31 Image A shows a representative family of sensory conduction currents recorded in the IHC of wild-type mice (left), Tmc1- / - mice (middle), and Tmc1- / - mice injected with Anc80 Tmc1 (right). Cochleas were collected 8 days after injection.

[0101] Figure 31 B is Figure 31 The graph in A shows the recovery rate of the mice. Figure 32 Figure B shows the percentage of functional cells in wild-type mice (left), Tmc1- / - mice (middle), and Tmc1- / - mice injected with Anc80 Tmc1 (right).

[0102] Part 1: Efficient cochlear gene transferis a graph showing the distortion product otoacoustic emission (DPOAE) threshold as a function of stimulation frequency for wild type, Tmcl- / - mice and Tmcl- / - mice injected with Anc80 Tmcl. DETAILED DESCRIPTION

[0103] Because sensory cells of the adult mammalian cochlea lack the ability to self-repair, current treatment strategies, depending on the level and exact location of the lesion, rely on amplification (hearing aids), better sound propagation (middle ear prostheses / active implants) or direct neuronal stimulation (cochlear implants) to compensate for permanent damage to the primary sensory hair cells or spiral ganglion neurons, which form the auditory nerve and transmit acoustic information to the brain. Although these approaches have revolutionized the field, they are still far from optimal in restoring the complex human hearing function that is important for modern life. Specifically, major problems still include limited frequency sensitivity, unnatural sound perception and limited speech discrimination in noisy environments.

[0104] Transferring therapeutic genes to the cochlea has been considered to further improve the current standard of care ranging from age-related and environmentally caused hearing loss to genetic forms of deafness. Over 300 genetic loci are associated with genetic hearing loss, over 70 causative genes have been described (Parker & Bitner-Glindzicz, 2015, Arch. Dis. Childhood, 100:271-8). Therapeutic success of these approaches clearly depends on safe and efficient delivery of exogenous gene constructs to the relevant therapeutic cellular targets in the cochlear organ of Corti (OC).

[0105] The OC contains two types of sensory hair cells: IHCs, which transduce mechanical information carried by sound into electrical signals to the neuronal structures, and OHCs, which are used for amplification and tuning of the cochlear response, a process required for complex hearing function. Other potential targets in the inner ear include spiral ganglion neurons, pillar cells of the spiral limbus, which are important for maintaining the adjacent tectorial or supporting cells, which have a protective function and can be triggered to transdifferentiate into hair cells up to the early neonatal stage.

[0106] Injection into the cochlear duct, which is filled with high-potassium endolymph, can directly access hair cells. However, this change in the microfluidic environment can disrupt the inner ear cochlea, increasing the risk of injection-related toxicity. The middle ear can access the cochlear duct, the scala tympani and the scala vestibuli space filled with perilymph through the round or oval window membrane (RWM). The RWM is the only non-osseous opening into the inner ear, is relatively easy to access in many animal models, and the administration of viral vectors using this route is well tolerated. In humans, cochlear implantation usually relies on the insertion of a surgical electrode through the RWM.

[0107] Previous studies evaluating AAV serotypes in organotypic cochlear explants and in vivo inner ear injections showed that they only rescued partial hearing in a mouse model of genetic deafness. Surprisingly, adeno-associated viruses (AAVs) containing parental AAV capsid proteins efficiently transduced OHCs. This finding overcame low transduction rates that limited the successful development of cochlear gene therapy using conventional AAV serotypes. AAVs containing parental AAV capsid proteins as described herein provide a valuable platform for delivering inner ear genes to IHCs and OHCs, as well as other inner ear cell arrays that are damaged by genetic hearing and balance disorders. In addition to providing high transduction rates, AAVs containing parental AAV capsid proteins as described herein showed similar safety profiles in mice and non-human primates after systemic injection and were antigenically distinct from circulating AAVs, which provided potential benefits in limiting pre-existing immunity that limits the effectiveness of conventional AAV vectors.

[0108] However, the compositions and methods described herein are capable of efficiently delivering nucleic acids to cells, particularly in the inner ear, such as in the cochlea (or cells of the cochlea or cochlear cells). As used herein, inner ear cells refer to, but are not limited to, inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis. Supporting cells refer to non-excitable cells in the ear, such as cells that are not hair cells or neurons. An example of a supporting cell is a Schwann cell.

[0109] Delivery of one or more nucleic acids described herein to inner ear cells can be used to treat any number of genetic or acquired hearing disorders, which are generally defined by partial hearing loss or complete deafness. The methods described herein can be used to treat hearing disorders such as, but not limited to, recessive deafness, dominant deafness, Usher syndrome, and other syndromic deafness, and hearing loss due to trauma or aging.

[0110] As used herein, adeno-associated viruses (AAVs) containing parental AAV capsid proteins are particularly effective at delivering nucleic acids (e.g., transgenes) to inner ear cells, and one class of parental AAV capsid proteins that is particularly effective is a parental scaffold called Anc80, as shown in SEQ ID NO: 1. One particular parental capsid protein in the Anc80 parental capsid protein classification is Anc80-0065 (SEQ ID NO: 2), however, a number of other parental capsid proteins in the Anc80 parental capsid protein classification are described in WO 2015 / 054653.

[0111] The Anc80 capsid protein-containing viruses described herein can be used to deliver a variety of nucleic acids to cells of the inner ear. Nucleic acid sequences that are typically delivered to cells for the purpose of expression are referred to as transgenes. Representative transgenes that can be delivered to and expressed in cells of the inner ear include, but are not limited to, transgenes encoding neurotrophic factors (e.g., glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), or heat shock protein (HSP)-70), immunomodulatory proteins, or anti-oncogenic transcripts. In addition, representative transgenes that can be delivered to and expressed in cells of the inner ear also include, but are not limited to, transgenes encoding antibodies or fragments thereof, antisense, silencing, or long non-coding RNA species, or genome editing systems (e.g., genetically modified zinc finger nucleases, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)).In addition, representative transgenes that can be delivered to and expressed in inner ear cells include nucleic acids referred to as ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP. Descriptions and definitions of the nomenclature used in this application can be found at hereditaryhearingloss.org / on the World Wide Web.

[0112] Expression of the transgene can be directed by a native promoter of the transgene (e.g., a promoter naturally occurring in the transgene coding sequence) or expression of the transgene can be directed by a heterologous promoter. For example, any of the transgenes described herein can be used with its native promoter. Alternatively, any of the transgenes described herein can be used with a heterologous promoter. As used herein, a heterologous promoter refers to a promoter that does not naturally direct expression of the sequence (i.e., is not found in nature with the sequence). Representative heterologous promoters that can be used to direct expression of any of the transgenes described herein include, for example, a cytomegalovirus (CMV) promoter, a chicken beta actin (CBA) promoter, a synthetic CASI promoter, a phosphoglycerate kinase (PGK) promoter, an elongation factor (EF)-1 promoter, an alpha nicotine receptor promoter, a dynein promoter, a growth factor independent (GFI1) promoter, and a vesicular glutamate transporter 3 (VGLUT3) promoter. In addition, a promoter that naturally directs expression of one of the above reference transgenes (e.g., a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, or an ATOH1 promoter) can be used as a heterologous promoter to direct expression of a transgene.

[0113] Methods of making transgenes for packaging into viruses containing Anc80 capsid proteins are well known in the art and utilize routine molecular biology and recombinant nucleic acid techniques. In one embodiment, a construct is provided that comprises a nucleic acid sequence encoding an Anc80 capsid protein and the construct carries a transgene flanked by suitable inverted terminal repeats (ITRs) that allow the transgene to be packaged in an Anc80 capsid protein.

[0114] A transgene can be packaged in an AAV containing Anc80 capsid proteins using, for example, a packaging host cell. The components of the viral particle (e.g., rep sequences, cap sequences, inverted terminal repeat (ITR) sequences) can be introduced into the packaging host cell transiently or stably using one or more constructs as described herein. The virus described herein contains at least an Anc80 capsid protein; the other components of the viral particle (e.g., rep sequences, ITR sequences) can be based on a parental sequence or a contemporary sequence. In some cases, for example, the entire viral particle can be based on a parental sequence. Such viruses can be purified using routine methods.

[0115] It will be appreciated that one or more than one transgene can be delivered to the inner ear. It will also be appreciated that more than one transgene can be delivered to the inner ear using a single AAV vector comprising Anc80 capsid proteins or using multiple AAV vectors comprising Anc80 capsid proteins.

[0116] In general, as used herein, "nucleic acid" can include DNA and RNA, and can also include nucleic acids containing one or more nucleotide analogs or backbone modifications. Nucleic acids can be single-stranded or double-stranded, depending on their intended use. Nucleic acids that can be used in the methods described herein can be identical to known nucleic acid sequences, or nucleic acids that can be used in the methods described herein can be sequences that are different from such known sequences. For example, a nucleic acid (or encoded polypeptide) can have at least 75% sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a known sequence.

[0117] In calculating the percent sequence identity, the two sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment with a second sequence). The number of identical matches is then ascertained, and the percent sequence identity can be calculated as the number of identical matches divided by the length of the alignment (i.e., the number of nucleotides or amino acids which are aligned) and multiplied by 100. It will be appreciated that the length of the alignment can be one of the sequences or a portion of one of the sequences up to the size of the shortest sequence. It will also be appreciated that a single sequence can be compared to one or more other sequences, and thus have different percent sequence identity values on each comparison.

[0118] ClustalW and default parameters to determine percent sequence identity between two or more sequences, which enables alignment of nucleic acid or polypeptide sequences over their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW calculates the optimal match between a query sequence and one or more target sequences and aligns them to determine identity, similarity, and difference. Gap(s) of one or more residues can be inserted into the query sequence, the target sequence, or both to maximize sequence alignment. For pairwise alignment of nucleic acid sequences, the default parameters are used (i.e., word size: 2; window size: 4; scoring method: percent; top diagonal line number: 4; and gap penalty: 5); for alignment of multiple nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transition: yes. For pairwise alignment of polypeptide sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percent; top diagonal line number: 5; and gap penalty: 3. For multiple alignment of polypeptide sequences, the following parameters are used: weight matrix: BLOSUM (Block Substitution Matrix); gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; and residue specific gap penalties: on. ClustalW is available on the Baylor College of Medicine Search Launcher website or the European Bioinformatics Institute website on the World Wide Web.

[0119] Changes can be introduced into the nucleic acid sequence, which, if the nucleic acid sequence is a coding sequence, can result in changes to the amino acid sequence of the encoded polypeptide. Changes can be introduced into the nucleic acid encoding sequence, for example, using mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis) or by chemically synthesizing the nucleic acid molecule with such changes. Such nucleic acid changes can result in conservative and / or non-conservative amino acid substitutions at one or more amino acid residues. A “conservative amino acid substitution” refers to the substitution of one amino acid residue for another residue with similar side chain (see, e.g., Dayhoff et al., (1978, in Atlas of Protein Sequence and Structure, 5 (Suppl. 3): 345-352), which provides a table of amino acid substitutions frequencies), and a non-conservative substitution refers to the substitution of an amino acid residue for another residue that does not have a similar side chain.

[0120] Nucleic acids can be included in constructs, which can also be referred to as vectors or plasmids. Constructs are commercially available or can be produced using recombinant techniques that are routine in the art. Constructs containing nucleic acids can have expression elements that direct and / or modulate expression of such nucleic acids, and can also contain sequences such as those used to maintain the construct (e.g., origin of replication, selectable marker). Expression elements are well known in the art and include, for example, promoters, introns, enhancer sequences, response elements, or inducible elements.

[0121] Methods of delivering nucleic acids to cells of the inner ear

[0122] Methods of delivering nucleic acids to cells are generally known in the art, and methods of delivering viruses (which can also be referred to as viral particles) containing transgenes to cells of the inner ear in vivo are described herein. As used herein, about 10 8 to about 10 12 viral particles can be administered to a subject, and the viruses can be suspended in a suitable volume (e.g., 10 μL, 50 μL, 100 μL, 500 μL, or 1000 μL) of, for example, artificial perilymph fluid.

[0123] Any number of mechanisms can be used to deliver viruses containing transgenes as described herein to cells of the inner ear (e.g., cells in the cochlea). For example, a therapeutically effective amount of a composition comprising viral particles containing one or more different types of transgenes as described herein can generally be injected through the round window or oval window in a relatively simple (e.g., outpatient) procedure. In some embodiments, a composition comprising a therapeutically effective amount of viral particles containing one transgene, or a collection of one or more different viral particles, wherein each particle in each collection contains the same type of transgene, but wherein each collection of particles contains a different type of transgene than the other collections, as described herein can be delivered to an appropriate location in the ear during a surgical procedure (e.g., an endolymphatic fenestration or canalostomy).

[0124] In addition, delivery vehicles (e.g., polymers) can be used to facilitate the transfer of a pharmaceutical agent across the tympanic membrane and / or through the round window, and any such delivery vehicles can be used to deliver viruses described herein. See, e.g., Arnold et al., 2005, Audiol. Neurootol., 10:53-63.

[0125] The compositions and methods described herein enable efficient delivery of nucleic acids to cells of the inner ear (e.g., cochlear cells). For example, the compositions and methods described herein enable delivery of at least 80% (e.g., at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of a transgene to and expression in inner hair cells, and at least 80% (e.g., at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of a transgene to and expression in outer hair cells.

[0126] As shown herein, expression of a transgene delivered using an AAV containing Anc80 capsid proteins can enable regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons (e.g., Atohl, NF2) such that hearing or vestibular function is restored for an extended period of time (e.g., months, years, decades, a lifetime).

[0127] As discussed in WO 2015 / 054653, AAVs containing Anc80 capsid proteins can be characterized using a seroprevalence and / or a degree of neutralization relative to a conventional AAV (i.e., an AAV not containing Anc80 capsid proteins). Seroprevalence, as understood in the art, refers to the proportion of subjects in a seropositive population (i.e., who have been exposed to a particular pathogen or immunogen), and is calculated as the number of subjects in a population who have produced antibodies against a particular pathogen or immunogen divided by the total number of individuals in the population under consideration. Determining the seroprevalence of a virus is routinely performed in the art and generally involves determining the positive rate of one or more antibodies in samples (e.g., blood samples) from a particular population of individuals using immunoassays. In addition, several methods can be used to determine the degree of neutralizing antibodies in serum samples. For example, a neutralizing antibody assay measures the titer of an experimental sample containing an antibody concentration that neutralizes 50% or more of an infection compared to a control sample that does not contain the antibody. See also, Fisher et al. (1997, Nature Med., 3:306-12) and Manning et al. (1998, Human Gene Ther., 9:477-85). Representative conventional AAVs include, but are not limited to, AAV8 (or a virus containing AAV8 capsid proteins) and / or AAV2 (or a virus containing AAV2 capsid proteins).

[0128] Usher Syndrome

[0129] Human Usher syndrome (USH) is a rare genetic disease that can lead to deafness and blindness. As an autosomal recessive genetic disease, it affects 16,000 to 20,000 people in the United States and causes 3 to 6% of early childhood deafness, depending on the severity of the symptoms. Usher syndrome is divided into three clinical subtypes (USH-1, -2, and -3) according to the severity of the symptoms. USH1 is the most severe form. Patients affected by USH1 have congenital bilateral profound sensorineural hearing loss, vestibular areflexia, and prepubertal retinitis pigmentosa (progressive, bilateral, symmetric degeneration of the retinal rod and cone function). Unless equipped with a cochlear implant, individuals generally do not have the ability to generate speech. Although there is currently no biological treatment for Usher patients, early reintroduction of wild-type defective genes can reverse the disease.

[0130] Six Usher genes are associated with USH1: MY07A (myosin 7a), USH1C (harmonin), CDH23 (cadherin 23), PCDH15 (pre-cadherin 15), SANS (sans), and CIB2 (calcium and integrin binding protein 2). These genes encode proteins involved in the morphogenesis of hair bundles in the inner ear and are part of an interacting group (see, e.g., Mathur & Yang, 2015, Biochim. Biophys. Acta, 1852:406-20). Harmonin is at the center of the USH1 interacting group, where it binds to other Usher 1 proteins. Harmonin has been proposed to function as a scaffold protein due to its PDZ (PSD-59 95 / Dlg / ZO-1) interaction domain. In vitro binding studies have shown that all other known USH1 proteins bind to the PDZ domain of harmonin, as do two USH2 proteins, usherin and VLGR1. The USH1C gene consists of 28 exons, which encode 10 alternative splice forms of harmonin, divided into three different subclasses (a, b, and c) according to the composition of the protein domains. The three isoforms differ in the number of PDZ protein-protein interaction domains, coiled coil (CC) domains, and proline-serine-threonine (PST)-rich domains.

[0131] USH1 proteins are located at the apical part of the hair cells in the mechanosensory bundle, which is composed of hundreds of stereocilia interconnected by numerous extracellular links. Cadherin 23 and proto-cadherin 15 are products of Usher genes (USH1D and USH1E, respectively) that form tip links located at the distal end of the stereocilia. Harmonin-b binds to CDH23, PCDH15, F-actin and itself. It is found at the stereocilia tips near the insertion point of the tip links in the hair cells and is thought to play a role in transduction and adaptation in the hair cells. Harmonin-b is expressed in the early postnatal stages, but its expression in the cochlea and vestibule decreases around postnatal day 30 (P30). Harmonin-a also binds to cadherin 23 and is found in the stereocilia. A recent report revealed another role for harmonin-a in the synapse, where it limits the availability of the Cav1.3 Ca2+ channel through a ubiquitin-dependent pathway.

[0132] Over the past decade, several mouse models for Usher syndrome have been identified or designed, of which seven affect harmonin. Of these, only one model (Ush1c c.216G>A model) recapitulates the features of human Usher syndrome auditory and retinal defects. Ush1c c.216G>A is a knock-in mouse model that, due to the presence of a similar point mutation found in a French-Acadian USH1C patient cohort, affects expression of all conventional harmonin isoforms. This mutation introduces a cryptic splice site at the end of exon 3 of the Ush1c gene. Use of this cryptic splice site generates a frameshift transcript with a 35 bp deletion and leads to translation of a severely truncated protein that lacks the PDZ, PST, and CC domains. Homozygous c.216AA knock-in mice have severe hearing loss by 1 month of age, while heterozygous c.216GA mice do not have any abnormal phenotypes. Cochlear histological examination of c.216AA mice found bundle disorganization, abnormal cell rows, and loss of inner and outer hair cells in the mid and basal turns at P30.

[0133] In particular, the parental AAV capsid proteins described herein can be used in conjunction with a harmonin transgene to treat patients diagnosed with Usher syndrome-associated deafness (e.g., USH1C-associated deafness) to successfully transduce hair cells and drive expression and correct localization of a harmonin splice form, thereby reintroducing wild-type harmonin. Moreover, the present application has demonstrated that early postnatal round window membrane injection of AAV containing parental AAV capsid proteins as described herein successfully restored auditory and vestibular function in homozygous c.216AA mice. Restoration of auditory function in the injected mice was associated with restoration of mRNA expression of wild-type harmonin-encoding and preservation of bundle morphology and mechanical transduction.

[0134] TMC1 / TMC2

[0135] Over 40 different mutations have been identified in TMC1 that cause deafness. These are subdivided into 35 recessive mutations and 5 dominant mutations. Most of the recessive mutations cause severe, congenital hearing loss (e.g., DFNB7 / 11), but a few cause late-onset, moderate to severe hearing loss. All of the dominant mutations cause progressive hearing loss (e.g., DFNA36), which begins in adolescence. In particular, AAV vectors comprising Anc80 capsid proteins as described herein can be used to deliver a non-mutated (e.g., wild-type) TMC1 sequence or a TMC2 sequence, thereby preventing hearing loss (e.g., further hearing loss) and / or restoring hearing function.

[0136] Conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques are used in accordance with this disclosure, which are within the skill of the art. Such techniques are explained fully in the literature and are exemplified in certain examples below. The following examples further describe the application, but are not limited in scope to the methods and compositions described in the claims.

[0137] Examples

[0138] Example 1 - Adeno-associated virus (AAV) containing parental AAV capsid proteins results in safe and effective cochlear gene

[0139] transfer Example 1A - Histological analysis

[0140] The following methods and materials were used in Example 1.

[0141] Viral vectors

[0142] AAV2 / 1, 2 / 2, 2 / 6, 2 / 8, 2 / 9, and AAV2 / Anc80L65 with a CMV-driven eGFP transgene and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) expression cassette were prepared at the Gene Transfer Vector Core at Massachusetts Eye and Ear (vector.meei.harvard.edu) as previously described (Zinn et al., 2015, Cell Reports, 12:1056-68). The AAV2 / Anc80L65 plasmid reagent was obtained through addgene.com.

[0143] In vitro plant culture

[0144] To assess as described in an earlier publication (Dilwali et al., 2015, Scientific Reports, 5: 18599), a total of 156 ex vivo cochlea explants were prepared using mouse pups of both strains on postnatal day 4. Briefly, after decapitation, the temporal bones of the mice were removed and the cochlea, connected to the region of the spiral ganglion neurons, were dissected out as organotypic explants for culture. Two specimens were obtained from each cochlea, one consisting of the apical portion ("apical") and one of the basal turn ("basal"). For each serotype, a minimum of 4 (CBA / CaJ, 48h), 2 (CBA / CaJ, 48h+5d), 3 (C57BL / 6, 48h) and 2 (C57BL / 6, 48h+5d) basal and apical specimens were inoculated. Specimens that did not maintain the cochlea morphology during culture were discarded. Sample numbers were chosen to show variability in transduction and as a basis for selection for further in vivo evaluation. Using 50 μl of AAV-containing medium (98% Dulbecco's Modified Eagle Medium (DMEM), 1% ampicillin, and 1% N2 supplement for the first 12 hours, plus 1% fetal bovine serum (FBS)), the explants were cultured for 48h. For the 48h+5d condition, the AAV-containing medium was replaced with fresh medium without AAV for an additional 5 days. Human otic epithelium from 4 consenting adult patients undergoing vestibular schwannoma resection was exposed to 10 10 GC AAV 24 hours and cultured in medium for 10 days, after which the tissue was fixed and stained and imaged using phalloidin. Studies were approved by the Surrey Borders NRES Committee London (Health Research Authority), number 11 / LO / 0475. 10 GC AAV 24 hours and cultured in medium for 10 days, after which the tissue was fixed and stained and imaged using phalloidin. Studies were approved by the Surrey Borders NRES Committee London (Health Research Authority), number 11 / LO / 0475.

[0145] Animal models and general methods

[0146] Wild-type C57BL / 6J and CBA / CaJ mice were obtained from Jackson Laboratory (Bar Harbor, ME) and animals of either sex were used in experiments at an estimated 50 / 50 ratio. Group size for each experiment for in vitro and in vivo transduction assays and subsequent endpoints was determined by availability of specimens and technical feasibility. Qualitative validation of observations reported for Anc80 transduction was performed in experiments using different vector lots (human otic tissue transduction was excluded due to the unique and limited nature of the specimens obtained). Statistical analysis of transduction efficiency between serotypes was not performed due to the limited nature of the specimens obtained and the qualitative nature of the results reported.

[0147] CSF and blood sampling

[0148] Cerebrospinal fluid (CSF) was collected from the cisterna magna (Lui & Duff, 2008, J. Visualized Exp., 21 :e960) and intracardial blood was taken by thoracotomy in a terminal procedure. The maximum amount of clear CSF (up to 5 μΐ^) per animal was collected by microcapillary into 60 μΐ^ of PBS, which was subsequently normalized with control PBS before the start of the experiment due to slight differences in initial dilution. Blood samples were obtained in 1.1 mL Z-Gel microtubes (Sarstedt, Numbrecht, Germany) and centrifuged at 8,000 rpm for 8 minutes, and the serum was stored at -80°C together with the CSF samples (in PBS) until further use.

[0149] Figure 1

[0150] After a follow-up period of 5 to 29 days, animals were sacrificed and whole cochlea tissue specimen-embedments were prepared according to previous reports (Sergeyenko et al., 2013, J. Neurosci., 33: 13686-94). Whole cochlea tissue specimen-embedments and outer plants were stained using antibodies against myosin 7A (Myo7A, #25-6790 Proteus Biosciences, Ramona, CA, 1 :400) and beta-tubulin (TuJl, #MMS-435P Biolegend, San Diego, CA, 1 :200), and their respective secondary antibodies (Alexa Fluor 555 anti-mouse antibody and Alexa Fluor 647 anti-rabbit antibody, #A-21422 and #A-21245 Thermo Fisher Scientific, Waltham, MA, 1 :1000) (Dilwali et al., 2015, Scientific Reports, 5: 18599). Slides of the specimens were observed by confocal microscopy. The same experimental settings were used to obtain each image of a given experimental series, and laser intensities were chosen according to the specimen with the strongest eGFP signal to prevent fluorescence saturation. Z-protocol stacks for overview images and magnifications of the organ of Corti and spiral ganglion neuron (SGN) regions were obtained. 3D-reconstruction was performed using AMIRA in order to more accurately determine SGN transfection status.

[0151] Figure 4 and Example 1B - Quantification of eGFP expressionResults in FIG. 6 show the tropism of the 5 serotypes obtained by monitoring AAV expression of eGFP in C57BL / 6 and CBA / CaJ, respectively. It is notable that eGFP expression in cochlear cultures exposed to Anc80 was qualitatively more robust, with apparent expression in many cochlear cell types.

[0152] Figure 1

[0153] For in vitro data, cochleas were manually quantified for the percentage of eGFP-positive inner (IHC) and outer hair cells (OHC) by counting the number of eGFP-positive cells in one or two 100 μm sections at the base and apex of each specimen, respectively, divided by the total number of hair cells. All visible SGNs in the organ of Corti were evaluated for their eGFP expression. The area of the spiral limbus and supporting cells was assessed in a quantitative manner scored from 0 (no expression) to 3 (strongest signal) (corrected for each experimental series as indicated above). Control samples without AAV were used to exclude autofluorescence. The data indicate that Anc80 targets IHC and OHC with an efficiency between 60 and 100% in the apex and base of both mouse strains tested. Anc80 shows consistent and qualitatively brighter IHC and OHC eGFP expression compared to AAV2 Figure 4 and Figure 1 ).

[0154] To control for potential underestimation of the differences in expression onset between different AAVs at the 2-day (early) time point, a longer experiment was performed. A new set of cochleas was transduced under the same conditions, and after 48 h of incubation with AAVs, the medium containing the vector was discarded and replaced with fresh medium to maintain the survival of the cultures for an additional 5 days (referred to as 48 h + 5 d). Similar expression patterns were observed in this longer-term study for AAV2 and Anc80. A modest increase in expression of AAV6, 8, and 9 was noted in CBA / CaJ mice, particularly in the basal turn Figure 4 I, J, and Figure 5 I, J) of FIG. 7. Other cell types were targeted by all serotypes, with the limbus more readily than the supporting cells, followed by SGNs Example 1C - In vivo injections , 6 and 7). Consistently, Anc80 transduction was more efficient and expressed more strongly, as evidenced by brighter eGFP fluorescence.

[0155] Figure 2

[0156] Glass microinjection pipettes were used to inject through the round window membrane (RWM) in mouse pups (P0 to P2). Pipettes were pulled from glass capillary tubes on a P-2000 pipette puller (Sutter Instrument, Novato, CA) and beveled (tip diameter ~20 μm, 28° angle) using a micropeeler beveler (Sutter Instrument, Novato, CA). The surgical site (left mastoid prominence) was anesthetized using EMLA cream (lidocaine 2.5% and prilocaine 2.5%) outside use with a sterile swab. Body temperature was maintained on a 38°C warming pad prior to surgery. Pups were anesthetized by rapid induction of hypothermia by placing in ice / water until loss of consciousness and maintained in this state on a cold plate for 5-10 minutes during surgery. The surgical site was disinfected by swabbing with betadine and repeated swabbing with 70% ethanol three times. An incision behind the ear was made to expose the tympanic bulla, the micropipette was manually advanced through the bulla and overlying fascia, and the RWM was pierced with the micropipette tip. Approximately 1 μL of virus was injected unilaterally into the left ear of 5 (AAV1), 4 (AAV2), 2 (AAV8), 1 (AAV6), 3 (Anc80) C57BL / 6 animals manually over 1 min. To control for factors related to specific vector preparation (e.g., mass and purity), Anc80 results were confirmed in subsequent studies using different vector lots from independent preparations, which confirmed the qualitative results we present here (data not shown). Injections were performed in a non-blinded fashion for each group. Occasionally, injections were inserted too deep, too shallow, or at the wrong angle. If there was visible damage to the middle or inner ear structures, the sample was excluded from further analysis. The success rate of injections ranged from ~50% to ~80%, depending on the level of experience of the injector. After injection, the skin incision was closed using 6-0 black monofilament suture (Surgical Specialties, Wyomissing, PA). Pups were then placed back on the 38°C warming pad for 5-10 min before being returned to the mother for nursing.

[0157] Consistent with previous reports, AAV1 transduced IHCs with moderate to higher efficiency ( Figure 2 A, B). These studies showed that AAV2, 6, and 8 targeted lower numbers of IHCs, with only AAV8 showing roughly equivalent transduction at the apical and basal ( Figure 2 B). Also, consistent with previous reports, OHC transduction was lowest (<5%) for all conventional AAV serotypes tested. However, at doses 20-fold (for AAV1) to 3-fold (for AAV2) lower, Anc80 transduced nearly 100% of IHCs and ~90% of OHCs ( Figure 8 A-C). For all serotypes, at the same dose 1.36 x 1012 Transduction under GC resulted in a large number of IHCs and OHCs being transduced by Anc80, but live cell imaging by epifluorescence microscopy revealed that AAV1, 2, and 8 targeted only a very small number of IHCs and no OHCs were observed to be transduced by these vectors Figure 8

[0158] Subsequently, Anc80-transduced samples were fixed, stained, and imaged by confocal microscopy, which revealed a dose-dependent transduction of hair cells Figure 2 Figure 8 Figure 2 ) indicates that Anc80 has a unique transduction biology compared to other AAVs. Similar levels of Anc80 transduction were found throughout the cochlea, from the base to the apex, in a total of 3 mice injected with Anc80 Figure 8 Figure 8 Figure 9 A low magnification view of the apex of the cochlea (Fig. 1 A) shows strong eGFP expression at a location distal to the injection site. High magnification images of the base reveal that 100% of IHCs and 95% of OHCs were transduced (Fig. 1 B).

[0159] In some animals, strong eGFP expression was found in the contralateral uninjected ear (Fig. 1 C). In mice, the cochlear aqueduct specifically provides a fluid pathway from the perilymph of the cochlea into the CSF, the contralateral aqueduct, and the contralateral cochlea. Therefore, it was also investigated whether Anc80-eGFP injected through the RWM could transduce neurons in the brain. Indeed, cross-sections of the cerebellum showed strong eGFP expression in Purkinje neurons of the cerebellum (Fig. 1 A, B). Figure 10 Figure 3

[0160] Since some forms of genetic deafness also result in vestibular dysfunction, Anc80 can be a useful vector for delivering genes to human vestibular organs. To investigate this possibility, human vestibular epithelium was collected from 4 adult patients who underwent vestibular schwannoma resection; sensory epithelium was placed in culture medium as previously described (Kesser et al., 2007, Gene Ther., 14:1121-31). For transduced samples, Figure 3 Example 1D - Immunological assays

[0161] Figure 10 ​​​​​​​​​

[0162] The antibody titers of Anc80 in CSF and serum were determined by neutralization assay (Zinn et al., 2015, CellReports, 12:1056-68). Heat-inactivated CSF or serum samples (collected as described above) were serially diluted in serum-free medium (Life Technologies, Carlsbad, CA) using 96-well plates, and then neutralized using Anc80-luciferase (10... 6 GC / well was incubated at 37°C for 1 hour. The sample / Anc80-luciferase mixture was then transferred to HEK293 cells, which had been treated with adenovirus the previous day (MOI 20). After incubation at 37°C for 1 hour, diluted serum medium (1 part serum-free, 2 parts serum-containing) was added to each well.

[0163] Two days later, cells were treated with lysis buffer (Promega, Madison, WI) and frozen at -80°C for 30 minutes. Cells were then thawed at 37°C for 15 minutes and subsequently treated with substrate buffer (Tris-HCl, MgCl2, ATP (Life Technologies, Carlsbad, CA), D-luciferin (Caliper Life Sciences, Hopkinton, MA)). The luminescence output was read using a Synergy BioTek Plate Reader (BioTek, Winooski, VT).

[0164] At the level of assay and sampling sensitivity, low levels of neutralization against the vector were detected in the serum of injected mice, but not in CSF. Example 1E - Hair cell electrophysiology (C).

[0165] Figure 2

[0166] The cochlea was excised, encapsulated on a glass coverslip, and observed under an Axio Examiner A1 upright microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 63x water immersion objective and differential interference contrast optics. Electrophysiological recordings were performed at room temperature (22℃–24℃) in standard solutions (in mM) containing 137 NaCl, 5.8 KCl, 10 HEPES, 0.7 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, and 5.6 D-glucose, vitamins (1:100), and amino acids (1:50) (pH 7.4; ~310 mOsm / kg).

[0167] Recording electrodes (3-4 MΩ) were pulled using R-6 glass (King Precision Glass, Claremont, CA) and filled with intracellular solution containing (in mM): 140 CsCl, 5 EGTA-KOH, 5 HEPES, 2.5 Na2ATP, 3.5 MgCl2, and 0.1 CaCl2(pH 7.4; ~280 mOsm / kg). Whole-cell, tight-seal patch-clamp technique was used to record mechanically evoked currents using an Axopatch 200B (Molecular Devices, Sunnyvale, CA). Hair cells were held at -84 mV. Currents were filtered at 5 kHz using a low-pass Bessel filter, digitized at >20 kHz using a 12-bit acquisition board (Digidata 1440A, Molecular Devices, Sunnyvale, CA), and recorded using pCLAMP 10 software (Molecular Devices, Sunnyvale, CA).

[0168] Bundles from IHCs and OHCs were deflected using rigid glass probes mounted on a PICMA chip piezoelectric actuator (Physik Instrumente, Karlsruhe, Germany) driven by a LVPZT amplifier (E-500.00, Physik Instrumente, Karlsruhe, Germany) and filtered at 40 kHz using an 8-pole Bessel filter (Model 3384 filter, Krohn-Hite Corporation, Brockton, MA) to eliminate residual pipette resonance. Rigid glass probes were designed to fit the concave surface of the stereociliary array of whole-bundle recording suitable for IHCs (4-5 pm in diameter) and OHCs (3-4 pm in diameter). For whole-cell electrophysiological recordings at >P10, cochlear tissue was dissected at P5-7 and incubated at 37°C, 5% CO2for up to 30 days using MEM (1X) + GlutaMAX™-I media containing 1% FBS.

[0169] Representative currents evoked by bundle deflection from P7 OHCs and P35 IHCs show no differences in amplitude, sensitivity, or kinetics between eGFP-positive and eGFP-negative control cells Figure 2 D). Fifty-one eGFP-positive and 52 eGFP-negative hair cells were noted from all regions of the cochlea and from ages between 1 week and 5 weeks after exposure to Anc80. In all cases, the responses were indistinguishable from wild type Example 1F - Hearing tests E), demonstrating that Anc80 transduction had no adverse effects on sensory cell function.

[0170] Figure 2

[0171] Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) data were collected as previously described (Askew et al., 2015, Science Translational Med., 7:285ra108). DPOAE is a measure of proper cochlear amplification and tuning and is a sensitive measure of outer hair cell viability (Guinan et al., 2012, Hearing Res., 293:12-20). Stimuli were detected in anesthetized mice at frequencies of 5.6, 8, 11.3, 16, 22.6, and 32 kHz at sound pressure levels ranging from 10 to 90 dB. Four Anc80-injected ears and four uninjected ears, as well as one negative control ear with an injection lesion but no EGFP fluorescence, were analyzed at P28-P30.

[0172] The minimum sound thresholds required to elicit ABRs were plotted (F of Figure 2 ) and the results indicated no difference in thresholds between injected and uninjected ears. Histological analysis revealed strong eGFP fluorescence in all four injected ears (data not shown). In one case, there were no eFGP-positive cells and an elevated ABR threshold (F of Figure 2 ), suggesting that the injection failed and the needle had likely damaged the cochlear duct and caused permanent damage. Despite the robust outer hair cell transduction of Anc80-eGFP, there was no difference in DPOAE thresholds relative to uninjected control ears (G of Example 1G - Rotarod test ). Thus, data from both ABR and DPOAE indicated that RWM injection, Anc80 transduction, and transgene expression in IHCs and OHCs were safe with respect to auditory function.

[0173] Figure 3

[0174] Balance behavior was tested on a rotarod apparatus in 5 C57BL / 6 mice. Mice known to have impaired vestibular function perform poorly on a rotating apparatus (Parker & Bitner-Glindzicz, 2015, Archives Dis. Childhood, 100:271-8). Previous studies have emphasized that this rotarod test can detect balance impairment only when one ear is affected (Fukui & Raphael, 2013, Hearing Res., 297:99-105; Geleoc & Holt, 2014, Science, 344: 1241062). 3 mice were injected at P1 and tested at P36, and 2 non-injected control mice were tested at P79. All mice were tested using the following rotarod protocol. On day 1, mice were placed on the rotarod, which was rotating at 4 RPM, for 5 minutes to train them to balance. On day 2, mice were tested 5 times, with 5 minutes between each test. For each test, the rotarod was accelerated by 1 RPM (Fukui & Rapheal, 2013, Hearing Res., 297:99-105), and the initial speed of the rotarod was 2 RPM. The time until the mouse fell off the apparatus was recorded in seconds.

[0175] Since the perilymph solution of the cochlea is continuous with the perilymph solution of the vestibular labyrinth, it was assessed whether injection of Anc80-eGFP through the cochlear RWM would transduce vestibular sensory organs. Indeed, whole mount preparations of the vestibular epithelium showed strong eGFP expression in type I and type II hair cells of the utricle, a vestibular organ sensitive to gravitational and linear head movements, and in the semicircular canals, which are sensitive to rotational head movements Figure 11 A, B). Thus, to address the safety concern that Anc80 transduction might affect balance, rotarod tests were performed in injected mice that had confirmed vestibular expression, and the results showed that their vestibular function was similar to that of non-injected controls Part 2 - Gene therapy to restore function in a mouse model of Usher syndrome ).

[0176] Example 2 - Mouse model of Usher syndrome

[0177] Example 2A - Scanning electron microscopy (SEM) in a mouse Usher model

[0178] The following methods and materials were used in Example 2.

[0179] Tissue preparation

[0180] Otic capsules and Corti's organs were collected from Ushlc c.216G>A heterozygous or homozygous mutant mice from postnatal day 0 to 8 (P0 to P8) for electrophysiological studies. Postnatal pups were sacrificed by rapid decapitation. The temporal bones were excised and immersed in MEM (Invitrogen, Carlsbad, CA) supplemented with 10 mM HEPES (pH 7.4). The Corti's organs were isolated without the use of enzymes as previously described. The otic capsules were removed after a 10 min treatment with 0.1 mg / ml of protease (Protease XXIV, Sigma). The excised organs were mounted on a circular glass coverslip. A pair of thin glass fibers, previously glued to the coverslip, were placed on the edges of the tissue to stabilize it in a flat position. The tissue was used immediately or stored in culture medium containing 1% fetal bovine serum. Cultures were maintained for 7 to 8 days and the medium was changed every 2 to 3 days, and used for in vitro experiments involving viral vector infection.

[0181] Animals

[0182] Ushlc c.216G>A knock-in mice were obtained from the Louisiana State University Health Science Center. This inbred line in the C57BL6 background previously had age-related hearing loss due to the Cdh23 (Ahl) mutation. Mice were genotyped using toe clips (before P8) or ear punches (after P8) and PCR as previously described (Lentz et al., 2007, Mutat. Res., 616: 139-44). The ratio of male and female mice used was approximately equal for all studies. No randomization pattern was otherwise applied.

[0183] Production of viral vectors

[0184] Total RNA was isolated from the cochlea of c.216AA mutant mice (RNAqueous Micro Kit, Ambion) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The cDNA of trunc-harmonin was amplified by PCR using high fidelity Platinum Taq DNA polymerase (Invitrogen) and the following primers: Trunc-harmonin.F (Kpnl) GAG GTACCA TGG ACC GGA AGG TGG CCC GAG (SEQ ID NO: 9); Trunc-harmoin.RV (BamHI) CAG GAT CCG GAC AAT TTC ATC CCC TAC (SEQ ID NO: 10). The 387 bp PCR product was cloned using the TA Cloning Kit (Invitrogen) and confirmed by sequencing. To generate the GFP fusion construct, the truncated harmonin fragment was subcloned into pEGFP-C1 using Kpnl and BamHI. The NheI-XbaI EGFP::trunc-harmonin cDNA was transferred into an AAV shuttle vector. The custom vector was packaged into AAV1 capsid with AAV2 inverted terminal repeats (ITRs) with the transgene expression cassette driven by the CMV promoter (AAV2 / 1.CMV.EGFP::trunc-harmoin.hGH, 1.92E14 gc / m, BCH).

[0185] Harmonin-a1 and Harmonin-b1 plasmids were prepared from EGFP-tagged constructs previously kindly provided by Lily Zheng and James Bartles (Zheng et al., 2010, J. Neurosci., 30:7187-201) (Department of Cell and Molecular Biology, Northwestern University, Feinberg School of medicine, Chicago, IL) in our laboratory. Harmonin-a1 was originally from mouse kidney and Harmonin-b1 was isolated from mouse cochlear sensory epithelium. Harmonin-a1 construct was further modified to replace the EGFP tag with tdTomato at its N-terminus. Fluorescently tagged and untagged constructs were packaged into AAV vectors. Viral vectors were produced by the Viral Core Laboratory at Boston Children's Hospital and the Gene Transfer Vector Core at Massachusetts Eye and Ear Infirmary. The following vectors were generated: AAV2 / 1.CMV.tdTomato::harmonin-a1 4.33 10^13gc / ml (BCH); AAV2 / 1.CMV.EGFP::harmonin-b1 2.73 564 10^14gc / ml (BCH); AAV2 / 1.CMV.EGFP-harmonin-a1 2.81 10^12gc / ml (MEEI); AAV2 / 1.CMV.EGFP-trunc-harmonin 1.92 10^14gc / ml (BCH); AAV2 / Anc80.CMV.harmonin-a1 1.93 10^12gc / ml (MEEI); AAV2 / Anc80.CMV.harmonin-b1 1.74 10^12gc / ml (MEEI); AAV2 / Anc80.CMV.trunc-harm.WPRE 9.02 567 10^12gc / ml (MEEI). For in vitro experiments, 10 μΐ of concentrated vector was added to 1 ml of MEM supplemented culture medium on freshly dissected tissue in the presence of 1% fetal bovine serum for 24 h. Cultures were then maintained for up to 10 days.

[0186] Round window membrane (RWM) injection

[0187] RWM injections were performed in accordance with an animal experimental protocol #15-01-2878R approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital. Newborn mice were injected with 0.8-1 pl of AAV vector at P0-P1 and P10-P12. P0-P1 mice were first anesthetized using hypothermic exposure and P10-P12 mice were anesthetized using isoflurane. After anesthesia, a postauricular incision was made to expose the otic bulla and make the cochlea visible. Injections were performed through the RWM using a glass micropipette controlled by a micromanipulator (Askew et al., 2015, Sci. Transl. Med., 7:295ral08). The volume of injected material was controlled at -0.02 pl / min and injections were performed for 10 min. Standard postoperative care was employed. Sample size for in vivo studies was determined on a continuous basis to optimize sample size and reduce variance.

[0188] Electrophysiological recordings

[0189] Recordings were performed in standard artificial perilymph solution (in mM): 144 NaCl, 0.7 NaH2P04, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 5.6 D-glucose, and 10 HEPES-NaOH, adjusted to pH 7.4 and 320 mOsmol / kg. Vitamins (1:50) and amino acids (1:100) were added using a concentrate (Invitrogen, Carlsbad, CA). Hair cells were observed from the apical surface using an upright Axioskop FS microscope (Zeiss, Oberkochen, Germany) fitted with a 63x water-immersion objective and differential interference contrast optics. Recording pipettes (3-5 MΩ) were pulled using borosilicate capillary glass (Garner Glass, Claremont, CA) and filled with intracellular solution (in mM): 135 KCl, 5 EGTA-KOH, 10 HEPES, 2.5 K2ATP, 3.5 MgCl2, 0.1 CaCl2, pH 7.4. Currents were recorded using whole-cell voltage-clamp at room temperature at a holding potential of -64 mV. Data were acquired using an Axopatch Multiclamp 700A or Axopatch 200A (Molecular devices, Palo Alto, CA), filtered with a low-pass Bessel filter at 10 kHz, digitized by a 12-bit acquisition board (Digidata 1322) at >20 kHz, and using pClamp 8.2 and 10.5 (Molecular Devices, Palo Alto, CA). Data were analyzed offline using OriginLab software and are expressed as mean ± standard deviation unless otherwise stated.

[0190] Statistical analysis

[0191] At each time point, the test and control vectors were evaluated in at least three mice per group to ensure reproducibility. Sample sizes are noted in the figure legends. All animals that successfully underwent RWM injection were included in the study analysis. Those animals that did not successfully inject were excluded from the mean, but included in the figure legends for full disclosure. Injection success was determined by ABR recovery (threshold > 90 dB SPL). Statistical analysis was performed using Origin 2016 (OriginLab Corporation). Data are presented as mean ± standard deviation (SD) or standard error of the mean (SEM) as described in the text and figure legends. Significance of differences between means was determined using one-way analysis of variance (ANOVA).

[0192] Figure 12

[0193] Corti's organs of control and mutant mice were prepared for SEM at P7, P18, and ~P42 (6 weeks). P18 SEM was performed in collaboration with Dr. Edwin Rubel at the University of Washington. The inner ear was fixed overnight at 4°C in 0.1 M sodium phosphate buffer containing 4% glutaraldehyde. The next day, the specimen was washed three times in 0.1 M sodium phosphate buffer (PB) and post-fixed in 0.1 M PB containing 1% osmium tetroxide for 30 min on ice. The specimen was then washed in 0.1 M PB and dehydrated through a graded ethanol series: 35%, 70%, 95%, and 100% (x2). The sample was critical point dried, mounted on a SEM stub, and coated with Au / Pd sputter. SEM analysis was performed using a JEOL JSM-840A scanning electron microscope. Similar preparations were performed for P8 and 6-week stages. The outer plant of the Corti's organ was fixed in 0.1 M cacodylate buffer (Electron Microscopy Sciences) containing 2.5% glutaraldehyde supplemented with 2 mM CaCl2for 1 h at room temperature. The specimen was dehydrated through an acetone series, critical point dried using liquid CO2, sputter coated with 4-5 nm of platinum (Q150T, Quorum Technologies, United Kingdom), and observed using a field emission scanning electron microscope (S-4800, Hitachi, Japan).

[0194] Homozygous c.216AA mutant mice are deaf and show vestibular impairment characterized by circling and head shaking behavior. Previous work (Lentz et al., 2010, Dev., Neurobiol., 70:253-67) described significant inner and outer hair cell degeneration at the base of the cochlea at P30. At 1 month of age, degeneration and hair cell death were also observed at the mid-turn, but the apical region of the organ remained well preserved. Assuming that hair cell degeneration gradually occurs during development of the inner ear organ, to assess hair cell survival at early stages, SEM analysis of the Corti's organ was performed at P8 and P18. At these ages, outer hair cells (OHCs) and inner hair cells (IHCs) were preserved and their bundles well oriented in heterozygous c.216GA mice ( Figure 19 A-C, G, I and Figure 12 A-C, K). However, at both ages analyzed, homozygous c.216AA mice showed significant disorganization of the hair bundles throughout the length of the Corti's organ ( Figure 19 D-F, H, J-L and Figure 12 D-J, L). At P8, IHC bundles were slightly disorganized in the basal, mid and apical regions ( Figure 12 D-F, J). Many IHC bundles presented a wavy pattern and the rows of stereocilia were slightly disorganized ( Figure 12 J). Although many OHCs of c.216AA mutant mice had well preserved hair bundles ( Figure 12 H, K), obvious fragmented and disorganized hair bundles were sporadically observed along the organ ( Figure 19 D-F, L). Despite the presence of most hair cells as previously reported (Lentz et al., 2013, Nat. Med., 19:345-50), the disruption was more pronounced at P18 ( Figure 18 D-F).

[0195] To assess the morphology of the hair bundles in mice using harmonin-b1 gene therapy, temporal bones of 6-week-old untreated (or non-injected) and treated (or injected) mice were prepared for SEM analysis. Untreated c.216AA mice showed severe hair cell loss in the basal and mid regions of the organ ( Figure 18In the basal region, OHCs were mostly absent in the first row and sporadically present in the second and third rows. In the central region of the organ, OHCs were also largely absent in the first row. A milder phenotype was observed in the apical region. High-magnification SEM also revealed severe tuft disintegration of hair follicles throughout the entire length of the organ in c.216AA mutant mice. Notably, in 6-week-old c.216AA mice, hair follicles were not observed in the typical stepwise structure where all three rows of cilia were preserved. In contrast, hair cells in c.216AA mice showed disordered tufts, with retracted cilia along the first row, and the second row was an abnormal and very well-preserved row. In contrast, reduced hair cell loss and normal hair follicles were observed in c.216AA mice treated with harmonin-b1. Hair cell estimation was performed by estimating the presence or absence of hair follicles in a typical field of view.

[0196] Data showed that the number of hair cells from the base to the top of the organ was remarkably well preserved in injected mice, ranging from 40% to 79% at the base, 68% to 95% in the middle, and 93% to 99% at the top (n=1824 cells from the ears of n=4 c.216AA mice and n=792 cells from the ears of n=2 salvaged c.216AA mice). Although abnormal hair tufts were still evident in mice injected with harmonin-b1, most tufts had three rows of static cilia and their morphology was almost indistinguishable from that of their heterozygous controls. Example 2B - FM1-43 imaging in a Usher mouse model ).

[0197] Figure 13

[0198] 5 μmol of FM1-43 (Invitrogen) was prepared by diluting with extracellular recording solution and applied to tissue for 10 seconds. The tissue was then washed three times with the extracellular recording solution to remove excess dye and prevent uptake via endocytosis. After 5 minutes, intracellular FM1-43 was imaged using a Zeiss Axioscope FS equipped with an epifluorescence light source, differential interference contrast optics, and an FM1-43 filter set (Chroma Technologies) through immersion 20x, 40x, and 63x objectives. Images were captured in 16-bit format after background fluorescence subtraction using a CCD camera and an Argus-20 image processor (Hamamatsu). All acquired images were analyzed offline using Adobe Photoshop or Image-J software, maintaining the same gain and contrast settings.

[0199] To assess hair cell function in the early stages, the uptake of FM1-43 in the inner ear organs immediately after dissection at P4 was analyzed. Following brief application (<10 seconds), FM1-43 infiltrated hair cells with functional mechanosensitive channels. Uniform FM1-43 uptake was observed in the hair cells of c.216GA mice. Figure 13 However, the uptake levels of OHC in c.216AA mice differed, indicating that some, but not all, cells retained functional transduction pathways. Figure 13 (B). Similar observations were made along the entire length of the cochlea. No differences in tone quality distribution were found. FM1-43 uptake in the IHC of c.216AA mice was also reduced in the first week after birth (data not shown). FM1-43 uptake was also assessed in the follicular hair cells of mutant mice. Interestingly, in c.216AA mutant mice, uptake at P6 was limited to areas outside the grooves, indicating a lack of mechanosensitive channel opening in hair cells in the groove region at rest. Example 2C - Mechanical stimulation in a Usher mouse model (C, D).

[0200] Figure 12

[0201] OHC and IHC: Mechanical stimulation was delivered via a rigid glass probe mounted on a 524-dimensional PICMA chip piezoelectric actuator (Physik Instruments, Waldbronn, Germany) driven by a 400mA ENV400 amplifier (Piezosystem Jena Germany). The probe tip was fired and polished (Fire polisher, H602, WorldPrecision Instruments Inc., Sarasota, FL) to suit the resting ciliary tract (Stauffer & Holt, 2007, J. Neurophysiol., 98:3360-9). Deflection was induced by applying a voltage step, filtered at 50 kHz using an 8-pole Bessel filter (Khron-Hite, 528 Brockton, MA) to eliminate residual pipette resonance. Hair cell deflection was monitored using a C2400CCD camera (Hamamatsu, Japan). The movement of the stimulation probe within ±2 μm of its resting position was corrected using a voltage step. Record video images of the probe to confirm the absence of off-axis motion and correct probe movement (spatial resolution ~4nm). The rise time of 10-90% of probes is ~20μsec.

[0202] VHC: Mechanical stimuli were delivered via a rigid glass probe mounted on a piezoelectric bimorph element. Coupling was performed by gently aspirating the motile hair bundles into the stimulus pipette. Deflections were induced by applying a voltage step to the piezoelectric device, which consisted of two bimorphs mounted in series and directly coupled to the stimulus probe. Voltage steps were controlled by pClamp 8.0 software and filtered with an 8-pole Bessel filter (Khron-Hite, Brockton, MA) at 1 kHz. Bundle deflections were monitored using a C2400 CCD camera (Hamamatsu, Japan). Prior to experiments, the motion of the stimulus probe was calibrated to be near its resting position (± 2 μm).

[0203] During the first postnatal period, the auditory and vestibular epithelia retain mechanosensitive hair cells. Included are some hair cells with relatively normal morphology Figure 13 ). In the organ of Corti, recordings were obtained from the middle and apical turns of the c.216AA mouse cochlea from P3 to P6 from hair cells with apparently normal bundles and from hair cells with more severely disrupted bundles. In the c.216AA mutant, OHCs retain mechanosensitivity, but their responses are significantly reduced in amplitude, from ~63% to 170 ± 80 pA (n = 24; p < 0.001, Figure 13 Wide response amplitudes were observed in OHCs of c.216AA mice, ranging from 31 to 292 pA. Significant differences were observed when data were grouped according to bundle morphology (p < 0.01): currents evoked in mutant hair cells from severely disorganized bundles were smaller than those evoked in mutant cells that more closely resembled normal bundles, 120 ± 65 pA (n = 9) and 201 ± 74 pA (n = 15), respectively. Despite the reduced current amplitudes, the responses of the hair cells to mechanical displacement retained similar properties to those of the heterozygous c.216GA mice. Stimulus response [I(X)] curves were fitted with a second order Boltzmann equation (F of Figure 20 and the operating range (10-90%) was determined using the fit (B of Figure 13 No significant differences in operating range were observed between OHCs recorded from c.216GA and c.216AA (p = 0.054). Similarly, although the bundles of IHCs from c.216AA mutant mice were observed to be mildly disrupted under DIC microscopy, the transduction currents were significantly reduced at P6 Figure 20E, F, G). The maximum conductance current (P6-P7) in hybrid c.216GA IHCs averaged 587 ± 96 pA (n = 21) at a holding potential of -64 mV, but was reduced by 46% in c.216AA IHCs to 316 ± 127 pA (n = 19; p < 0.001). The operating range was significantly reduced in IHCs of c.216AA mutant mice (p < 0.01) Figure 20 G).

[0204] Adaptation was defined as the reduction in conductance current in the presence of a constant beam deflection, which was also present in c.216AA mutant mice. Adaptation kinetics were analyzed using a bi-exponential fit to determine fast and slow components. Although both components were slowed in c.216AA mutant IHCs and OHCs, only the difference in the slow component was significant (p < 0.05 in OHCs and p < 0.001 in IHCs; Figure 20 C, D, H, I). On the other hand, the degree of adaptation measured at Popen= 0.5 was significantly lower in OHCs and IHCs of c.216AA than in hair cells of c.216GA Figure 13 E, J; p < 0.001). Altogether, these results indicate that the mechanosensitivity is slightly impaired in inner and outer hair cells of c.216AA mice and importantly that both cell types survive during the first postnatal week, a prerequisite for gene therapy and restoration of cell function.

[0205] A reduction in the mechanical conductance current was also observed in vestibular hair cells in c.216AA mice. In areas outside the strial groove, the current was significantly reduced from 231 ± 53 pA (n = 8, P6-P7) in c.216GA to 109 ± 30 pA (n = 9, P5-P7) in c.216AA (p < 0.001) Figure 13 E, F, H). In the strial area, only very small currents or no current were recorded (6 ± 13 pA, n = 6, P5-P7), which is consistent with the lack of FM1-43 uptake in this area (see below; Figure 15 C, D). Although the oval recess hair bundle appeared largely intact by DIC microscopy, the conductance current of hair cells outside and in the strial groove was significantly reduced or absent, respectively. Thus, these results indicate that the transduction apparatus is correctly assembled and targeted in mutant mice, except in the strial area, but the number of functional complexes is reduced in newborn mice.

[0206] Next, the function of c.216AA hair cells exposed to AAV vectors driving expression of harmonin was evaluated. To enhance the possibility of functional rescue using exogenous harmonin, untagged harmonin-al or harmonin-bl coding sequences driven by the CMV promoter were packaged into AAV capsid proteins called Anc80 (Zinn et al., 2015, Cell Rep., 12:1056-68). As shown herein, Anc80 capsid proteins transduce 100% of IHCs and 80-90% of OHCs in vivo. It is hypothesized that harmonin-b is required for mechanical transduction in IHCs and OHCs and is necessary for auditory function in both cell types. RWM injections of AAV2 / Anc80.CMV.harmonin-bl (0.8 μΐ, 1.9 x 1012gc / ml) and a mixture of AAV2 / Anc80.CMV.harmonin-al (1.7 x 1012gc / ml) + AAV2 / Anc80.CMV.harmonin-bl (0.5 μΐ + 0.5 μΐ) alone and in combination were performed and mechanical transduction responses were evaluated 2 weeks post-treatment.

[0207] Prior to cochlear ossification, tissue was extracted at P5-P6 and maintained in culture for 10 days. Although mature OHCs (>P10) cannot survive under ex vivo recording conditions, stable electrophysiological recordings were obtained from IHCs at a time equivalent to P14-P16. Results are shown in Figure 15 Although IHCs from non-injected mice showed a significant reduction in transduction current at P16 (79 ± 43 pA, n = 8), a clear recovery of sensory transduction was evident in AAV-treated mice. A significant recovery was observed at P1 in injected mice (***P < 0.001) with an average maximum transduction of 388 ± 66 pA (n = 15) and 352 ± 28 pA (n = 7; Example 2D - Confocal imaging in a Usher mouse model using harmonin-bl alone. Co-injection of harmonin-bl and harmonin-al did not significantly change the level of recovery. These results indicate that delivery of exogenous harmonin-bl by RWM injection at an early stage is able to restore mechanical transduction in IHCs.

[0208] Figure 14

[0209] To prepare tissues for confocal imaging from postnatal mice P0-P8, fixation was performed using 4% paraformaldehyde (PFA) for 15 min. Triton 0.01% was used for permeabilization and Alexa Fluor phalloidin (Invitrogen, 1 / 200) was used for counterstaining to label actin filaments. Images were acquired on a LSM700 Zeiss confocal microscope. In slightly older mice (4 to 8 weeks), the temporal bones were removed after sacrifice and placed in 4% PFA for 1 hour, followed by decalcification using 120 mM EDTA for 24 to 36 hours. The sensory epithelium was then isolated and injected for immunostaining as described above. Mouse anti-CTBP2 (BD bioscience #612044, 1 / 200) was used for incubation for 48 hours and Alexa Fluor goat anti-mouse (1 / 200) was used for counterstaining overnight at 4°C to label ribbon synapses. Images were acquired using a Zeiss LSM 710 laser confocal microscope (IDDRC Imaging Core grant P30 HD18655) and processed using Zeiss LSM Image Browser 4.2.

[0210] Previous work revealed that harmonin is expressed in sensory hair cells in two alternative splice forms. To assess the ability of AAV vectors to drive expression of exogenous harmonin splice forms, the otocysts and organ of Corti from newborn c.216AA and wild-type (C57BL / 6J) mice were exposed to AAV2 / 1 vectors encoding eGFP fused to the N-terminus of harmonin-b1 (eGFP::harmonin-b1) or tdTomato fused to the N-terminus of harmonin-a1 (tdTomato::harmonin-a1). Vectors were applied in vitro or in vivo by RWM injection (1 μΐ) at PI. When applied in vitro, P0-P1 tissues were incubated with vectors for 24 hours and maintained in culture for 1 week. Confocal images indicated that hair cells of wild-type, c.216GA and c.216AA mice were successfully transduced with both eGFP::harmonin-b1 and tdTomato::harmonin-a1 (Figures 1A-C, E). Figure 14 The eGFP::harmonin-b1 signal was apparent at the tip of the stereocilia in VHC (Figures 1A), IHC and OHC (Figures 1B, C). The tdTomato::harmonin-a1 signal was detected in the OHCs and IHCs of the basal turn of the cochlea of wild-type mice (Figures 1D, E). Figure 14 The eGFP::harmonin-b1 signal was apparent at the tip of the stereocilia in VHC (Figures 1A), IHC and OHC (Figures 1B, C). The tdTomato::harmonin-a1 signal was detected in the OHCs and IHCs of the basal turn of the cochlea of wild-type mice (Figures 1D, E). Figure 14 The eGFP::harmonin-b1 signal was apparent at the tip of the stereocilia in VHC (Figures 1A), IHC and OHC (Figures 1B, C). The tdTomato::harmonin-a1 signal was detected in the OHCs and IHCs of the basal turn of the cochlea of wild-type mice (Figures 1D, E). Figure 14 The eGFP::harmonin-b1 signal was apparent at the tip of the stereocilia in VHC (Figures 1A), IHC and OHC (Figures 1B, C). The tdTomato::harmonin-a1 signal was detected in the OHCs and IHCs of the basal turn of the cochlea of wild-type mice (Figures 1D, E). Figure 14 The eGFP::harmonin-b1 signal was apparent at the tip of the stereocilia in VHC (Figures 1A), IHC and OHC (Figures 1B, C). The tdTomato::harmonin-a1 signal was detected in the OHCs and IHCs of the basal turn of the cochlea of wild-type mice (Figures 1D, E). Example 2E - Auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE)harmonin-a and -b were co-localized in the P7 otocysts (Fig. 6E), but not in the P7 utricles (data not shown).

[0211] The localization of the exogenous fusion constructs is consistent with previous work in which harmonin-b was localized to the distal end of the stereocilia, near the tip link insertion, and harmonin-a was localized to the synapse.

[0212] Figure 23

[0213] ABRs and DPOAEs were recorded from mice anesthetized with xylazine (5-10 mg / kg i.p.) and ketamine (60-100 mg / kg i.p.). Subcutaneous needle electrodes were inserted into the skin: a) dorsally between the two ears (reference electrode); b) posterior to the left pinna (recording electrode); and c) dorsally on the animal’s hips (ground electrode). The channel at the base of the pinna was trimmed to expose the ear canal. For ABR recordings, a 5-msec tone was presented to the ear canal and a hearing device (EPL Acoustic system, MEEI, Boston). The responses were amplified (10,000x), filtered (0.1-3 kHz), and averaged using a PC-based data acquisition system (EPL, Cochlear function test suite, MEEI, Boston). Sound levels were raised from 0 to 110 dB sound pressure level (SPL) in 5- to 10-dB steps. In each level, 512 to 1024 responses were averaged (alternating with stimulus polarity) after “artifact rejection.” Thresholds were determined by visual inspection. Data were analyzed and plotted using Origin-2015 (OriginLab Corporation, MA). Thresholds are expressed as mean ± standard deviation unless otherwise noted. For DPOAEs, f1 and f2 primary tones (f2 / f1 = 1.2) were presented with f2 varying between 5.6 and 45.2 kHz in half-octave steps, and L1-L2 = 10 dB SPL. In each f2, L2 was varied between 10 and 80 dB SPL in 10-dB SPL increments. The DPOAE threshold was defined as the L2-level that evoked a DPOAE of 5 dB SPL above the noise floor. The average noise floor across all frequencies was consistently below 0 dB SPL. In our custom-built acoustic system, stimuli were generated using a 24-bit digital I-O card (National Instruments PXI-4461) in a PXI-1042Q chassis, amplified by a SA-1 speaker driver (Tucker-Davis Technologies, Inc.), and delivered by two electrostatic drivers (CUI CDMG15008-03A). A microphonic electret microphone (Knowles FG-23329-P07) at the end of a small probe tube was used to monitor ear canal sound pressure. Most of these experiments were not performed under blind conditions.

[0214] To determine if the truncated harmonin interferes with normal auditory function, the Anc80.CMV.trunc-harm vector was generated to overexpress the truncated protein. The vector was injected into the inner ear of c.216GA mice by RWM. ABRs and DPOAES were measured at 4, 6, and 12 weeks and no difference between the thresholds of injected and uninjected c.216GA mice was found (recordings from 6 week old mice shown in C-D of FIG. 6). This data, as a control for injection technique and vector, is important in that it suggests that the exogenous truncated harmonin does not compete with the endogenous full-length harmonin, suggesting that the endogenous truncated form in c.216AA hair cells is unlikely to interfere with the exogenous full-length harmonin expressed by the gene therapy vector. Figure 16

[0215] To determine if the harmonin gene augmentation can rescue auditory and balance function in Ushlc mice, RWM injections of AAV2 / Anc80.CMV.harmonin-al (0.8 μl, 1.7 x 1012gc / ml) or AAV2 / Anc80.CMV.harmonin-bl (0.8 μl, 1.9 x 1012gc / ml) were performed at P0-P1 and auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAES), acoustic startle reflex, open field and rotarod behavior were assessed. Mice were evaluated at 6 weeks, a time when c.216AA mice have severe hearing loss and vestibular impairment. Some mice were further tested at 3 and 6 months.

[0216] Twelve mice injected with AAV2 / Anc80.CMV.harmonin-al did not recover auditory function at 6 weeks (A-C of FIG. 7), indicating that exogenous expression of harmonin-al was not sufficient for auditory rescue. However, 19 of 25 mice injected with AAV2 / Anc80.CMV.harmonin-bl had a clear recovery of auditory function at 6 weeks. At low frequencies (5.6 to 16 kHz), the best ABR thresholds in the AAV2 / Anc80.CMV.harmonin-bl injected ear were 25-30 dB SPL, which is very similar to the thresholds of wild type mice (A-B of FIG. 7). Partial rescue was observed at 22.6 kHz and almost no rescue at 32 kHz. Rescue of DPOAE thresholds was also clear, which is consistent with rescue of function in OHCs (C-D of FIG. 7). Figure 16 Figure 16 Figure 16 ​​​C). In the late stage, 8 mice with thresholds < 45 dB SPL at 8-11.3 kHz were tested to assess the duration of rescue. From 6 weeks to 3 months, a ~10 dB SPL ABR threshold shift was observed in the low frequency range and a ~30 dB SPL ABR threshold shift was observed in the high frequency range Figure 16 D). Similar shifts were also observed in DPOAE thresholds Figure 16 E). After this time point, ABR thresholds and DPOAEs remained stable up to 6 months of age Figure 21 D-E), which was the last time point tested.

[0217] To assess whether both harmonin-a1 and harmonin-b1 are required for more complete hearing rescue (especially at the high frequency end), AAV2 / Anc80.CMV.tdTomato::harmonin-a1 (0.5 μl; 2.38 4.1E^12 gc / ml) and AAV2 / Anc80.CMV.eGFP::harmonin-b1 (0.5 μl; 3.0E^12 gc / ml) were co-injected. From the two fluorescent tags positive cells, 65% of the hair cells expressed both harmonin-a1 and harmonin-b1 Figure 16 ). Fluorescently labeled harmonin-a1 was sometimes observed in the stereocilia of AAV2 / Anc80.CMV.tdTomato::harmonin-a1 exposed mice, perhaps due to overexpression. ABR and DPOAE thresholds in mice co-injected with untagged harmonin-a1 and harmonin-b1 vectors Figure 16 ) were similar to mice injected with harmonin-b1 alone, which did not provide further improvement, suggesting that harmonin-a1 can not be necessary for hearing function. Importantly, the data indicate that harmonin-b1 alone is sufficient to significantly restore hearing thresholds at low frequencies Figure 22

[0218] To further evaluate the extent of rescue, ABR waveforms were analyzed for mice with thresholds < 45 dB SPL and compared between 8 control c.216GA mice and 5 c.216AA mice injected with AAV2 / Anc80.CMV.harmonin-b1. Analysis of responses at 8-11.3 kHz and 16 kHz showed normal 1stwave amplitude (no significant difference, P > 0.2, Student t-test) and longer 1stpeak latency (P > 0.001) ​ ​), suggesting that neurotransmission in the synapse can be delayed. Hearing rescue was also observed in the contralateral ear in many animals, which had ABR thresholds as low as 20 dB SPL at 11.3 kHz (harmonin-b1 : mean 59.7 ± 5.3 dB SPL, n = 15 / 25; harmonin-a1+-b1 : mean 76.2 ± 10.3 dB SPL, n = 4-6). Diffusion of AAV vectors to the contralateral ear has been observed previously, possibly occurring through the perilymphatic channels that remain continuous with the subarachnoid space in neonatal mice.

[0219] We also investigated whether injection at a late stage of development could result in partial hearing rescue. RWM injections of AAV2 / Anc80.CMV.harmonin-b1 (0.8 μl) were performed at P10-P12 and auditory thresholds were evaluated at 6 weeks. Mice injected at P10-P12 had no detectable DPOAE and their ABR thresholds were not different from un-injected c.216AA control mice (n = 10; data not shown), indicating that the window of opportunity for intervention can be limited to the early postnatal period, possibly due to lower viral transduction efficiency in older tissues or to the degeneration of the organ of Corti in late stages of development.

[0220] Example 2F - RT-PCR in Usher mouse model

[0221] Using cDNA was prepared from 6 auditory organs of P2-P3 wild-type, heterozygous and homozygous Ush1c c.216G>A mice using a reverse transcription kit (Qiagen). cDNA encoding full-length (450 bp) or truncated harmonin (-35 bp) was amplified using the following primers: forward primer mUsh1c_Ex2F: 5' CTC ATT GAA AAT GAC GCA GAG AAG G 3' (SEQ ID NO: 11), reverse primer mUsh1c_Ex5R: 5' TCT CAC TTT GAT GGA CAC GGT CTT 3' (SEQ ID NO: 12). These primers are specific for mouse Ush1c sequence and will amplify both endogenous and AAV2-derived Ush1c, as the target sequence is outside the region where the human knock-in Ush1c c.216A allele is part of. DNA and RNA levels in mouse tissues collected 6 weeks post-treatment were also assessed. DNA and RNA were isolated from the cochlea using TRIzol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. RNA was reverse transcribed using the GoScript reverse transcription system (Promega, Madison, WI). Radiolabeled PCR was performed using GoTaq Green Master Mix (Promega, Madison, WI). For viral DNA amplification, primers specific for mouse Ush1c were used: mUsh1c_Ex3F (5'-GAA CCC AAC CGC CTG CCG (SEQ ID NO: 13)) and mUsh1c_Ex4WTR (5'-TGC AGA CGG TCC AAG CGT-3' (SEQ ID NO: 14)).

[0222] These primers will amplify only viral Ush1c DNA, as homozygous Ush1c.216AA mice have a knock-in of the human USH1C c.216A gene in exons 3 and 4, which replaces the mouse sequence (Lentz et al., 2007, Mutat. Res., 616: 139-44). For amplification of full-length (450 bp) and aberrantly spliced / truncated harmonin (415 bp) cDNAs, the same primers as described above ((mUsh1c_Ex2F and mUsh1c_Ex5R) were used. Gapdh primers were: mGapdh_Ex3F (5'-611 GTG AGG CCG GTG CTG AGT ATG -3' (SEQ ID NO: 15)) and mGapdh_Ex4R (5'-GCC AAA GTT GTC ATG GAT GAC -3' (SEQ ID NO: 16)). Products were resolved using 6% non-denaturing polyacrylamide gels and quantified using a Typhoon 9400 phosphoimager (GE Healthcare).

[0223] As previous studies suggested the possibility that truncated harmonin might disrupt the function of full-length harmonin by competing with it as an endogenous binding partner, it was explored whether the persistent expression of the truncated protein might limit the recovery of c.216AA mice injected with a vector expressing endogenous full-length harmonin (Fig. 1A). To address this question, the expression of Ush1c transcripts in c.216GA and c.216AA mice was detected using RT-PCR assays. In agreement with previous reports, Ush1c transcripts encoding both full-length and truncated harmonin were detected in c.216GA cochleae, and only transcripts encoding truncated harmonin were detected in c.216AA cochleae (Fig. 1B). Figure 23 Figure 23

[0224] ​​To confirm the expression of AAV2 / Anc80.CMV.harmonin-b1 and explore the relationship between viral expression levels and the ABR threshold, DNA and RNA were isolated from injected and contralateral cochleas and quantified by PCR and RT-PCR, respectively. Expression was assessed in 6-week-old c.216GA mice and AAV2 / Anc80.CMV.harmonin-b1 (0.8 μl; 1.93 10^12 gc / ml) injected and uninjected c.216AA mice. Samples included two injected mice with good ABR rescue (threshold ≤35 dB SPL at 11.3 kHz) and two injected mice with poor ABR rescue (threshold ≥90 dB SPL at 11.3 kHz). RNA encoding the correctly spliced ​​form of harmonin was detected in all injected cochleas and, to a lesser extent, in the contralateral cochleas of all tested animals. Figure 24 A) and AAV2 / Anc80.CMV.harmonin-b1 DNA ( Figure 24 (B).

[0225] ABR thresholds and the expression levels of DNA and RNA vary among animals. Figure 24 However, a strong correlation was found between AAV2 / Anc80.CMV.harmonin-b1 DNA levels, the amount of RNA encoding the correctly spliced ​​form of harboron, and ABR threshold levels, suggesting that variability in ABR data may be a direct result of AAV expression. To assess the long-term survival of hair cells in mice that had successfully restored their ABR threshold, tissue preparation and IHC and OHC counting were performed on five 6-month-old mice. Figure 25 Although the number of IHCs did not change in either cohort, 50% or more of OHCs were retained in the three mice that demonstrated long-term ABR rescue. OHC survival was observed in all organs except the basal gyrus. Figure 25 ).

[0226] Example 2G - Acoustic startle response in Usher mouse model

[0227] Aural startle response (ASR) was measured using a startle monitor (Kinder Scientific). Mice were placed in small, unrestricted cubic resin-glass recording chambers (27 cm × 10 cm × 12.5 cm) fixed to a piezoelectric / plexiglass sensing assembly and allowed to acclimate to 60 dB SPL background white noise for 5 minutes. Each group consisted of 35 trials, during which single noise pulses ranging from 60 to 120 dB SPL at 10 dB SPL intensity were provided at trial intervals of an average of 30 s (range 25–35 s). The pulses were arranged in a pseudo-random order against a constant 60 dB SPL background noise to limit external noise interference. The startle monitor reduced the response to each pulse to measuring the first N, the maximum N, and the longest duration of the response (ms) to calculate the peak startle response (ASR amplitude) and the time from stimulus to peak startle response (ASR latency). ASR was performed in a blinded state.

[0228] To assess whether ABR / DPOAE recovery resulted in behavior-related auditory function recovery, startle responses to sound were measured in mice injected with AAV2 / Anc80.CMV.harmonin-a1, AAV2 / Anc80.CMV.harmonin-b1, and mice co-injected with both vectors. Startle response analysis to white noise showed partial rescue of the response in 6-week-old mice injected with AAV2 / Anc80.CMV.harmonin-b1 and in mice co-injected with both vectors. Figure 17 Mice receiving only harmonin-a1 were similar to those not injected with c.216AA and did not show a recovery of startle response.

[0229] Example 2H - Vestibular evaluation in Usher mouse model

[0230] Vestibular function was assessed using open field and rotarod balance tests. Open field tests were conducted using a circular frame with a diameter of 42 cm, placed in a sound chamber with LED lighting at the top, a central setting of 30 lux, and in a dimly lit room. One mouse was placed in the circular open field at a time and allowed to explore for 5 minutes. Behavior was recorded and tracked using an Ethovision XT, allowing for the measurement of walking distance and speed. All open field assessments were performed in a blind state. The rotarod performance test involved placing the mouse on a rod within a closed enclosure, which was initially rotated at 4 rpm and then at 0.1 rpm for s. -1 The speed was accelerated. On day 1, mice were placed on the stick for 5 minutes to familiarize them with the device. The following day, animals were placed on the stick for a total of 5 trials. A 5-minute rest period was set between trials. The timer displayed the length of time the animal was able to remain on the device before falling onto the instrument housing plate and was recorded after each test batch.

[0231] Because the perilymphatic space between the cochlea and the vestibular labyrinth is continuous, transduction of vestibular sensory organs via RWM injection of AAV vectors may also be possible. To assess vestibular behavior, mouse performance on a rotarod was tested. Although poor rotarod performance was observed in c.216AA mice and c.216AA mice injected with AAV2 / Anc80.CMV.harmonin-a1 (mean latency to fall <22 sec), c.216AA mice injected with AAV2 / Anc80.CMV.harmonin-b1 and those co-injected with both harmonin-a1 and -b1 vectors maintained balance on the rotarod for 60–120 seconds, consistent with control c.216GA mice. Figure 17 (B).

[0232] Recovery of open field behavior was also observed in c.216AA mice injected with both harmonin-b1 and harmonin a1 and b1. Representative open field exploration trajectories are shown below. Figure 17 As shown in C. c.216GA mice explored the boundaries of the area and showed minimal whole-body rotation, while c.216AA mice showed more activity throughout the room and an increase in whole-body rotation quantified in rotations / min ( Figure 17 Surprisingly, although ABR rescue was not observed in mice injected with AAV2 / Anc80.CMV.harmonin-a1, open field data indicated that vestibular function recovered to the level of control mice. There was no difference in behavior between c.216GA mice injected with AAV2 / Anc80.CMV.trunc-harmonin and control c.216GA mice, again demonstrating that there is no interference between truncated and wild-type harmonin. Figure 17 (CE).

[0233] Behavioral assays showed that the use of harmonin-a1 produced partial vestibular rescue due to the elimination of rotational behavior, but mice injected with harmonin-a1 failed the rotarod test. On the other hand, mice injected with harmonin-b1 showed functional recovery in both tests. Figure 17 The absence of transduction and FM1-43 uptake in the groove region suggests that hair cell and possibly type I cell function in the groove region may depend on proper harmonin expression. Figure 13 ).

[0234] Although hearing recovery is significant at low frequencies rather than high frequencies ( Figure 16 However, preservation of hair tuft morphology was observed throughout the organ at 6 weeks. Figure 18). The absence of rescue at high frequencies is unlikely to be due to injection damage. High frequency hearing loss was observed in any c.216GA injected with AAV vectors Figure 23 As an explanation, the targeting of AAV throughout the length of the cochlea argues against a lack of transduction efficiency at the base. One possibility is that there are other harmonin isoforms, such as short harmonin-c, that can be necessary for rescue of function at the high frequency end of the cochlear base. Alternatively, since cochlear development begins at the basal end, it is possible that hair cells at the base high frequency end are already mature at P0, beyond their repair time point. If this is the case, then embryonic intervention can produce better rescue in the high frequency region.

[0235] Part 3 - Gene therapy of other mutations involved in hearing loss

[0236] Example 3A - In vivo experiments

[0237] Anc80 vectors carrying mouse TMC1 coding sequence driven by a modified CMV promoter were produced using the helper-free system and dual transfection method as previously described (Grimm et al., 2003, Mol. Ther., 7:839:50). A triple flag-tag (FLAG) sequence was fused to the C-terminus of the TMC coding sequence to enable visualization of the expressed protein. Anc80-CMV-Tmc vectors were purified using an iodixanol discontinuous gradient followed by ion exchange chromatography. Titer ranged from 1 x 1010 12 to 1 x 1010 13 gc / ml was measured by quantitative PCR using primer pairs specific for the human beta-globin intron element. Virus aliquots were stored at -80°C and thawed prior to use.

[0238] Age P0-P2 mice were used for in vivo delivery of viral vectors as described below according to protocols approved by the Institutional Animal Care and Use Committee of Boston Children's Hospital (protocols #2659, #2146). C57BL / 6J (Jackson Laboratories) or Swiss Webster strain (Taconic) mice were used as wild type control mice, and mice carrying TMC1 mutant alleles (TMC1Δ / Δ or Tmc1- / -) were on C57BL / 6J background as previously described (Kawashima et al., 2011, J. Clin. Invest., 121:4796-809).

[0239] For preparation of tissue for evaluation, temporal bones were collected from mouse pups at P0-P10. Pups were sacrificed by rapid decapitation and the temporal bones were dissected in MEM (Invitrogen) supplemented with 10 mM HEPES, 0.05 mg / ml ampicillin, and 0.01 mg / ml ciprofloxacin at pH 7.40. The membranous labyrinth was isolated under a dissecting microscope, the Reissner's membrane was peeled off, and the tectorial membrane and vascular stria were mechanically removed. The organ of Corti cultures were placed flat under a pair of thin glass fibers, which were adhered at one end to an 18 mm circular glass coverslip with Sylgard. The tissue was immediately used for electrophysiological studies. For mice older than P10, the temporal bones were collected after the animals were sacrificed by inhalation of CO2, and whole tissue preparations of the cochlea were prepared for embedding.

[0240] All mean values and error bars in the figures represent mean ± SD. Statistical significance between injected and non-injected ears was compared using a two-tailed paired t-test. P < 0.05 was considered significant.

[0241] Example 3B - In vivo injection of viral vectors

[0242] Injections were performed in mouse pups (P0-P2) through the round window membrane (RWM) using a beveled glass microinjection pipette. Pipettes were pulled from glass capillary tubes on a P-2000 pipette puller (Sutter Instrument) and beveled (tip diameter ~20 μm, 28° angle) using a micro-manipulator beveler (Sutter Instrument). The surgical site (left mastoid process) was anesthetized using EMLA cream (lidocaine 2.5% and prilocaine 2.5%) outside application using a sterile swab. The body temperature was maintained on a 37°C warming pad for 30-60 minutes prior to surgery.

[0243] Pups were anesthetized by rapid induction of hypothermia for 2-3 minutes until loss of consciousness and maintained in this state on a cold plate for 10-15 minutes during surgery. The surgical site was sterilized by scrubbing with Betadyne and repeated scrubbing with 70% ethanol three times. A post-auricular incision was made to expose the tympanic bulla, the micro-manipulator (MP-30, Sutter Instrument Company) was advanced through the bulla and overlying fascia, and the RWM was pierced with the micro-manipulator tip.

[0244] A pneumatic microinjector (WPI Nanoliter 2010) was used to inject titers at 10 12 to 10 14 gc / mL (total viral particles at 10 9 to 10 11Approximately 1 μl of virus was injected unilaterally into the left ear. The skin incision was closed using a 6-0 monofilament suture (Ethicon). The pup was then placed back on a warming pad to recover.

[0245] Example 3C - Immunofluorescence

[0246] Immunostaining was performed to determine the distribution of transgene expression delivered by the viral vector. For this purpose, immunostaining was performed on newly dissected Corti organs, which were fixed by immersion in 4% paraformaldehyde diluted in PBS at room temperature for 1 h. The tissues were then washed in PBS, permeated in 0.01–0.1% Triton X-100 for 30 min, and counterstained with AlexaFluor546-phalloidin (Molecular Probes, 1:200 dilution) for 1 h to label actin filaments.

[0247] For localization of the exogenously expressed TMC::FLAG fusion protein, tissues were blocked for 1 hour with 2% BSA and 5% normal goat serum, and incubated overnight at 4°C with an antibody against the FLAG motif (BD Biosciences, 1:200 dilution). For hair cell counting, tissues were blocked in normal goat serum for 1 hour, stained overnight at 4°C with rabbit anti-myosin VIIa primary antibody (Proteus Biosciences, 1:1000 dilution), and labeled for 1 hour with goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Life Technologies, 1:200 dilution). Samples were fixed on glass coverslips with Vectashield mounting medium (Vector Laboratories) and imaged using a Zeiss LSM700 confocal microscope at magnifications of 10X–63X.

[0248] Figure 26 Immunofluorescence was shown to demonstrate uniform Anc80 delivery of Harmonin to Ush1c mutant mice, and Figure 28 Immunofluorescence was shown indicating the delivery of KCNQ4 to Anc80 cells in KCNQ4 mutant mice. Therefore, Anc80 is an effective vector for treating a variety of different genetic defects (at multiple different loci) leading to hearing loss.

[0249] Example 3D - Hair cell electrophysiology

[0250] Organotypic cochlea cultures were bathed in standard artificial perilymph containing 137 mM NaCl, 0.7 mM NaH2P04, 5.8 mM KC1, 1.3 mM CaCl2, 0.9 mM MgCl2, 10 mM Hepes, and 5.6 mM D-glucose. Vitamins (1 :50) and amino acids (1 :100) were added to the solution using concentrates (Invitrogen), and the final pH was adjusted to 7.40 (310 mosmol / kg) using NaOH. Patch pipettes (3-5 MΩ) were pulled using R6 capillary glass (King Precision Glass) and filled with intracellular solution containing 135 mM CsCl, 5 mM Hepes, 5 mM EGTA, 2.5 mM MgCl2, 2.5 mM Na2-adenosine triphosphate, and 0.1 mM CaCl2, with the final pH adjusted to 7.40 using CsOH (285 mosmol / kg). Whole-cell, tight-seal voltage-clamp recordings were made using an Axopatch 200B (Molecular Devices) at room temperature (22°-24°C) and -84 mV. Sensory transduction currents were filtered at 10 kHz using a low-pass Bessel filter, digitized using a 16-bit acquisition board (Digidata 1440A) at >20 kHz, and recorded using pCLAMP 10 software (Molecular Devices). Data were stored and analyzed offline using OriginPro 8 (OriginLab).

[0251] Figure 29 shows that potassium currents are restored to near wild-type levels in Anc80-KCNQ4-transfected KCNQ4- / - cells (C) compared to mutant mice (B), indicating that gene therapy using Anc80 is able to restore function (A). Figure 29 Figure 10 Figure 29

[0252] Example 3E - Auditory brainstem response (ABR)

[0253] ​​​ABR recordings were performed as previously described (Maison et al., 2010, J. Neurosci., 30:6751-62). Briefly, P25-P30 mice were anesthetized by IP injection (0.1 ml / 10 g-body weight) of ketamine 50 mg and xylazine 5 mg diluted in 5 ml of 0.9% saline. ABR experiments were performed in a soundproof room at 32°C. To test the hearing function, mice were presented with pure tone stimuli at 5.6 kHz, 8 kHz, 11.3 kHz, 16 kHz, 22.6 kHz, or 32 kHz at sound pressure levels ranging between 10 and 115 dB in 5 dB steps until the threshold intensity was detected that elicited reproducible ABR waveforms (peaks I-IV). Using alternating polarity stimuli, 512 to 1024 responses were collected and averaged for each sound pressure level. Waveforms with amplitudes greater than 15 μν (peak to trough) were rejected by the "reject artifact" function.

[0254] Prior to starting the ABR test, the skin and cartilage that normally covers the entrance of the external ear canal was trimmed with dissecting scissors and the sound pressure at the entrance of the ear canal was calibrated for each test subject at all stimulation frequencies. Acoustic stimuli were delivered directly to the ear under study through a custom probe tube speaker / microphone assembly (EPL PXI system) consisting of two electrostatic earphones (CUI Miniature Dynamics) for generating the primary tone and a Knowles microelectronic condenser (Electret Condenser) for recording the ear canal sound pressure. The sound stimuli consisted of 5-ms tone bursts (delivered at 40 / s) with a 0.5-ms rise-fall at the beginning. 2 The sound stimuli consisted of 5-ms tone bursts (delivered at 40 / s) with a 0.5-ms rise-fall at the beginning.

[0255] ABR signals were collected using subcutaneous needle electrodes inserted in the pinna (active electrode), vertex (reference electrode), and buttocks (ground electrode). ABR potentials were amplified (10,000x), filtered (0.3-10 kHz), and digitized using custom data acquisition software (LabVIEW). The sound stimuli and electrode voltages were sampled at 40-μs intervals using a digital I-O board (National Instruments) and stored for offline analysis. Thresholds were defined as the lowest decibel level at which any wave (I-IV) could be detected and reproduced with increasing sound intensity. ABR thresholds were averaged across each experimental group and used for statistical analysis.

[0256] Figure 27 The graphs indicate that delivery of Anc80 viral vectors encoding and expressing Harmonin can provide almost complete restoration of hearing function, particularly at lower frequencies (e.g., about 5 to about 22 kHz).

[0257] Example 3F - Quantitative RT-PCR analysis

[0258] This experiment was performed to evaluate the amount of virus present in the cochlea after in vivo administration. Two TMC1- / - mice were injected in the left ear at PI. Cochleas were isolated from both left and right ears and maintained in culture for 3 days (equivalent to P10). RNA was extracted and its quality confirmed using an Agilent Bioanalyzer (Agilent Technologies), reverse-transcribed into cDNA and used for quantitative RT-PCR analysis using specific primer pairs for TMC1 and SYBR GreenER qPCR reagents (Invitrogen) as previously described (Kawashima et al., 2011, J. Clin. Invest., 121 :4796-809).

[0259] To amplify TMC1 fragments, the following primers were used: 5'-CAT CTG CAG CCA ACT TTG GTG TGT-3' (SEQ ID NO: 17) and 5'-AGA GGT AGC CGG AAA TTC AGC CAT-3' (SEQ ID NO: 18). Expression levels were normalized to Actb (encoding β-actin) amplified using 5'-TGA GCG CAA GTA CTC TGT GTG GAT-3' (SEQ ID NO: 19) and 5'-ACT CAT CGT ACT CCT GCT TGC TGA-3' (SEQ ID NO: 20). All primers were designed to span introns and validated using melt curve analysis and negative controls. Data were analyzed using the ΔΔCT method to determine changes relative to Actb and differences between injected and uninjected ears.

[0260] These results indicate that TMC1 mRNA expression in the injected ear was 12-fold higher than in the uninjected ear.

[0261] Example 3G - FM1-43 labelling

[0262] FM1-43 dye loading experiments were performed as previously described (Gale et al., 2001, J. Neurosci., 21 :7013-25; Meyers et al., 2003, J. Neurosci., 23:4054-65; and Geleoc & Holt, 2003, Nat. Neurosci., 10:1019-20). Coverslips with adhered cochlear cultures were placed on a glass bottom chamber of an upright microscope (Zeiss Axioscope FS Plus). 5-μΜ FM1-43FX (Invitrogen) diluted in artificial perilymph was applied for 10 sec and the tissue was washed 3 times in artificial perilymph to remove dye from the outer leaflet of the cell membrane. After 5 minutes, intracellular FM1-43 was imaged using a FM1-43 filter set and epifluorescence light source with a 63X water immersion objective. Tissues were fixed and processed for immunofluorescence detection as described above.

[0263] Figure 30 is an immunostaining image showing FM1-43 dye uptake in cells exposed to Anc80 viral vectors as used in the present application, and Figure 31 FIGURE 11 illustrates that TMC1 delivered by Anc80 viral vectors as described herein is capable of restoring sensory transduction in Tmc1 -deficient hair cells in vivo.

[0264] Example 3H - Distortion product otoacoustic emissions (DPOAE)

[0265] DPOAE data were collected under the same conditions as ABR data and in the same recording session. To produce DPOAEs at 2f1-f2, primary tones were produced at a frequency ratio of 1.2 (f2 / f1), with f2 levels of 10 dB, sound pressure level below f1 levels, for each f2 / f1 pair. f2 levels were swept from 20 to 80 dB in 5-dB steps. Waveform and spectral averaging were used at each level to increase the signal-to-noise ratio of the recorded ear canal sound pressure. The amplitude of the DPOAE at 2f1-f2 was extracted from the averaged spectrum, as well as the noise floor at nearby points in the spectrum. An equal response curve was interpolated in the DPOAE amplitude versus sound level curve. Threshold was defined as the f2 level required to produce a DPOAE at 0 dB.

[0266] Figure 32 FIGURE 11 illustrates that TMC1 delivered by Anc80 viral vectors as described herein is capable of restoring sensory transduction in Tmc1 -deficient hair cells in vivo.

[0267] Other Embodiments

[0268] It is to be understood that, even though many of the aspects of the methods and compositions of matter have been described above with the aid of this detailed description, the descriptions are merely meant to illustrate and not to limit aspects of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0269] Disclosed are methods and compositions which can be used, can be used in combination, can be used to make, or are products of the disclosed methods and compositions. The present application discloses these and other compositions of matter, and it should be understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference can not have been made to each and every individual and collective combination and permutation of these compositions and methods, each is specifically contemplated herein. For example, if a particular composition of matter or a particular method is disclosed and discussed, and a number of compositions or methods are discussed, it is contemplated that each combination and permutation of the compositions and methods is specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Claims

1. Use of an adeno-associated viral (AAV) particle in the manufacture of a medicament for treating a hearing disorder, wherein the AAV particle comprises (i) Anc80 capsid proteins consisting of the amino acid sequence of SEQ ID NO: 2, and (ii) one or more transgenes, wherein the one or more transgenes are delivered into the inner ear of a subject into at least 80% of inner hair cells (IHCs) and at least 80% of outer hair cells (OHCs) of the inner ear by administering the AAV particle to the inner ear to treat the hearing disorder.

2. The use of claim 1, wherein the transgene is selected from ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOX L1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNRI, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPTL, POLRID, POLRIC, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, or XIAP.

3. The use of claim 1, wherein the transgene encodes a neurotrophic factor, an antibody or fragment thereof, an immunomodulatory protein, or an anti-oncogenic transcript.

4. The use of claim 1, wherein: (i) the transgene encodes an antisense, a silencing, or a long non-coding RNA species; or (ii) the transgene encodes a genome editing system selected from a genetically engineered zinc finger nuclease, a TALEN, and a CRISPR.

5. The use of claim 1, wherein the one or more transgenes are under the control of a heterologous promoter sequence.

6. The use of claim 5, wherein the heterologous promoter sequence is selected from a CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, an EF-1 promoter, an alpha 9 nicotine receptor promoter, a dynein promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, or an Atohl promoter.

7. The use of claim 1, wherein the one or more transgenes are further delivered to one or more spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, or cells in the stria vascularis, or any combination thereof, in the inner ear of the subject.

8. The use of claim 1, wherein the medicament is for administration by injection from the round window in the subject.

9. The use of claim 1, wherein the medicament is for administration during a cochleostomy.

10. The use of claim 1, wherein the medicament is for administration during a canalostomy.

11. The use of claim 1, wherein the medicament is for administration by one or more drug delivery vehicles.

12. The use of claim 1, wherein expression of the transgene results in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons, thereby restoring hearing or vestibular function.

13. Use of an adeno-associated virus (AAV) particle in the manufacture of a medicament for treating a hearing disorder, wherein the AAV particle comprises (i) an Anc80 capsid protein consisting of the amino acid sequence of SEQ ID NO: 2, and (ii) a TMC1 or TMC2 transgene, wherein the TMC1 or TMC2 transgene is delivered to at least 80% of inner hair cells (IHCs) and at least 80% of outer hair cells (OHCs) in the inner ear of a subject.

14. Use of an adeno-associated virus (AAV) particle in the manufacture of a medicament for treating a hearing disorder, wherein the AAV particle comprises (i) an Anc80 capsid protein consisting of the amino acid sequence of SEQ ID NO: 2, and (ii) an Usher transgene, wherein the Usher transgene is delivered to at least 80% of inner hair cells (IHCs) and at least 80% of outer hair cells (OHCs) in the inner ear of a subject.

15. The use of claim 14, wherein the Usher transgene is selected from MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, or PDZD7.

16. The use of claim 13 or claim 14, wherein the transgene is further delivered to one or more spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, or cells in the stria vascularis, or any combination thereof, in the inner ear of the subject.

17. The use of claim 13 or claim 14, wherein the transgene is under the control of a heterologous promoter sequence.

18. The use of claim 17, wherein the heterologous promoter sequence is selected from a CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, an EF-1 promoter, an alpha 9 nicotine receptor promoter, a dynein promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, or an Atohl promoter.

19. The use of claim 13 or claim 14, wherein the medicament is for administration by injection from the round window in the subject.

20. The use of claim 13 or claim 14, wherein the medicament is for administration during a cochleostomy.

21. The use of claim 13 or claim 14, wherein the medicament is for administration during a canalostomy.

22. The use of claim 13 or claim 14, wherein the medicament is for administration by one or more drug delivery vehicles.

23. The use of claim 13 or claim 14, wherein expression of the transgene results in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons, thereby restoring hearing or vestibular function.

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