HK40003977B - Materials and methods for delivering nucleic acids to cochlear and vestibular cells - Google Patents

Description

Materials and methods for delivery of nucleic acids to cochlear and vestibular cells

Technical Field

The present disclosure relates generally to materials and methods for delivering nucleic acids to the cochlea and vestibular cells.

Background

Genetic-based hearing loss is an important issue, with fewer treatment options than cochlear implants. Genetic hearing problems are often due to single gene defects. A1/500 th of the infants were diagnosed with presbycusis, of which about 50% had a genetic etiology. Usher syndrome accounts for 3 to 6% of presbycusis, and is associated with a number of different clinical subtypes, each of which may be caused by any one of a number of different mutations in the various genes, whereas a genetic defect occurring in the TMC1 gene is more prevalent and is estimated to account for 1-2% of all hereditary deafness.

The inner ear (e.g. the cochlea, especially the inner and outer hair cells (IHC and OHC) in the cochlea) is an attractive target for gene therapy for intervention in hearing loss and deafness of various etiologies (most directly, genetic deafness in a monogenic form). However, effective targeting and transduction of IHC and OHC, as well as other inner ear cells that may be associated with gene therapy approaches, has been a challenge.

Disclosure of Invention

Hearing loss is the most common sensory disorder worldwide, and most of the presbycusis is caused by genetic factors. Nevertheless, the transformation of cochlear gene therapy to clinical has slowed due to the lack of safe, clinically relevant and effective delivery modalities. However, the novel gene delivery modalities described herein, which include novel compositions and methods based on adeno-associated virus (AAV) containing the Anc80 capsid protein, provide efficient gene transfer to inner ear cells, including IHC and OHC. As shown herein, adeno-associated virus (AAV) containing a parent scaffold (ancetral scaffold) capsid protein, referred to as Anc80 or a specific Anc80 capsid protein (e.g., anc 80-0065), is surprisingly effective in targeting different cells in the inner ear, including IHC and OHC, in vivo.

In one aspect, an AAV vector is provided comprising an Anc80 capsid protein and a TMC1 or TMC2 transgene. In another aspect, an AAV vector is provided comprising an Anc80 capsid protein and one or more transgenes selected from the group consisting of: MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7. In one embodiment, the AAV vector further comprises a heterologous promoter.

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.

In yet 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.

In one embodiment, the one or more cells in the inner ear are selected from the group consisting of: inner Hair Cells (IHC) and Outer Hair Cells (OHC). 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 blood vessel veins.

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, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNJ1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOTOL, MARVELD2, MCM 2L 2, ESPN, ESRRRB, EYA1, EYA4, GRXCR2, HGF 2, HOME 2, HORT, and HORT 2 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, PKs, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PRPTQ, RDX, S1PR2, SANS, SARS, NARS, and so on SEMA3E, SERPINB6, SLC17A8, SLC22A4, 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.

In some embodiments, the transgene encodes a neurotrophic factor (e.g., GDNF, BDNF, NT3, and HSP 70). 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, silent, or long non-coding RNA species. In some embodiments, the transgene encodes a genome editing system selected from the group consisting of: zinc finger nucleases, TALENs and CRISPR which are modified by genetic engineering.

In some embodiments, the Anc80 capsid protein has the amino acid sequence of SEQ ID NO:1, or a pharmaceutically acceptable salt thereof. In some embodiments, the Anc80 capsid protein has the amino acid sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. In some embodiments, the transgene is under the control of a heterologous promoter sequence. Representative heterologous promoter sequences include, but are not limited to, CMV promoter, CBA promoter, CASI promoter, PGK promoter, EF-1 promoter, alpha 9 nicotinic receptor promoter, dynein promoter, KCNQ4 promoter, myo7a promoter, myo6 promoter, gfi1 promoter, vglut3 promoter, and Atoh1 promoter.

In some embodiments, the administering step comprises administering the pharmaceutical composition by injection of Anc AAV 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 a cochleostomy or during a tracheostomy. In some embodiments, the Anc AAV is administered to the middle ear and/or round window via one or more drug delivery vehicles.

In some embodiments, expression of the transgene results in regeneration of Inner Hair Cells (IHCs), outer Hair Cells (OHCs), spiral ganglion neurons, blood vessels veins, vestibular hair cells, and/or vestibular ganglion neurons, thereby restoring hearing or vestibular function.

In one aspect, an article of manufacture is provided, the article of manufacture comprising an AAV vector and a pharmaceutical composition. In such preparations, the AAV vector comprises an Anc80 capsid protein and a transgene operably linked to a promoter. In some embodiments, the transgene is selected from the group consisting of: <xnotran> 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 XIAP. </xnotran>

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 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. In yet another embodiment, a method of treating a hearing disorder in a subject is provided. Such methods typically comprise 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.

In yet another aspect, a method of delivering a Usher 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. In yet another embodiment, a method of treating a hearing disorder in a subject is provided. Such methods can comprise administering an AAV to a subject, wherein the AAV comprises an Anc80 capsid protein and a Usher transgene that, when expressed in one or more cells of the inner ear, restores hearing in the subject. Representative Usher transgenes include, but are not limited to, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7.

In one embodiment, the one or more cells in the inner ear are selected from the group consisting of: inner Hair Cells (IHC) and Outer Hair Cells (OHC). 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 blood vessels veins.

In one embodiment, the Anc80 capsid protein has the amino acid sequence of SEQ ID NO:1, or a pharmaceutically acceptable salt thereof. In one embodiment, the Anc80 capsid protein has the amino acid sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. In one embodiment, the transgene is under the control of a heterologous promoter sequence. Representative heterologous promoter sequences include, but are not limited to, CMV promoter, CBA promoter, CASI promoter, PGK promoter, EF-1 promoter, alpha 9 nicotinic receptor promoter, dynein promoter, KCNQ4 promoter, myo7a promoter, myo6 promoter, gfi1 promoter, vglut3 promoter, and Atoh1 promoter.

In one embodiment, the administering step comprises administering the pharmaceutical composition by injection of Anc AAV from the round window. In one embodiment, the Anc AAV is administered by injection from the round window. In one embodiment, the Anc AAV is administered during a cochleostomy or during a tracheostomy. In one embodiment, the Anc AAV is administered to the middle ear and/or round window via one or more drug delivery vehicles.

In one embodiment, expression of the transgene results in regeneration of Inner Hair Cells (IHC), outer Hair Cells (OHC), spiral ganglion neurons, blood vessels veins, vestibular hair cells, and/or vestibular ganglion neurons (e.g., atoh1, NF 2), thereby restoring hearing or vestibular function and/or preventing hearing loss (e.g., further hearing loss).

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 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned in this application are incorporated by reference in their entirety.

Drawings

Part 1: efficient cochlear gene transfer

Figures 1A-1G are images showing representative confocal projections comparing in vitro several AAV serotypes of eGFP transgene expression in C57BL/6 mouse cochlear explants. FIGS. 1A-1G show expression of the basal cochlea for all serotypes, as well as the apical and basal expression of the indicated AAV. Scale bar =100 μ M. The upper diagram: myo7A + TuJ1; the following figures: eGFP only; the middle graph is as follows: overlapping; s cell: supporting cells, OHC: outer hair cells, IHC: inner hair cells. FIGS. 1H-1K are graphs showing the percentage of eGFP-positive hair cells per 100 μ M after incubation for 48H or 48h +5 days. N =3 for 48h, and N =2 for 48h + 5d. Error bars represent standard error of the mean (SEM).

Figures 2A-2G are images showing AAV serotype in vivo cochlear transduction at titers indicated in each of the above panels. FIG. 2A is a confocal image of organ of Corti mice counterstained with Alexa-546-phalloidin (red) and imaged eGFP (green). Scale bar =50 μm. Fig. 2B is a graph showing quantification of eGFP-positive IHC in the base and apical of AAV-eGFP injected cochlea. Fig. 2C is a graph showing quantification of eGFP-positive OHCs in the base and top of the AAV-eGFP injected cochlea. Fig. 2D is an image showing sensory conduction current clusters recorded at P7 (left panel) of eGFP-negative OHC (black) and eGFP-positive OHC (green). Vertical scale represents 200pA; the vertical scale bar represents 20msec. The currents from eGFP negative (black) and eGFP-positive (green) P35IHC are shown on the right panel. The vertical scale represents 100pA; the horizontal scale bar represents 20msec. Fig. 2E is a graph showing the magnitude of the sensory conduction current plotted against 103 IHCs and OHCs at the ages shown at the bottom. Data from eGFP-negative (black) and eGFP-positive (green) are shown. The number of cells in each group is shown in the figure. Fig. 2F is a graph showing the mean ± Standard Deviation (SD). ABR thresholds were plotted for 4 Anc 80-injected ears (green) and 4 non-injected ears (black) and data from 1 injected ear with injection-related lesions but no eGFP fluorescence (red). Fig. 2G is a graph showing the mean ± SD. DPOAE thresholds were plotted for 4 Anc 80-injected ears (green) and 4 non-injected ears (black) and 1 negative control ear with injected lesions but no eGFP fluorescence (red). The injection titers for the data points in FIGS. 2B-2G are shown in FIG. 2A.

FIGS. 3A-3D are images showing the transduction of Anc80-eGFP in forecourt sensory epithelial cells. FIG. 3A is a graph showing results from using 1. Mu.L of Anc80-eGFP (1.7X 10) ¹² GC/mL) of P1 miceAn image of the elliptical capsule. The tissue was collected, fixed, stained with Alexa 546-phalloidin (red) and imaged for eGFP (green). Scale bar =100 μm. Fig. 3B is an image showing the ridges from the posterior semicircular canal of the same mouse shown in fig. 3A. Scale bar =50 μm. Fig. 3C is an image showing the sensory epithelium of a human elliptic sac. The tissue was exposed to an Anc80-eGFP vector, cultured, fixed, stained with Alexa 546-phalloidin (red) and imaged for eGFP fluorescence (green). Scale bar =100 μm. Fig. 3D is an image showing a high magnification view of human oocyst epithelium stained with Alexa 546-phalloidin (red) and Myo7A (blue), which is an image of eGFP (green) transduced under the same conditions as fig. 3C. White arrows in the overlay indicate selected eGFP-positive/Myo 7A-positive cells. Scale bar =20 μm.

Fig. 4A-4J are representative images showing in vitro comparisons of several AAV serotypes of eGFP transgene expression in CBA/CaJ mouse cochlear explants. Figures 4A-4F show images of the results obtained after incubation with equal doses of AAV serotype. Scale bar =200 μm. Error bars shown in FIGS. 4G-4J represent SEM.

Fig. 5A-5H are images showing an eGFP quantitative expression scoring system ranging from 0 (fig. 5d, 5h) (lowest expression) to 3 (fig. 5a, 5e) (highest level expression) to illustrate the intensity range in terms of intensity and number of cells infected, where "0" indicates no noticeable expression (fig. 5d, 5h), "1" indicates that the number of cells selected has ambiguous expression (fig. 5c, 5g), "2" indicates that there is a significant number of cells in each microscopic field with lower to medium level expression (fig. 5b, 5f) and "3" indicates that a higher percentage of cells have medium to higher level eGFP expression (fig. 5a, 5e). The scales shown in fig. 5A (for fig. 5A-5D) and fig. 5E (for fig. 5E-5H) =20 μm.

FIG. 6 shows a graph of eGFP expression in limbic, supportive and spiral ganglion neurons of C57BL/6 mice using the eGFP scoring system detailed in FIG. 5 above. Error bars represent SEM. The transduction of SGN was assessed by eGFP-positive cell counts per microscope field.

FIG. 7 shows a graph of eGFP expression in limbic, supportive, and spiral ganglion neurons of CBA/CaJ mice using the eGFP scoring system detailed in FIG. 5 above. Error bars represent SEM. The transduction of SGN was assessed by eGFP-positive cell counts per microscope field.

Fig. 8A-8E are images showing the extensive transduction of inner and outer hair cells in the mouse cochlea using Anc80. Fig. 8A is an image showing low magnification of the entire apical portion of the cochlea of a mouse injected with Anc 80-eGFP. The cochlea was collected, stained with Alexa 546-phalloidin (red) and imaged for eGFP (green). Scale bar =100 μm. Fig. 8B is an image showing a high magnification view of the base of the cochlea of different mice injected with Anc 80-eGFP. The cochlea was collected, stained with Alexa 546-phalloidin (red) and imaged for eGFP (green). Scale bar =20 μm. Fig. 8C and 8D are graphs showing quantitative comparison of transduction efficiency of inner and outer hair cells after round window injection in C57BL/6 mice. Fig. 8E is an image showing the dose dependence of Anc80 hair cell transduction. The cochlea was exposed to two different Anc80-eGFP titers, fixed, stained with Alexa 546-phalloidin (red) and imaged for eGFP (green). Scale bar =20 μm.

Fig. 9A-9H are images showing evaluation of bilateral cochlear transduction from base to top in mouse cochlea for expression of eGFP transgene in tissue sections stained with TuJ1 (red) and Myo7A (pomegranate red). Efficient Anc80 transduction was observed in the injected cochlea extending to the apex (fig. 9A/9F) and also in the contralateral uninjected ear (fig. 9B-9E = from apex to base). Close-up images of eGFP-positive and TuJ 1-positive helical ganglion neurons (fig. 9G). Reconstructed 3D images for SGN evaluation of Anc80 transduction (fig. 9H). The scales were 100 μm (FIGS. 9A-9E) and 20 μm (FIG. 9F/9G).

Fig. 10A-10B show microscopic images of longitudinal sections of mouse brain after unilateral cochlear injection of Anc80 (fig. 10A). Dominant expression was observed in the cerebellum, particularly in purkinje cells (white arrows) (fig. 10B). The scales were 1mm (FIG. 10A) and 300 μm (FIG. 10B). Fig. 10C is a graph showing anti-AAV Neutralizing Antibody (NAB) titers in serum and cerebrospinal fluid (CSF) of animals that were not injected and injected with Anc80 RWM. Titers reflect dilutions of serum or CSF at which 50% inhibition of transduction was observed in NAB assays. Due to the limited sample volume, the sensitivity limits for serum and CSF NAB were 1/4 and 1/52.5.

Fig. 11A-11B are images and graphs, respectively, showing vestibular function following Anc80 cochlear transduction. Mice were given Anc80-eGFP by injection and evaluated for expression and for equilibration function on a rotarod device. Fig. 11A is an image showing eGFP (green) expression in vestibular tissues as determined by confocal microscopy using Myo7A (red) immunofluorescence staining. FIG. 11B is a graph showing the mean time until the mice fell off the device +/-SEM. Scale bar =50 μm.

Part 2 Gene therapy for restoring function to the Usher syndrome mouse model

FIGS. 12A-12L show scanning electron microscope images of organ Corti in Ush1 c.216G > A mutant mice. Fig. 12A-12F show images of the basal, medial and apical areas of the organ of Corti imaged in c.216ga and c.216aa mutant mice. FIGS. 12G-12L are high magnification images of OHC (FIGS. 12G-12H) and IHC (FIGS. 12I-12J). Asterisks indicate protected tufts; arrows indicate random tufts; and arrows indicate wavy IHC beams. Scale bar is low magnification: 5 μm (FIGS. 12A-12F); high magnification: 2 μm (FIG. 12G), 3 μm (FIG. 12H), 2 μm (FIGS. 12I-12J) and 1 μm (FIG. 12K, 12L).

Fig. 13A-13H are images showing mechanical conductance in hair cells of Ush1 c.216g > a newborn mutant mice. Fig. 13A-13D are images showing FM1-43 staining to assess the presence of open conductive pathways in hair cells of c.216ga and c.216aa mice. The IHC FM1-43 fluorescence appears darker because the IHC is at a different focal plane. Left panel: DIC, right panel: FM1-43; the scale bar is 10 mu m; FIG. 13C, scale bar 50 μm; FIG. 13D, scale bar 10 μm. The white lines on fig. 13D indicate the furrows (no uptake) and the extra-furrows regions (uptake). FIGS. 13E-13H are graphs showing the mechanistic transduction profiles evaluated in OHC, IHC and VHC of neonatal c.216GA and c.216AA mice. Typical conduction currents (fig. 13E), their associated current/displacement curves fitted by a second-order boltzmann function (fig. 13F), and average peak conduction currents (fig. 13G-13H) are plotted. The mean peak conductance for OHCs, IHCs and VHCs was significantly different between the two genotypes (× P <0.01, one-way ANOVA).

FIGS. 14A-14E are images showing the expression and localization of fluorescently labeled harmonin in tissues exposed to adeno-associated viral vectors in vitro and in vivo. FIGS. 14A-14C show that inner ear tissue exposed to AAV2/1 vector was rapidly dissected, cultured, fixed, counterstained (Alexa Fluor phalloidin, invitrogen) and imaged using confocal microscopy. Scale bar of fig. 14A: 10 μm-upper panel; 5 μm-lower panel; scale bar of fig. 14B: 10 mu m; scale bar of fig. 14C: 3 μm; scale bar of fig. 14D: 30 mu m; scale bar of fig. 14E: 5 μm.

Fig. 15A-15C are images showing the recovery of mechanical conduction in mouse hair cells injected with Anc80 harmoni vector. FIGS. 15A-15C show the mechanical conduction current recorded in IHC of c.216AA mice injected with either the c.216AA non-injected control mouse and the C.216AA mice injected with either Anc80 harmonin-b1 or a combination of Anc80 harmonin-b1 and Anc80 harmonin-a 1. Organotypic cultures were prepared and recorded. Each data set records a corresponding I/X curve and a dual boltzmann fit function. The respective maximum mechanical conduction current Imax =102.1pA (c.216aa); 424.3pA (c.216AA + harmonin-B1) and 341.1pA (c.216AA + harmonin-a1& -B1) (FIG. 15B). The mean responses (mean ± SD) of the injections of harmonin-b1 and harmonin-a1+ -b1 showed a clear restoration of conduction (. + -. P < 0.001) compared to the non-injected mice. There was no significant difference in mean conduction current between the harmonin-b1 injected mice and the c.216GA control mice (NS P > 0.5). There was also no significant improvement in mechanical conduction recovery when harmonin-a and harmonin-b were used in combination. Figure 15C shows one-way ANOVA.

FIGS. 16A-16E are images showing ABR and DPOAE threshold recovery in mice injected with Anc80 harmonin-b 1. Figure 16A shows images of typical ABR responses to 16kHz tone in c.216aa control mice as well as c.216aa mice injected with vectors encoding harmonin-a1, harmonin-b1, or a combination of the two. The threshold for ABR recovery measured in mice injected with either harmonin-b1 alone or harmonin-a1 and b1 was close to 30dB SPL. Figure 16B shows an image of the mean ABR response obtained from: c.216AA; c.216GA; 216AA + harmonin-a1; 216AA + Harmonin-b1; 216AA + Harmonin-a1& -b1. Mean ± SE, continuous line. Dotted line: ABR thresholds for mice recorded at 16kHz over the entire frequency range are shown in figure 16A. Figure 16C shows the average DPOAE response obtained from: c.216AA; c, 216GA; 216AA + harmonin-a1; 216AA + harmonin-b1; 216AA + Harmonin-a1& -b1. Mean ± SE, continuous line. Dotted line: DPOAE thresholds for 4 mice recorded are shown in fig. 16A. The arrow indicates that the threshold is above the maximum stimulation level tested. Figures 16D-16E show ABR and DPOAE responses obtained at 6 weeks and 3 months in mice with an initial ABR threshold less than or equal to 45 dB. 6 of 8 mice remained for 6 months and were evaluated for ABR and DPOAE (dashed line). Mean. + -. SE. Although the threshold changes in ABR and DPOAE were evident in the first three months, hearing rescue at 6 months of age in the lower frequency range was still significant.

FIGS. 17A-17E are images showing startle response, rotarod capability and recovery from open field behavior in mice injected with Anc80 harmonin-a1 and Anc80 harmonin-b 1. Fig. 17A 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-b1 but not harmonin-a 1. Mean values are shown as ± SE. Fig. 17B shows rotarod capacity in control c.216ga, c.216aa, and c.216aa-injected mice. Complete recovery was observed in mice injected with harmonin-b1 and harmonin-a1/b 1; no recovery was observed in mice injected with harmonin-a1 alone. Mean values are shown as ± SE. FIGS. 17C-17E show the open field observations for 5min in controls c.216GA, c.216AA, and c.216AA and c.216GA-injected mice. A typical trace of 2.5min is shown (fig. 17B). C.216aa mice injected with harmonin-a1, harmonin-b1, or a combination of these two vectors at P1 showed similar normal behavior as their heterozygote c.216ga counterparts or c.216ga mice injected with truncated vectors, when the c.216aa mutant mice explored the entire field and underwent repeated whole-body rotations. Fig. 17C shows a graph illustrating the number of rotations per minute and the average value of the covering distance ± SD. Significant recovery was observed between non-injected and injected mice P <0.001. Statistical analysis was performed using one-way ANOVA.

FIG. 18 is a scanning electron microscope image of the organ of Corti of mice injected with anti 80 harmonin-b 1. Basal, medial and apical portions of the c.216ga, c.216aa and c.216aa mice Corti's organs were imaged. OHC and IHC hair bundles were protected in c.216ga mice, while they appeared to be scrambled in the Corti organs of c.216aa mice. Significant hair cell loss (asterisk) and bundle disintegration were observed in c.216aa mice with more significant degradation at the basal end of the organ. The hair bundles of 216AA mice lack normal rows of static cilia. The shorter rows appeared to retract, while the highest row remained in c.216aa mice (arrow). Although hair cell loss and bundle disintegration were still evident in the rescued c.216aa mice, the survival of hair cells was significantly higher in the basal and mid regions of the organ. The hair cell counts are summarized in the histogram. A total of 1824 cells were counted in c.216aa mice and 792 cells were counted in rescued c.216aa mice. Mean ± SE. High magnification imaging showed that the ladder array was rescued in many but not all cells (arrows) of injected c.216aa mice (arrows). Scale bar low magnification: 5 μm; high magnification: 1 μm.

FIGS. 19A-19L are images showing analysis by SEM of hair bundle morphology in Ush1 c.216G > A mice. FIGS. 19A-19C show normal tuft morphology exhibited by heterozygous c.216GA mice. FIGS. 19D-19I show the disorganized hair bundles observed in the organs of homozygous c.216AA mutant mice. FIGS. 19J-19L show mild disruption of IHC hair bundles in c.216AA mice. The distance from the top tip measured as: the base part is 3.5-4mm; the middle part is 1.8-2.2mm; the top is 0.6-0.8mm. Scale low magnification: 5 μm; high magnification: 1 μm.

Fig. 20A-20J are images showing the mechanical conductivity properties in c.216aa mutant mice. Figures 20A-20E show mechanical conduction analysis of neonatal OHC in the middle and middle-apical gyrus of the cochlea. A typical current trace from-Po =0.5 was fitted using a bi-exponential decay function to assess the adaptation in the c.216ga and c.216aa mutants (fig. 20A). The fit was used to generate the degree of fast (fig. 20C) and slow (fig. 20D) time doneness and adaptation (fig. 20E). The 10-90% operating range did not change significantly (fig. 20B). As shown in the scatter plot, the degree of adaptation in c.216aa mice was significantly lower than that of heterozygous OHCs (fig. 20E). Fig. 20F-20J show mechanical conduction analysis in neonatal IHC. The 10-90% working range values were smaller in c.216ga IHC compared to c.216aa IHC (fig. 20G). Accommodation was always present in c.216aa IHC, although slightly slower and to a significantly lower extent (fig. 20H-20J). Statistical analysis shown in each figure: * P <0.05, P <0.01 and P <0.001, one-way ANOVA.

FIGS. 21A-21C are data showing the expression of fluorescently labeled harmonin-a and harmonin-b Anc80 vectors at 6 weeks in Corti's organ at c.216AA after P1 binary vector injection. FIGS. 21A-21C show confocal images of gyromagnet in 6-week-old c.216AA mice after P1 co-injection of AAV2/Anc80.CMV. TdTomato:: harmonin-a1 (0.5. Mu.l; 4.11E ^ 12gc/ml) and AAV2/Anc80.CMV. EGFP:harmonin-b 1 (0.5. Mu.l; 2.99E ^ 12gc/ml). 69% and 74% of the total cells expressed eGFP (FIG. 21A) and tdTomato (FIG. 21C) and 65% expressed both markers, indicating successful co-transduction. Scale bar: 20 μm.

FIGS. 22A-22F are data showing analysis of ABR response in control c.216GA and injection-rescued c.216AA mice. Fig. 22A and 22D show examples of ABR responses at 8 and 16kHz in control c.216ga and rescued c.216aa mice. FIGS. 22B-22C and 22E-22F show the mean peak amplitude 1 (FIGS. 22B-22D) and latency (FIGS. 22C-22D) at 8-11.3 and 16kHz in 6 week old mice with comparable thresholds (n =8 c.216GA, n =5 c.216AA + Harmonin-B1 RWM P1). Mean ± SE: one-way ANOVA.

Figures 23A-23D show that mutant forms of harmonin expressed in Ush1 c.216g > a mice did not alter hair cell or auditory function. FIG. 23A is a sequence alignment between a wild-type harmonin-b1 protein and a truncated harmonin, a cryptic splice and post-frameshift secreted product associated with acadian G > A mutation in exon 3 of the Ush1c gene. FIG. 23B shows that semi-quantitative RT-PCR of 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 c.216GA and c.216AA mice. Figures 23C-23D show auditory brainstem responses (ABR, figure 23C) and aberrated products (DPOAE, figure 23D) measured in c.216ga-injected mice as well as control c.216ga and c.216aa mice. Curves are expressed as mean ± SE.

FIGS. 24A-24C are images showing restoration of correct Ush1C splicing in the inner ear after injection of AAV 2/Anc80.CMV.harmanin-b 1 into 6 week old mice. FIG. 24A shows semiquantitative RT-PCR amplification of correctly spliced (450 bp) and aberrant (415 bp) mRNA from the Ush1c.216A allele, indicating that correct Ush1C splicing was restored in the c.216AA rescue mice #1 and #2 injected (I) and contralateral ears (FIG. 24C) (35 dB SPL response at 11.3kHz injected ears). Mouse #3 with a weaker ABR response (90 dB SPL at 11.3 kHz) showed recovery of moderately correct mRNA expression, and mouse #4 (100 dB SPL at 11.3 kHz) showed no recovery. No correct splicing was detected in non-injected c.216AA mice (mouse #5, 6), but correct and truncated spliced forms were detected in c.216GA mice (mouse #7,8, 9). GAPDH of the corresponding mice amplified is shown in the lower panel to confirm the relative amounts of material. FIG. 24B is a image showing a semi-quantitative radiolabel PCR assay demonstrating the presence of AAV-mUsh1c in the injected and contralateral ears of Ush1c.216AA mice. AAV-mUsh1c DNA was present in mice #3 and #4, but at reduced relative levels. FIG. 24C shows the relative amounts of AAV-mUsh1C corrected using ABR thresholds. The analysis for 11.3 and 16kHz is shown. Linear regression indicates that there is a high correlation between the two.

Figure 25 is a graph showing long term ABR threshold recovery associated with the survival of OHCs in the mid to top region of the auditory organ. Hair cell counts of the entire organ of Corti were performed after left ear dissection of 3 non-injected c.216aa and 5 injected c.216aa. The total number of IHC and OHC hair cells increased in the injected mice. Comparing the rescued injected mice with the injected but poorly rescued mice found no difference in IHC numbers, but significant numbers of OHCs were noted in the rescued mice. Analysis over the entire length of the organ showed that the difference could be explained as an increase in hair cell survival from the middle to the apical region of the organ. Inserting a drawing: 2 mice (mouse #1, 2) showed poor ABR response (. Gtoreq.95 dB SPL) over the entire range tested, while 3 mice (mouse #3,4, 5) showed responses with a threshold range from 35 to 55dB SPL against acoustic stimuli between 5.6 and 16 kHz.

Part 3 Gene therapy for other mutations involved in hearing loss

FIGS. 26A-26D are representative confocal images from Ush1C mutant mouse cochlea injected by RWM with Anc80-Harmonin:: GFP (i.e., fusion of GFP to the Harmonin polypeptide), collected cochlea, stained for actin (red; FIG. 26A) and Myo7a (blue; FIG. 26B) and imaged against GFP (green; FIG. 26C). The superimposed images of fig. 26A, 26B, and 26C are as shown in fig. 26D.

FIG. 27 is a graph showing ABR thresholds plotted as a function of acoustic frequency for Ush1c mutant mice (squares) and Ush1c mutant mice injected with the Anc80-Harmonin: (circles) GFP vector.

Fig. 28A-28C show representative confocal images of KCNQ 4-/-cochlea injected by RWM using Anc80-KCNQ4 at low (fig. 28A) or high (fig. 28B) magnification relative to non-injected cochlea at high magnification (fig. 28C), cochlea collected and stained with Alexa 546-phalloidin (red) and an antibody against KCNQ4 (green).

FIGS. 29A-29C are a series of graphs showing KCNQ4 current in wild-type mice (FIG. 29A), P10 KCNQ 4-/-mice (FIG. 29B), and P10 KCNQ 4-/-mice injected with Anc80-KCNQ4 (FIG. 29C). Cochlea were collected 8 days after injection.

FIG. 30 is a series of three images showing FM1-43 uptake (FM 1-43 penetrating only functional Tmc1 channels) in Tmc 1-/-tissues injected with an Anc80 Tmc1 vector.

Fig. 31A is an image showing representative families of sensory conduction currents recorded from IHC of wild-type mice (left panel), tmc 1-/-mice (middle panel), and Anc80 Tmc 1-injected Tmc1 (right panel). Cochlea were collected 8 days after injection.

Fig. 31B is a graphical representation of the recovery rate of the mice shown in fig. 31A. The graph in fig. 31B represents the percentage of functional cells in wild type mice (left panel), tmc 1-/-mice (middle panel), and Anc80 Tmc1 injected Tmc1 (right panel).

FIG. 32 is a graph showing distortion product otoacoustic emission (DPOAE) thresholds as a function of stimulation frequency for wild-type, tmc 1-/-mice, and Tmc 1-/-mice injected with Ant 80 Tmc 1.

Detailed Description

Because of the lack of self-repair ability of the sensory cells of the cochlea of adult mammals, current treatment strategies (depending on the level and exact location of the injury) rely on amplification (hearing aids), better sound transmission (middle ear prosthesis/active implant) or direct neuronal stimulation (cochlear implant) to compensate for the permanent damage to the primary sensory hair cells or spiral ganglion neurons that form the auditory nerve and transmit acoustic information to the brain. Although these methods have revolutionized, they are far from optimal in restoring the complex human auditory functions important to modern life. In particular, major problems still include limited frequency sensitivity, unnatural sound perception and limited speech discrimination capability in noisy environments.

The transfer of therapeutic genes to the cochlea has been considered to further improve the current standard of care for hearing loss ranging from age-related and environmentally induced hearing loss to the hereditary form of deafness. Over 300 loci are associated with inherited hearing loss, and over 70 causative genes have been described (Parker & Bitner-Glindzicz,2015, arch, dis. Childhood,100, 271-8. The therapeutic success of these approaches apparently relies on the safe and effective delivery of exogenous gene constructs to relevant therapeutic cellular targets in cochlear Corti's Organ (OC).

The OC contains two types of sensory hair cells: IHC, which converts mechanical information carried by sound into electrical signals that are passed on to neuronal structures, and OHC, which are used to amplify and tune cochlear responses, a process required for complex hearing functions. Other potential targets in the inner ear include spiral ganglion neurons, columnar cells of the spiral limbus, which are important for maintaining adjacent membrane-covering or supporting cells, which have protective functions and can be triggered to transdifferentiate into hair cells until the early neonatal stage.

Injection into a cochlear duct filled with high potassium endolymph fluid can directly access hair cells. However, this change in the micro-fluid environment may damage the intra-cochlear, increasing the risk of injection-related toxicity. The space around the cochlear duct, scala tympani and scala vestibuli, which is filled with perilymph fluid, is accessible from the middle ear through an oval or circular window membrane (RWM). RWM is the only non-bony opening into the inner ear, is relatively accessible in many animal models, and administration of viral vectors using this route is well tolerated. In humans, cochlear implant generally relies on the insertion of a surgical electrode by the RWM.

Previous studies evaluating AAV serotypes in organotypic extracochlear plants and in vivo inner ear injections have shown that they can only rescue some of the hearing in a mouse model of hereditary hearing loss. Unexpectedly, adeno-associated virus (AAV) containing parental AAV capsid proteins transduces OHCs with high efficiency. This finding overcomes the low transduction rate, which limits the successful development of cochlear gene therapy using conventional AAV serotypes. AAV containing parental AAV capsid proteins as described herein provides a valuable platform for delivering inner ear genes to IHC and OHC and other inner ear cell arrays damaged by inherited hearing and balance disorders. In addition to providing high transduction rates, AAV containing parental AAV capsid proteins as described herein have been shown to have similar safety profile in mice and non-human primates following systemic injection, and are antigenically distinct from circulating AAV, which provides potential benefits in terms of preexisting immunity that limits the effectiveness of conventional AAV vectors.

However, the compositions and methods described herein are capable of delivering nucleic acids to cells with high efficiency, particularly cells 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 (IHC), outer Hair Cells (OHC), spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the blood vessel veins. Support cells refer to cells in the ear that are not excitable, e.g., cells that are not hair cells or neurons. An example of a supporting cell is a schwann cell.

Delivery of one or more nucleic acids described herein to the 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, occult deafness, dominant deafness, usher syndrome, and other syndromic deafness, as well as hearing loss due to trauma or aging.

Method for preparing virus carrying specific transgenes

As used herein, adeno-associated virus (AAV) containing parental AAV capsid proteins are particularly effective in delivering nucleic acids (e.g., transgenes) to the inner ear cells, and one particularly effective class of parental AAV capsid proteins is the parental scaffold designated Anc80, which is set forth in SEQ ID NO:1, respectively. One particular parent capsid protein in the Anc80 parent capsid protein classification is Anc80-0065 (SEQ ID NO: 2), however, a number of other parent capsid proteins in the Anc80 parent capsid protein classification are described in WO 2015/054653.

The viruses comprising the Anc80 capsid protein described herein can be used to deliver a variety of nucleic acids to inner ear cells. Nucleic acid sequences that are delivered to cells for the purpose of expression are often 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 a neurotrophic factor (e.g., glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT 3), or Heat Shock Protein (HSP) -70), an immunomodulatory protein, or an anti-oncogenic transcript. In addition, representative transgenes that can be delivered to and expressed in inner ear cells also include, but are not limited to, transgenes encoding antibodies or fragments thereof, antisense, silent 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 (CRISPRs)). In addition, representative transgenes capable of being 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, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNJ1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOTOL, MARVELD2, MCM 2L 2, ESPN, ESRRRB, EYA1, EYA4, GRXCR2, HGF 2, HOME 2, HORT, and HORT 2 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, PJKK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PRPTQ, RDX, S1PR2, SANS, PSO 2, and so forth SEMA3E, SERPINB6, SLC17A8, SLC22A4, 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. The nomenclature used in this application is described and defined in hereditaryheartingloss.

Expression of the transgene may be directed by the native promoter of the transgene (e.g., a promoter naturally present in the transgene coding sequence) or expression of the transgene may 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 may be used with a heterologous promoter. As used herein, a heterologous promoter refers to a promoter that does not naturally direct the expression of the sequence (i.e., is not found with the sequence in nature). Representative heterologous promoters that can be used to direct expression of any of the transgenes set forth herein include, for example, the Cytomegalovirus (CMV) promoter, the chicken β actin (CBA) promoter, the synthetic CASI promoter, the phosphoglycerate kinase (PGK) promoter, the Elongation Factor (EF) -1 promoter, the alpha nicotine receptor promoter, the dynein promoter, the growth factor independent (GFI 1) promoter, and the vesicular glutamate transporter 3 (VGLUT 3) promoter. In addition, a promoter that naturally directs expression of one of the above-described reference transgenes (e.g., the KCNQ4 promoter, myo7a promoter, myo6 promoter, or ATOH1 promoter) can be used as a heterologous promoter to direct expression of the transgene.

Methods for making transgenes for packaging into viruses containing the Anc80 capsid protein are well known in the art and utilize conventional molecular biology and recombinant nucleic acid techniques. In one embodiment, a construct is provided comprising a nucleic acid sequence encoding an Anc80 capsid protein and carrying a transgene flanked by suitable Inverted Terminal Repeats (ITRs), which allows packaging of the transgene in the Anc80 capsid protein.

The transgene can be packaged in an AAV containing the Anc80 capsid protein using, for example, a packaging host cell. Components of the viral particles (e.g., rep sequences, cap sequences, inverted Terminal Repeat (ITR) sequences) can be transiently or stably introduced into packaging host cells using one or more constructs as described herein. The viruses described herein contain at least an Anc80 capsid protein; other components of the viral particle (e.g., rep sequences, ITR sequences) can be based on the parental or contemporary sequences. In some cases, for example, the entire viral particle may be based on the parental sequence. Such viruses can be purified using conventional methods.

It is understood that one or more transgenes may be delivered to the inner ear. It is also understood that more than one transgene may be delivered to the inner ear using a single AAV vector comprising an Anc80 capsid protein or using multiple AAV vectors comprising an Anc80 capsid protein.

In general, as used herein, "nucleic acid" can include DNA and RNA, and can also include nucleic acids that contain one or more nucleotide analogs or backbone modifications. Nucleic acids may be single-stranded or double-stranded, depending generally on their intended use. The nucleic acids that can be used in the methods described herein can be identical to known nucleic acid sequences, or the nucleic acids that can be used in the methods described herein can be sequences that differ 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.

In calculating percent sequence identity, two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned regions (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to give a percentage sequence identity value. It will be appreciated that the length of the aligned regions may be a fraction of one or both sequences up to the full length of the shortest sequence. It will also be appreciated that a single sequence may be aligned with more than one other sequence and may therefore have a different percentage value of sequence identity over each aligned region.

Two or more sequences are aligned using the computer program ClustalW and default parameters to determine percent sequence identity, 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 computes and aligns the best matches between the query sequence and one or more target sequences to determine identity, similarity, and differences. Gaps in one or more residues may be inserted in the query sequence, the target sequence, or both to maximize sequence alignment. For pairwise alignments of nucleic acid sequences, default parameters (i.e., font size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5) are used; for alignment of multiple nucleic acid sequences, the following parameters were used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transfer: is. For pairwise alignment of polypeptide sequences, the following parameters were used: the character size is as follows: 1; window size: 5; the scoring method comprises the following steps: percent; number of top diagonal lines: 5; and gap penalties: 3. for multiple alignments of polypeptide sequences, the following parameters were used: the weight matrix is: BLOSUM (module replacement matrix); gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic vacancies: opening; hydrophilic residue: gly, pro, ser, asn, asp, gln, glu, arg and Lys; and residue-specific gap penalties: and opening. ClustalW may run on the Baylor College of Medicine Search Launcher website or the European bioinformatics institute website on the world Wide Web.

Changes can be introduced into a nucleic acid sequence which, if the nucleic acid sequence is a coding sequence, can result in a change in the amino acid sequence of the encoded polypeptide. For example, changes can be introduced into a nucleic acid coding sequence using mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis) or by chemical synthesis of nucleic acid molecules having such changes. Such nucleic acid changes may result in conservative and/or non-conservative amino acid substitutions at one or more amino acid residues. "conservative amino acid substitutions" refer to substitutions in which an amino acid residue is replaced with a different amino acid residue having a similar side chain (see, e.g., dayhoff et al, (1978, atlas of Protein Sequence and Structure, 5 (supply.3): 345-352), which provides a table of the frequency of amino acid substitutions), and non-conservative substitutions refer to substitutions in which an amino acid residue is replaced with an amino acid residue that does not have a similar side chain.

The nucleic acid may be contained in a construct, which may also be referred to as a vector or plasmid. The constructs are commercially available or can be produced using recombinant techniques conventional in the art. Constructs containing nucleic acids can have expression elements that direct and/or regulate the expression of such nucleic acids, and can also contain sequences such as those used to maintain the construct (e.g., origins of replication, selectable markers). Expression elements are well known in the art and include, for example, promoters, introns, enhancer sequences, response elements or induction elements.

Methods for delivering nucleic acids to inner ear cells

Methods of delivering nucleic acids to cells are generally known in the art, and methods of delivering a virus containing a transgene (which may also be referred to as a viral particle) to an inner ear cell in vivo are described herein. As used herein, about 10 can be administered to a subject ⁸ To about 10 ¹² Individual viral particles, and the virus may be suspended in a suitable volume (e.g., 10 μ L, 50 μ L, 100 μ L, 500 μ L, or 1000 μ L), e.g., artificial perilymph fluid.

Any number of mechanisms can be used to deliver a virus containing a transgene as described herein to an inner ear cell (e.g., a cell 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 or oval window in a relatively simple manner (e.g., an outpatient). In some embodiments, a composition as described herein comprising a therapeutically effective number 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 as compared to the other collection, can be delivered to a suitable location within the ear during surgery (e.g., inner ear windowing or canalostomy).

In addition, a delivery vehicle (e.g., a polymer) can be used to facilitate transfer of the agent across the tympanic membrane and/or through the round window, and any such delivery vehicle can be used to deliver the viruses described herein. See, e.g., arnold et al, 2005, audio. Neurool., 10.

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

As shown herein, expression of a transgene delivered using an AAV containing an Anc80 capsid protein can regenerate Inner Hair Cells (IHCs), outer Hair Cells (OHCs), spiral ganglion neurons, blood vessels, vestibular hair cells, and/or vestibular ganglion neurons (e.g., atoh1, NF 2) such that auditory or vestibular function is restored over an extended period of time (e.g., months, years, decades, lifetime).

AAV containing the Anc80 capsid protein can be characterized using seropositivity and/or degree of neutralization relative to conventional AAV (i.e., AAV not containing an Anc80 capsid protein), as discussed in WO 2015/054653. Seropositivity as understood in the art refers to the proportion of subjects in a seropositive population (i.e., having been exposed to a particular pathogen or immunogen) and is calculated as the number of subjects in the population that produce antibodies to the particular pathogen or immunogen divided by the total number of individuals in the population under consideration. Determining the seropositivity rate of a virus is routinely performed in the art and typically involves determining the positivity rate of one or more antibodies in a sample (e.g., a blood sample) of a particular population of individuals using an immunoassay. In addition, several methods can be used to determine the extent of neutralizing antibodies in a serum sample. For example, a neutralizing antibody assay measures the titer of a test sample containing a concentration of antibodies that neutralize 50% or more of the infection as compared to a control sample without the antibodies. See also, fisher et al (1997, nature Med., 3) and Manning et al (1998, human Gene Ther., 9. Representative conventional AAVs include, but are not limited to, AAV8 (or a virus comprising an AAV8 capsid protein) and/or AAV2 (or a virus comprising an AAV2 capsid protein).

Usher syndrome

Human Usher Syndrome (USH) is a rare genetic disease that can lead to hearing 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 presbycusis. 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 suffer from congenital bilateral profound sensorineural hearing loss, vestibular nonreflection, and pre-pubertal retinal pigment degeneration (progressive, bilateral, symmetrical degeneration of rod and cone function of the retina). Individuals typically do not have the ability to generate speech unless fitted with a cochlear implant. Although there is currently no biological treatment for Usher patients, early reintroduction of the wild-type defective gene may reverse the disease.

The 6 Usher genes are associated with USH 1: MYO7A (myostatin 7A), USH1C (harmonin), CDH23 (cadherin 23), PCDH15 (procalcitonin 15), SANS (SANS), and CIB2 (calcium and opsin binding protein 2). These genes encode proteins involved in the morphogenesis of the inner ear and are part of the interaction group (see, e.g., mathur & Yang,2015, biochim. Biophys. Acta,1852, 406-20. Harmonin is located in the center of the USH1 interaction group where it binds to other Usher 1 proteins. Since it has a PDZ (PSD-59/Dlg/ZO-1) interaction domain, it has been proposed that harmonin functions as a scaffold protein. In vitro binding studies showed that all other known USH1 proteins bind to the PDZ domain of harmonin, as do the two USH2 proteins, usherin and VLGR 1. The USH1C gene consists of 28 exons, which encode 10 alternatively spliced forms of harmonin, which are divided into three distinct subclasses (a, b, and C) according to the composition of the protein domain. The three isoforms differ in the number of PDZ protein-protein interaction domains, coiled Coil (CC) domains and proline-serine-threonine (PST) -rich domains.

The USH1 protein is located on the top of the hair cells in the mechanosensory hair bundle and consists of hundreds of static cilia interconnected by many extracellular junctions. Cadherin 23 and procalcitonin 15 are the products of the Usher gene (USH 1D and USH1E, respectively) which form a tip junction distal to the cilia. Harmonin-b binds to CDH23, PCDH15, F-actin and itself. It was found in hair cells at the tip of the cilia near the point of insertion of the tip junction, and is thought to play a role in transduction and adaptation in hair cells. Harmonin-b is expressed in the early postnatal stage, 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 resting cilia. Recent reports have revealed another role for harmonin-a in synapses, which in combination with cav1.3 Ca2+, restricts the availability of channels through ubiquitin-dependent pathways.

Over the past decade, several mouse models for Usher syndrome have been identified or designed, seven of which affect harmonin. Of these, only one model (Ush 1 c.216g > a model) reproduces the features of human Usher syndrome auditory and retinal deficits. Ush1 c.216g > a is a knock-in mouse model that affects the expression of all conventional harmonin isoforms due to the presence of similar point mutations found in the French-Acadian Ush1C patient cohort. This mutation introduces a cryptic splice site at the 3-terminus of exon in the Ush1c gene. The use of this cryptic splice site results in a frameshift transcript with a 35bp deletion and results in translation of a severely truncated protein lacking the PDZ, PST and CC domains. Homozygous c.216aa knock-in mice had severe hearing loss at 1 month of age, while heterozygous c.216ga mice did not present any abnormal phenotype. Histological examination of the cochlea of 216aa mice revealed bundle dissociation, abnormal cell lines and loss of inner and outer hair cells in the middle and basal gyrus at P30.

In particular, patients diagnosed with Usher syndrome-associated deafness (e.g., USH 1C-associated deafness) can be treated with a parental AAV capsid protein described herein in combination with a harmin transgene to successfully transduce hair cells and drive expression and proper localization of a spliced form of harmin, thereby reintroducing wild-type harmin. Furthermore, the applicant has demonstrated that early postnatal round window membrane injection of AAV containing the parental AAV capsid proteins as described herein successfully restored auditory and vestibular function in homozygous c.216aa mice. Restoration of auditory function in injected mice correlates with the restoration of mRNA expression encoding wild-type harmonin and the maintenance of hair bundle morphology and mechanical conduction.

TMC1/TMC2

Over 40 different mutations have been identified in TMC1 leading to deafness. It is subdivided into 35 recessive mutations and 5 dominant mutations. Most recessive mutations result in severe, congenital hearing loss (e.g., DFNB 7/11), but a few result in late-onset, moderate-to-severe hearing loss. All dominant mutations cause progressive hearing loss (e.g., DFNA 36), which begins to develop during adolescence. In particular, AAV vectors comprising the Anc80 capsid protein as described herein can be used to deliver non-mutated (e.g., wild-type) TMC1 sequences or TMC2 sequences to prevent hearing loss (e.g., further hearing loss) and/or restore hearing function.

Conventional molecular biology, microbiology, biochemistry and recombinant DNA techniques well known in the art may be used in light of this disclosure. These techniques are fully described in the literature and exemplified in certain examples below. The invention will be further described by the following examples which do not limit the scope of the methods and compositions of matter described in the claims.

Examples

Part 1: efficient cochlear gene transfer

Example 1-adeno-associated virus (AAV) containing parental AAV capsid proteins results in safe and effective cochlear genes Transfer of

The following methods and materials were used in example 1.

Viral vectors

AAV2/1, 2/2, 2/6, 2/8, 2/9, and AAV2/Anc80L65 with CMV-driven eGFP transgene and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) expression cassettes were prepared in Gene Transfer Vector Core (Vector. Meei. Harvard. Edu) of Massachusetts Eye and Ear as previously described (Zinn et al, 2015, cell reports,12, 1056-68. Com to obtain AAV2/Anc80L65 plasmid reagent.

In vitro explant culture

For evaluation as described in earlier publications (Dilwali et al, 2015, scientific reports,5, 18599), a total of 156 cochlear explant cultures were prepared on postnatal day 4 using mouse pups of both strains. Briefly, temporal bones of mice were harvested after decapitation and cochlear grafts connected to the area of the spiral ganglion neurons were dissected out as organotypic explants for culture. Each cochlea obtains two specimens, one consisting of below the top ("top") and one consisting of above the base ("base"). For each serotype, a minimum of 4 (CBA/CaJ, 48 h), 2 (CBA/CaJ, 48h + 5d), 3 (C57 BL/6, 48 h) and 2 (C57 BL/6, 48h + 5d) base and top specimens were inoculated. Specimens that did not retain cochlear morphology during culture were culled. Sample numbers were selected to show changes in transductionHeterology and as a basis for selecting for further in vivo evaluation. 50 μ l of 10 was used ¹⁰ GC AAV medium (98% Dulbecco's Modified Eagle Medium (DMEM), 1% ampicillin, and 1% N2 supplements added over the first 12 hours plus 1% Fetal Bovine Serum (FBS)) was cultured for 48h in exo plants. For the condition of 48h +5d, medium containing AAV was replaced with fresh medium without AAV on the other 5 days. Human vestibular epithelium from the oocyst obtained from 4 consented adult patients with vestibular schwannomas resection was cultured and exposed to 10 according to the methods described previously (Kesser et al, 2007, gene Ther, 14 ¹⁰ GC AAV was cultured for 24 hours and in culture medium for 10 days, after which the tissue was fixed and stained and imaged with phalloidin. The study was approved by the Surrey Borders NRES Committee London (health research institute) under the number 11/LO/0475.

Animal models and general methods

Wild type C57BL/6J and CBA/CaJ mice were from Jackson Laboratory (Bar Harbor, ME) and animals of either sex were estimated to be used in the experiments at a 50/50 ratio. The group size of each experiment used for in vitro and in vivo transduction assays and subsequent endpoints was determined by specimen acquisition and technical feasibility. The observations reported for Anc80 transduction were qualitatively validated in subsequent experiments with different vector batches (except for human vestibular tissue transduction due to the unique and limited nature of the specimens obtained). Because the samples obtained were limited and the qualitative nature of the results reported, no statistical analysis was performed on transduction efficiency between serotypes.

CSF and blood sampling

Cerebrospinal fluid (CSF) was collected from the cisterna magna (Lui & Duff,2008, j. Visualized Exp., 21. The maximum amount of clear CSF (up to 5 μ L) per animal was collected via a microcapillary tube into a volume of 60 μ L PBS, which was then normalized by the addition of control PBS before the start of the experiment, due to slightly different initial dilutions. After obtaining a blood sample in a 1.1mL Z-Gel microtube (Sarstedt, N ü mbrecht, germany), it was centrifuged at 8,000rpm for 8 minutes and the serum was stored at-80 ℃ together with the CSF sample (in PBS) until further use.

Example 1A histological analysis

After a follow-up period of 5 to 29 days, animals were sacrificed and cochlear whole tissue specimen inlays were prepared according to previous reports (Sergeyenko et al,2013, j.neurosci, 33. The cochlear whole tissue specimen embedding and the explant are stained using antibodies against myosin 7A (Myo 7A, #25-6790 protein biosciences, ramona, ca, 1. The slides of the specimens were observed by confocal microscopy. Each image of a given experimental series was obtained using the same experimental setup, with the laser intensity selected to prevent fluorescence saturation based on the specimen with the strongest eGFP signal. A Z-protocol stack for overview images and an enlargement of the region for the Corti's organs and the Spiral Ganglion Neurons (SGNs) were obtained. 3D reconstruction was performed using AMIRA to more accurately determine SGN transfection.

The results in FIGS. 1 and 4 show tropism for the 5 serotypes obtained by monitoring the expression of AAV encoding eGFP in C57BL/6 and CBA/CaJ, respectively. Notably, eGFP expression was qualitatively more pronounced in cochlear cultures exposed to Anc80, with significant expression in many cochlear cell types.

Example 1B-quantification of eGFP expression

For in vitro data, the percentage of eGFP-positive Inner Hair Cells (IHC) and Outer Hair Cells (OHC) was determined manually and quantitatively by dividing the number of eGFP-positive cells by the total number of outer or inner hair cells in 1 or 2 100 μm sections per specimen base and top sample. All visible SGNs in extra-cochlear plants were evaluated for their eGFP expression. The area of the helical margin and supporting cells was assessed in a quantitative manner (corrected for each experimental series as shown above) scored from 0 (no expression) to 3 points (strongest signal). 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 top and basal of the two tested mouse strains. Anc80 showed consistent and qualitatively brighter IHC and OHC eGFP expression compared to AAV2 (fig. 1 and 4).

In order to control the potential differences in expression initiation between different AAVs that could result in underestimating expression at the 2 day (early) time point, longer experiments were performed. A new set of cochlea was transduced under the same conditions, and after 48h of culture with AAV, the medium of the vector-containing explant culture was discarded and replaced with fresh medium to maintain culture survival for an additional 5 days (called 48h + 5d). Similar expression patterns were observed in this longer-term study against AAV 2and Anc80. Moderate increases in AAV6, 8, and 9 expression, particularly at the basal gyrus, were noted in CBA/CaJ mice (fig. 1J, 1K and fig. 4I, 4J). Other cell types were targeted by all serotypes, with borders being easier than supporting cells, followed by SGNs (fig. 5,6 and 7). Consistently, anc80 transduction has higher efficiency and stronger expression as demonstrated by the brighter eGFP fluorescence.

EXAMPLE 1C in vivo injection

Mouse pups (P0 to P2) were injected through Round Window Membrane (RWM) using a beveled glass microinjection pipette. Pipettes were drawn out on a P-2000 pipette drawing Instrument (Sun Instrument, novato, CA) using a glass capillary and beveled (tip diameter 20 μm,28 ℃ angle) using a micropipette beveller (Sun Instrument, novato, CA). The surgical site (left mastoid) was covered with EMLA cream (lidocaine 2.5% and prilocaine 2.5%) using a sterile swab for analgesia. Body temperature was maintained on a 38 ℃ warming pad prior to surgery. The pups were anesthetized by rapid induction of hypothermia for 2-3 minutes in ice/water until loss of consciousness, and held in this state on a cold plate for 5-10 minutes during surgery. The surgical site was sterilized by scrubbing with biot iodine and rubbing three times with 70% ethanol. A retroauricular incision was made to expose the clear blebs, a micropipette was manually advanced through the blebs and overlying fascia, and passed through the RWM with the micropipette tip. Approximately 1 μ L of virus was injected manually, unilaterally, within 1min into the left ear of 5 (AAV 1), 4 (AAV 2), 2 (AAV 8), 1 (AAV 6), 3 (Anc 80) C57BL/6 animals. To control factors associated with the preparation of a particular support (such as quality and purity), the results of Anc80 were confirmed in subsequent studies using different support batches from independent preparations, confirming the qualitative results we present here (data not shown). Each group was injected in a non-blind manner. Sometimes the needle is inserted too deeply, too shallowly or at an incorrect angle. If there is visible damage to the middle or inner ear structure, the sample is excluded from further analysis. The success rate of injection ranges from-50% to-80%, depending on the level of experience of the injector. Following injection, the skin incision was closed using 6-0 black monofilament suture (Surgical Specialties, wyomissing, pa.). The pups were then returned to the 38 ℃ warm pad for 5-10min and then returned to the mother for nursing.

Consistent with previous reports, AAV1 transduced IHC with moderate to high efficiency (fig. 2a, 2b). These studies indicate that AAV2, 6 and 8 target a lower number of IHCs, only AAV8 showed approximately the same transduction at the top and base (fig. 2B). Furthermore, consistent with previous reports, there was minimal OHC transduction for all conventional AAV serotypes examined: (<5%). However, at 20-fold (against AAV 1) to 3-fold (against AAV 2) lower doses, anc80 transduced nearly 100% IHC and-90% OHC (fig. 2A-2C). 1.36x 10 at the same dose for all serotypes ¹² Transduction under GC resulted in the transduction of large amounts of IHC and OHC by Anc80, but AAV1, 2and 8 were found to target IHC only in very small amounts and were not observed in OHC after live cell imaging by epifluorescence microscopy (figure 8c,8 d).

Samples transduced with Anc80 were subsequently fixed, stained and imaged by confocal microscopy, revealing a dose-dependence of hair cell transduction (fig. 8E). OHC targeting without ethical ratios (figure 2C, figure 8) suggests that Anc80 has qualitatively unique transduction biology compared to other AAV. Similar levels of Anc80 transduction were found throughout the cochlea from base to apex in a total of 3 Anc80 injected mice (fig. 2a, b, c). A low magnification view of the top of the cochlea (fig. 8A) shows that locations distant from the injection site have stronger eGFP expression. High magnification images of the substrate revealed that 100% IHC and 95% OHC were transduced (FIG. 8B).

In some animals, strong eGFP expression was found in the contralateral non-injected ear (fig. 9). In mice, the cochlear aqueduct exclusively provides a fluid pathway from the pericochlear lymph into the CSF, the contralateral aqueduct, and the contralateral cochlea. Therefore, it was also investigated whether Anc80-eGFP injected by RWM could transduce neurons in the brain. Indeed, a cross-section of the cerebellum showed a strong eGFP expression in the cerebellum purkinje neurons (fig. 10a,10 b).

Since some forms of genetic deafness also lead to vestibular dysfunction, anc80 may be a useful vector for gene delivery to the human vestibular apparatus. To investigate this possibility, human vestibular epithelium was collected from 4 adult patients undergoing vestibular schwannomas resection; sensory epithelium was placed in culture as described previously (Kesser et al, 2007, gene Ther, 14. For the transduced samples, fig. 3C shows strong eGFP fluorescence throughout the human vestibular epithelium in hair cells and supporting cells. A high magnification view of the epithelium counterstained with Myo7A in fig. 3D shows that 83% (19/23) of Myo 7A-positive hair cells are also eGFP-positive, indicating that Anc80 is able to efficiently transduce mouse and human hair cells.

Example 1D immunological assay

Antibody titers to Anc80 in CSF and serum were determined by neutralization assays (Zinn et al, 2015, cellreports, 12. Using a 96-well plate format, heat-inactivated CSF or serum samples (collected as described above) were serially diluted in serum-free media (Life Technologies, carlsbad, calif.) followed by the use of Anc 80-luciferase (10) ⁶ GC/well) was treated at 37 ℃ for 1 hour. The sample/Anc 80-luciferase mixture was then transferred to HEK293 cells, which had been treated with adenovirus the previous day (MOI 20). After 1 hour at 37 ℃, diluted serum medium (1 part serum-free, 2 parts serum-containing) was added to each well.

After 2 days, cells were treated with lysis buffer (Promega, madison, wis.) and frozen at-80 ℃ for 30min. The cells were then thawed at 37 ℃ for 15 minutes, followed by treatment with substrate buffer (Tris-HCl, mgCl2, ATP (Life Technologies, carlsbad, calif.), D-fluorescein (Caliper Life Sciences, hopkinton, MA)). Luminescence output was read using a Synergy BioTek Plate Reader (BioTek, winooski, VT).

At the level of assay and sampling sensitivity, low levels of neutralization to the vector were detected in the serum of the injected mice, but not in the CSF (fig. 10C).

Example 1E-Hair cell electrophysiology

The cochlea was cut out, packaged on a glass cover slide and viewed on an Axio exaamino. 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 ℃ C. -24 ℃ C.) in standard solutions (in mM) containing: MEM (Life Technologies, carlsbad, calif.) contained 137NaCl, 5.8KCl, 10HEPES, 0.7NaH ₂ PO ₄ 、1.3CaCl ₂ 、0.9MgCl ₂ And 5.6D-glucose, vitamin (1) 100 and amino acid (1).

The recording electrode (3-4M Ω) was drawn using R-6 Glass (King Precision Glass, claremont, calif.) and filled with an intracellular solution (in mM) containing: 140CsCl, 5EGTA-KOH, 5HEPES, 2.5Na ₂ ATP、3.5MgCl ₂ And 0.1CaCl ₂ (pH 7.4; -280 mOsm/kg). Mechanical conduction currents were recorded using Axopatch 200B (Molecular Devices, sunnyvale, calif.) using a whole cell, tight seal technique. Hair cells were held at-84 mV. The current was filtered at 5kHz using a low pass Bessel filter, digitized at ≧ 20kHz using a 12-bit acquisition plate (Digidata 1440A, molecular Devices, sunnyvale, calif.), and recorded using pCLAMP 10 software (Molecular Devices, sunnyvale, calif.).

Deflection from IHC and O using a rigid glass probe mounted on a PICMA chip piezoelectric actuator (Physik Instrument, karlsruhe, germany) driven by an LVPZT amplifier (E-500.00, physik Instrument, karlsruhe, germany)The flock of HC was filtered at 40kHz using an 8-pole Bessel filter (Model 3384 filter, krohn-Hite Corporation, brockton, mass.) to eliminate residual pipette resonance. Rigid glass probes were designed to fit the concave surface of the hair cell cilia array for full bundle recording (diameter of OHC 3-4 μm and IHC 4-5 μm). For the>Whole cell electrophysiological recording at P10, dissection of cochlear tissue at P5-7 and 5% CO at 37 ℃% ₂ Next, 1% FBS-containing MEM (1X) + GlutaMAXTM-I medium was used for incubation for up to 30 days.

Representative currents resulting from the deflection of the hair bundles by P7OHC and P35IHC showed no difference in amplitude, sensitivity or kinetics between eGFP positive and eGFP negative control cells (fig. 2D). 51 eGFP-positive and 52 eGFP-negative hair cells were scored from all areas of the cochlea and from ages between 1 week and 5 weeks after exposure to Anc80. In all cases, the response was indistinguishable from wild type (fig. 2E), confirming that Anc80 transduction had no adverse effect on sensory cell function.

Example 1F Hearing test

Auditory Brainstem Response (ABR) and distortion product otoacoustic emission (DPOAE) data were collected as previously described (Askew et al, 2015, science relative Med, 7. DPOAE is a measure of the correct cochlear enlargement and tuning and is a sensitive measure of outer hair cell viability (Guinan et al, 2012, hearing Res, 293. The stimuli detected in the anesthetized mice vary at sound pressure levels between 10 and 90dB at frequencies of 5.6, 8, 11.3, 16, 22.6 and 32 kHz. 4 Anc 80-injected and 4 non-injected ears and 1 negative control ear with injection lesion but no EGFP fluorescence were analyzed at P28-P30.

The minimum acoustic threshold required to cause ABR is plotted (fig. 2F) and the results indicate that there is no difference in threshold between injected and non-injected ears. The histological analysis surface had strong eGFP fluorescence in all 4 injected ears (data not shown). In one case, there were no eFGP-positive cells and an elevated ABR threshold (fig. 2F), suggesting that the injection failed and that the needle may have destroyed the cochlear duct and caused permanent damage. Despite the strong outer hair cell transduction effect of Anc80-eGFP, no difference in DPOAE threshold was seen compared to the non-injected control ear (fig. 2G). Thus, data from ABR and DPOAE indicate that RWM injection, anc80 transduction, and transgene expression in IHC and OHC are safe for auditory function.

EXAMPLE 1G-rotating rod test

The equilibrium behavior of 5C 57BL/6 mice was tested on a rotarod apparatus. Mice with impaired vestibular function are known to perform poorly on rotating devices (Parker & Bitner-Glindzicz,2015, archives dis. Childhood,100 271-8. Previous studies highlighted that this rotarod test was able to detect dysbalance only when one ear was affected (Fukui & rapael, 2013, hearing Res, 297. 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 a rotarod rotating at 4RPM for 5 minutes to train their balance. On day 2, mice were tested 5 times, each 5 minutes apart. For each test, the wand was accelerated by 1RPM (Fukui & Rapheal,2013, hearing Res, 297. The time (in seconds) until the mouse dropped off the device was recorded.

Since the perilymphatic solution of the cochlea is continuous with that of the vestibular labyrinth, it was evaluated whether the Anc80-eGFP injection through the cochlear RWM transduced the vestibular sensory organs. In fact, the inclusion of whole tissue specimens of vestibular epithelium showed strong eGFP expression in hair cells type I and type II of the oval sac, a vestibular organ sensitive to gravitational and linear head movements and which is in the semicircular canal, sensitive to rotary head movements (fig. 3a,3 b). Therefore, to address the safety issue that Anc80 transduction may affect balance, rotarod testing was performed using injected mice that have been confirmed to have vestibular expression, which indicates that their vestibular function is similar to the non-injected control (fig. 11).

Section 2 Gene therapy to restore function to Usher syndrome mouse model

Example 2 mouse model of Usher syndrome

The following methods and materials were used in example 2.

Tissue preparation

Oocysts and Corti's organs of Ush1 c.216g > a heterozygotes or homozygous mutant mice were harvested from postnatal day 0 to day 8 (P0 to P8) for electrophysiological studies. Postnatal mouse pups were sacrificed by rapid decapitation. Temporal bones were excised and immersed in MEM (Invitrogen, carlsbad, CA) supplemented with 10mM HEPES (pH 7.4). As before, organ Corti was isolated without the use of enzymes. The oocysts were removed after 10min treatment with 0.1mg/ml protease (protease XXIV, sigma). The excised organs were packaged on circular glass coverslips. A pair of thin glass fibers pre-glued to a cover glass is placed on the edge of the tissue to stabilize it in a flat position. The tissue was used immediately or stored in medium containing 1% fetal bovine serum. Cultures were maintained for 7 to 8 days and the medium was changed every 2 to 3 days for experiments involving viral vector infection in vitro. Animal(s) production

Ush1 c.216G > A knock-in mice were from Louisiana State University Health Science Center. This input line in the C57BL6 background previously had age-related hearing loss due to Cdh23 (Ahl) mutation. Mice were genotyped using either toe clips (before P8) or ear holes (after P8) and PCR as described previously (Lentz et al, 2007, mutat. Res., 616. For all studies, the proportion of male and female mice used was approximately equal. No randomization pattern is otherwise performed.

Production of viral vectors

Total RNA was isolated from c.216aa mutant mouse cochlea (RNAqueous mini kit, ambion) and reverse transcribed using 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 (KpnI) GAG GTA CCA TGG ACC GGA AGG TGG CCC GAG (SEQ ID NO: 9); RV (BamHI) CAG GAT CCG GAC AAT TTC ATC CCC TAC (SEQ ID NO: 10). The 387bp 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 KpnI and BamHI. Trunc-harmonin cDNA was transferred into the AAV shuttle vector using NheI-XbaI EGFP. The custom vector was packaged into the AAV1 capsid with AAV2 Inverted Terminal Repeats (ITRs), in which the transgene expression cassette was driven by the CMV promoter (AAV 2/1.CMV. EGFP:: trunc-harmomin. HGH,1.92E14gc/m, BCH).

Harmonin-a1 and Harmonin-b1 plasmids were prepared in our laboratory from the EGFP-tagged constructs previously claimed by Lily Zheng and James Bartles (Zheng et al, 2010, J. Neurosci., 30. Harmonin-a1 was originally derived from mouse kidney and Harmonin-b1 was isolated from mouse cochlear sensory epithelium. The Harmonin-a1 construct was further modified to use tdTomato at its N-terminus instead of the EGFP tag. Fluorescently labeled and unlabeled constructs are packaged into AAV vectors. Viral vectors were generated by the virus Core laboratory at Boston Children's Hospital and by the Gene Transfer Vector Core at Massachusetts Eye and Ear Infirmy. 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. Mu.l of concentrated vehicle was added to 1ml MEM supplemented medium on immediate dissected tissue in the presence of 1% fetal bovine serum and cultured for 24h. The culture was then maintained for up to 10 days.

Round Window Membrane (RWM) injection

RWM injections were performed according to the animal protocol #15-01-2878R approved by the institutional animal care and use committee of the Boston Children hospital. Newborn mice were injected with 0.8. Mu.l to 1. Mu.l AAV vector at P0-P1 and P10-P12. P0-P1 mice were first anesthetized with cryoexposure and P10-P12 mice were anesthetized with isoflurane. After anesthesia, a retroauricular incision was made to expose the clear bleb and visualize the cochlea. Injections were performed by RWM using a glass microsyringe controlled by a micromanipulator (Askew et al 2015, sci. Trans. Med., 7. The volume of the injected material was controlled at about 0.02. Mu.l/min, and injection was carried out for 10min. Standard post-operative care was used. The sample size of the in vivo study is determined on a continuous basis to optimize sample size and reduce variance.

Electrophysiology recording

Recordings were made in standard artificial perilymph solutions (in mM) containing: 144NaCl, 0.7NaH ₂ PO ₄ 、5.8KCl、1.3CaCl ₂ 、0.9MgCl ₂ 5.6D-glucose and 10HEPES-NaOH, adjusted to pH 7.4 and 320mOsmol/kg. Using the concentrate (Invitrogen, carlsbad, CA) vitamins (1. The hair cells were observed from the top surface using an upright Axioskop FS microscope (Zeiss, oberkochen, germany) equipped with a 63x water immersion objective and differential interference contrast optics. Recording pipettes (3-5M Ω) were drawn using borosilicate capillary Glass (Garner Glass, claremont, CA) and filled with an intracellular solution (in mM) containing: 135KCl, 5EGTA-KOH, 10HEPES, 2.5K ₂ ATP、3.5MgCl ₂ 、0.1CaCl ₂ pH 7.4. The current was recorded at room temperature using a whole cell voltage clamp at a clamping potential of-64 mV. Data were acquired using Axoatch multiclad 700A or Axoatch 200A (Molecular Devices, palo Alto, calif.), filtered at 10kHz using a low-pass Bessel filter, digitized ≧ 20kHz by a 12-bit acquisition plate (Digidata 1322), and used with pClamp 8.2 and 10.5 (Molecular Devices, palo Alto, calif.). Data were analyzed offline using OriginLab software, expressed as mean ± standard deviation unless otherwise indicated.

Statistical analysis

Test and control vectors were evaluated in at least three mice per group at each time point to ensure reproducibility. The sample size is indicated in the legend. All animals that successfully received RWM injections were included in the study analysis. Those animals that failed injections were excluded from the mean values, but included in the legend for full disclosure. Injection success was determined from ABR recovery (threshold >90dB SPL). Statistical analysis was performed using Origin 2016 (Origin Lab Corporation). As described in text and legend, data are presented as mean ± Standard Deviation (SD) or standard error of the mean (SEM). Significance of differences between the means was determined using one-way analysis of variance (ANOVA).

Example 2A Scanning Electron Microscopy (SEM) in the mouse Usher model

SEM was performed on Corti's organs of control and mutant mice at P7, P18 and P42 (6 weeks). The P18SEM was performed in cooperation with doctor Edwin Rubel, university of Washington. The inner ear was fixed in 0.1M sodium phosphate containing 4% glutaraldehyde at 4 ℃ overnight. The next day the specimens were washed three times in 0.1M sodium Phosphate Buffer (PB) and postfixed in 0.1M PB containing 1% osmium tetroxide for 30min in an ice bath. The specimens were then washed in 0.1M PB and dehydrated stepwise by gradient ethanol: 35%, 70%, 95% and 100% (x 2). The samples were dried at the critical point, placed on a SEM column and sputter coated with Au/Pd. SEM analysis was performed using a JEOL JSM-840A scanning electron microscope. Similar preparations were performed for the P8 and 6 week phases. Use 2mM CaCl supplement ₂ 2.5% glutaraldehyde in 0.1M cacodylate buffer (Electron Microcopy Sciences) Corti's extra-organ plants were fixed for 1h at room temperature. Dehydrating the specimen in acetone step by step using liquid CO ₂ Critical point drying was performed, sputter coated with 4-5nm platinum (Q150T, quorum Technologies, united Kingdom) and observed using a field emission scanning electron microscope (S-4800, hitachi, japan).

Homozygous c.216aa mutant mice were deaf and showed vestibular disorder features manifested by circling and panning behaviors. Previous work (Lentz et al, 2010, dev., neurobiol., 70. At 1 month of age, degeneration and hair cell death were also observed at the point of return, but the top of the organ remained better. Assuming gradual hair cell degradation during inner ear organ development, SEM analysis of Corti organs was performed at P8 and P18 in order to assess early stage hair cell survival. At these ages, outer Hair Cells (OHC) and Inner Hair Cells (IHC) of heterozygous c.216GA mice were retained and their hair bundles were correctly oriented (FIGS. 12A-12C,12G,12I and FIGS. 19A-19C, 19K). However, at both ages analyzed, the homozygous c.216AA mice showed significant disassembly of the full-length hair bundles in the Corti organ (FIGS. 12D-12F,12H,12J-12L and FIGS. 19D-19J, 19L). At P8, the IHC bundles at the basal, medial and apical regions are slightly scrambled (FIGS. 12D-12F, 12J). Many IHC tracts exhibited a wavy pattern and slightly disorganized rows of cilia (fig. 12J). Although many OHCs of c.216aa mutant mice had well-preserved hair bundles (fig. 12h, 12k), distinct fragmented and disintegrated hair bundles were sporadically visible along the organs (fig. 12D-12f, 12l). Although most hair cells remained as reported previously (Lentz et al,2013, nat. Med., 19.

To assess the morphology of hair bundles in mice using the harmonin-b1 gene therapy, temporal bones of untreated (or non-injected) and treated (or injected) 6-week-old mice were prepared for SEM analysis. Untreated c.216aa mice showed severe hair cell loss at the basal and mid-regions of the organ (fig. 18). In the base area, OHCs are mostly absent in the first row, sporadically present in the second and third rows. In the middle region of the organ, the first row is also substantially free of OHCs. A more mild phenotype was observed in the apical end. High magnification SEM also showed that c.216aa mutant mice exhibited severe tuft breakdown throughout the length of the organ. Notably, in 6-week-old c.216aa mice, no tufts were observed in all three rows of the typical ladder structure of static cilia retention. In contrast, hair cells from c.216aa mice showed a disorganized bundle with retracted cilia along the first row, and the second row was abnormal and remained very intact. In contrast, hair cell loss reduction and normal hair bundles were observed in c.216AA mice treated with harmonin-b 1. Hair cell techniques are estimated from the presence or absence of hair bundles in a typical field of view.

The data showed that the hair cell number from the base to the top of the organ was very significantly maintained in the injected mice, 40 to 79% at the base, 68 to 95% in the middle and 93 to 99% at the top (n =1824 cells from n =4 c.216aa mouse ears and n =792 cells from n =2 rescued c.216aa ears). Although abnormal hair bundles were still evident in the mice injected with harmonin-b1, most of the hair bundles had three rows of static cilia and their morphology was hardly distinguishable from their heterozygous controls (FIG. 18).

Example 2B-FM 1-43 imaging in Usher mouse model

5 micromolar FM1-43 (Invitrogen) was prepared diluted with extracellular recording solution and applied to the tissue for 10 seconds, then washed 3 times in extracellular recording solution to remove excess dye and prevent uptake by endocytosis. After 5 minutes, intracellular FM1-43 was imaged by water immersion 20x, 40x and 63x objective using a Zeiss Axioscope FS equipped with an epi-fluorescent light source, differential interference contrast optics and FM1-43 filter set (Chroma Technologies). Images were captured at 16 bits after background fluorescence was subtracted using a CCD camera and Argus-20 image processor (Hamamatsu). All images taken were kept at the same gain and contrast settings and analyzed offline using Adobe Photoshop or Image-J software.

To assess hair cell function at an early stage, uptake of FM1-43 in the inner ear organs was analyzed immediately at P4. After a short application (< 10 seconds), FM1-43 permeates hair cells with functional mechanically sensitive channels. Uniform FM1-43 uptake was observed in hair cells of c.216ga mice (fig. 13A), but the level of uptake of OHCs was different in c.216aa mice, indicating that some but not all cells retained functional transduction channels (fig. 13B). Similar observations were made over the entire length of the cochlea. No difference in tone quality distribution was found. FM1-43 uptake in IHC was also reduced in c.216aa mice in the first week after birth (data not shown). The uptake of FM1-43 was also assessed in the oocyst hair cells of the mutant mice. Interestingly, in the c.216aa mutant mice, uptake was restricted to the region outside the furrow at P6, indicating that hair cells in the furrow region lack mechano-sensitive channel opening at rest (fig. 13c, 13d).

Example 2C mechanical stimulation in the Usher mouse model

OHC and IHC: mechanical stimulation was transmitted through a rigid glass probe mounted on a 524-dimensional PICMA chip piezoelectric actuator (Physik Instruments, waldbronn, gernamy) driven by a 400mA ENV400 amplifier (Piezosystem Jena Germany). The tip of the probe was Fire polished (Fire polish, H602, world Precision Instruments inc., sarasota, FL) to fit into the bundle of cilia (Stauffer & Holt,2007, j.neurophysiol, 98. The deflection is induced by applying a voltage step, which is filtered at 50kHz with an 8-pole bessel filter (Khron-Hite, 528 brockton, ma) to eliminate residual pipette resonance. The hair cell deflection was monitored using a C2400 CCD camera (Hamamatsu, japan). The voltage step was used to correct for the movement of the stimulation probe around its resting position ± 2 μm. Video images of the probe were recorded to confirm no off-axis motion and to correct for probe motion (spatial resolution 4 nm). The rise time of 10-90% of the probes was 20. Mu.sec.

VHC: mechanical stimulation was delivered through a rigid glass probe mounted on a piezoelectric bimorph element. Coupling was performed by gently aspirating the motile cilia into a stimulating pipette. The deflection is induced by applying a voltage step to a piezoelectric device consisting of two bimorphs mounted in series and coupled directly to the stimulation probe. The voltage steps were controlled by pClamp 8.0 software and filtered at 1kHz with an 8-pole Bessel filter (Khron-Hite, brockton, MA). The tuft deflection was monitored using a C2400 CCD camera (Hamamatsu, japan). Prior to the experiment, the motion of the stimulation probe was corrected to be near its resting position (± 2 μm).

During the first week after birth, the auditory and vestibular epithelia retain mechanically sensitive hair cells. Including some hair cells with relatively normal morphology (fig. 12). In the organ of Corti, recordings were obtained from the middle and apical gyrus of the cochlea of P3 to P6c.216aa mice from hair cells with apparently normal hair bundles and hair bundles with more severe destruction. In the c.216aa mutant, OHC retained mechano-sensitivity, but the magnitude of their responses was significantly reduced from-63% to 170 ± 80pA (n =24 p- <0.001, fig. 13e,13f, 13g). A broad response amplitude was observed in OHCs of c.216aa mice, which was between 31 and 292 pA. Significant differences were observed when grouping data according to tuft morphology (p < 0.01): the currents induced in mutant hair cells that severely disintegrate the hair bundles are less than those induced in mutant cells that retain more of the hair bundles, 120 ± 65pA (n = 9) and 201 ± 74pA (n = 15), respectively. The response of the capillaries to mechanical displacement retained similar properties to those of heterozygous c.216ga mice, despite the reduced current amplitude. The stimulation response [ I (X) ] curve was fitted using the second order boltzmann equation (fig. 13F), and the working range of 10-90% was determined using the fit (fig. 20B). No significant difference in working range was observed between OHCs recorded from c.216ga and c.216aa (p = 0.054). Similarly, although mild disruption of the hair bundles of IHC was observed in c.216aa mutant mice under DIC microscopy, the conduction current at P6 was significantly reduced (fig. 13e,13f, 13g). The maximum conducted current (P6-P7) in the heterozygous c.216ga IHC averaged 587 ± 96pA (n = 21) at a clamp potential of-64 mV, but decreased by 46% in c.216aa IHC to 316 ± 127pA (n = 19P < -0.001. A significant reduction in working range (p < 0.01) was measured in IHC of c.216aa mutant mice (fig. 20G).

Adaptation was defined as the drop in conduction current in the presence of a constant beam offset, which is also present in c.216aa mutant mice. The adaptation kinetics were analyzed using a bi-exponential fit to determine the fast and slow components. Although both components in c.216AA mutant mice IHC and OHC slowed down, the difference was only significant for the slow component (p <0.05 in OHC, and p <0.001 in IHC; FIG. 20C,20D,20H, 20I). On the other hand, the degree of adaptation measured at Popen =0.5 was significantly lower in OHC and IHC of c.216aa than in hair cells of c.216ga (fig. 20e,20j p-straw-0.001. Taken together, these results indicate that mechanical sensitivity is slightly impaired in the inner and outer hair cells of c.216aa mice, and it is important that both cell types survive the first week after birth, a prerequisite for gene therapy and restoration of cell function.

In the forecourt hair cells, a decrease in mechanically conductive current was also observed in c.216aa mice. In the region outside the grooves, the c.216aa current decreased significantly to 109 ± 30pA (n =9, P5-P7) (P < 0.001) compared to the current 231 ± 53pA (n =8, P6-P7) of c.216ga (fig. 13e,13f, 13h). Only very little or no current was recorded in the groove region (6. + -. 13pA, n =6, P5-P7), consistent with the lack of FM1-43 uptake in this region (see below; FIG. 13C, 13D). Although the oocyst hair bundles were shown to remain intact by DIC microscopy in general, there was a significant reduction or absence of conduction current in hair cells outside and in the furrow, respectively. Thus, these results indicate that the transduction device is correctly assembled and targeted in mutant mice, but that the number of functional complexes is reduced in neonatal mice, except for the groove region.

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, unlabeled harmonin-a1 or harmonin-b1 coding sequences guided by CMV promoters were packaged into AAV capsid proteins called Anc80 (Zinn et al 2015, cell Rep.,12 1056-68). As shown herein, the Anc80 capsid protein transduces 100% IHC and 80-90% OHC in vivo. It is speculated that harmonin-b is required for mechanical conduction in IHC and OHC and is essential for auditory function of both cell types. RWM was injected with a mixture of AAV2/Anc80.CMV.harmonin-b1 (0.8. Mu.l, 1.9X 10^ 12gc/ml) and AAV2/Anc80.CMV.harmonin-a1 (1.7X 10^ 12gc/ml) alone plus AAV2/Anc80.CMV. Harmonin-b1 (0.5. Mu.l + 0.5. Mu.l) and mechanical transduction response was evaluated 2 weeks after treatment.

Tissues were extracted at P5-P6 and maintained in culture for 10 days prior to cochlear ossification. Although mature OHC (> P10) did not survive ex vivo recording conditions, stable electrophysiological recordings were obtained from IHC at P14-P16 equivalents. The results are shown in FIG. 15. Although IHC from non-injected mice showed a significant reduction in conduction current at P16 (79 ± 43pa, n = 8), the recovery of sensory conduction was evident in mice receiving AAV treatment. Significant recovery was observed in injected mice at P1 (× P < 0.001), with mean maximum conduction of 388 ± 66pA (n = 15) and 352 ± 28pA (n =7; fig. 15C), respectively, using harmonin-b1 or a combination of b1 and a 1. There was no significant difference in the magnitude of the conduction current after treatment with harmonin-b1 compared to control c.216ga mice. Co-injection of harmonin-b1 and harmonin-a1 did not significantly alter the level of recovery. These results indicate that delivery of exogenous harmonin-b1 by RWM injection at an early stage can restore mechanical conduction in IHC.

Example 2D confocal imaging in Usher mouse model

To prepare tissues for confocal imaging from postnatal mice of P0-P8, 4% Paraformaldehyde (PFA) was used for fixation for 15 minutes. Triton permeabilization was performed using 0.01% and counterstained using Alexa Fluor phalloidin (Invitrogen, 1/200) to tag actin filaments. Images were obtained on a LSM700 Zeiss confocal microscope. In mice of slightly older weeks (4 to 8 weeks), the temporal bones were removed after sacrifice and placed in 4-vol pfa for 1 hour, followed by decalcification with 120mM EDTA for 24 to 36 hours. The sensory epithelium was then isolated and injected for immunostaining as described above. The banded synapses were labeled by incubation with mouse anti-CTBP 2 (BD bioscience #612044, 1/200) for 48 hours and counterstaining with Alexa Fluor goat anti-mouse (1/200) overnight at 4 ℃. Images were acquired using a Zeiss LSM 710 laser confocal microscope (IDDRC Imaging Core print P30 HD 18655) and processed using a Zeiss LSM image viewer 4.2.

Previous work revealed that in sensory hair cells, harmonin is expressed in two alternative spliced forms. To assess the ability of AAV vectors to drive expression of the exogenous harmonin splice form, the oocyst and Corti organs from neonatal c.216AA and wild-type (C57 BL/6J) mice were exposed to AAV2/1 vectors encoding eGFP (eGFP:: harmonin-b 1) fused to the N-terminus of harmonin-b1 or tdTomato (tdTomato:: harmonin-a 1) fused to the N-terminus of harmonin-a 1. The vector was applied at P1 by RWM injection (1. Mu.l) in vitro or in vivo. When applied in vitro, P0-P1 tissues were incubated with the vehicle for 24 hours and maintained in culture for 1 week. Confocal images indicated successful transduction of hair cells from wild-type, c.216GA, and c.216AA mice (FIGS. 14A-14C, 14E). The Hamonin-b 1 signal is evident at the tip of the quiet cilia in VHC (FIG. 14A), IHC and OHC (FIG. 14B, 14C). At P60, EGFP signals were also detected in OHC and IHC at the base of the cochlea of P1-injected mice (fig. 14D). TdTomato:harmonin-a 1 was detected at the base of auditory hair cells (FIG. 14E). Co-staining with the banded synaptic marker CTBP2 showed that there was general co-localization in P7IHC (fig. 14E), but no co-localization in P7 oval sacs (data not shown).

Localization of the exogenous fusion construct was consistent with previous work in which harmonin-b was localized to the distal end of the static cilia, near the tip junction insertion, and harmonin-a was localized to the synapse.

Example 2E Auditory Brainstem Response (ABR) and aberration products (DPOAE)

ABR and DPOAE were recorded from mice anesthetized with xylazine (5-10 mg/kg i.p.) and ketamine (60-100 mg/kg i.p.). Inserting a subcutaneous needle electrode into the skin: a) The back side between the ears (reference electrode); b) Behind the left auricle (recording electrode); and c) the back side of the animal's buttocks (ground electrode). The channel at the base of the pinna is trimmed to expose the ear canal. For ABR recordings, 5 milliseconds short tones are provided to the ear canal and hearing device (EPL Acoustic system, MEEI, boston). The responses were amplified (10,000 times), filtered (0.1-3 kHz) and averaged using analog-to-digital plates in a PC-based data acquisition system (EPL, cobalt function test suite, MEEI, boston). The sound level is raised from 0 to 110dB sound pressure level (decibel SPL) in steps of 5 to 10 dB. In each level, the average 512 to 1024 responses (alternating with stimulus polarity) after "artifact exclusion" occurred. The threshold was determined by visual inspection. Data were analyzed and plotted using Origin-2015 (Origin lab Corporation, MA). Unless otherwise stated, the threshold values are expressed as mean ± standard deviation. For DPOAE, f1 and f2 dominant tones (f 2/f1= 1.2) are provided, where f2 varies between 5.6 and 45.2 in chromatic steps, and L1-L2=10dB SPL. In each f2, L2 varies between 10 and 80dB SPL in increments of 10dB SPL. The DPOAE threshold is defined by the mean spectrum as the L2-level, i.e., the DPOAE that causes an SPL of the order of 5dB above the noise floor. The average noise floor level at all frequencies was below 0dBSPL. In our custom acoustic system, stimuli were generated using a 24-bit digital I-O card (National Instruments PXI-4461) in a PXI-1042Q cabinet, amplified by an SA-1 speaker driver (tuner-Davis Technologies, inc.) and delivered by two electrostatic drivers (CUI CDMG 15008-03A). An electret microphone (Knowles FG-23329-P07) at the end of a small probe tube was used to monitor the ear canal sound pressure. Most of these experiments were not performed under blind conditions.

To determine whether a truncated harmonin interferes with normal auditory function, an anc80.Cmv. Trunc-harm vector was generated to overexpress the truncated protein. The vector was injected by RWM into the inner ear of c.216ga mice. ABR and DPOAES were measured at 4,6 and 12 weeks, and no difference was found between the threshold values for injected and non-injected c.216ga mice (records from 6-week-old mice shown in fig. 23C-23D). Using this data as a control for injection technology and vector, it was important to consider that the exogenous truncated harmonin did not compete with the endogenous full-length harmonin, suggesting that the endogenous truncated forms in c.216aa hair cells are unlikely to interfere with the expression of the exogenous full-length harmonin by gene therapy vectors.

To determine whether harmonin gene amplification could rescue auditory and balance function in Ush1c mice, RWM injections of AAV2/Anc80.CMV.harmonin-a1 (0.8. Mu.l, 1.7X 10^ 12gc/ml) or AAV2/Anc80.CMV.harmonin-b1 (0.8. Mu.l, 1.9X 10^ 12gc/ml) were performed at P0-P1 and Auditory Brainstem Response (ABR), distortion product otoacoustic emission (DPOAE), acoustic startle reflex, field absence, and rotarod behavior were evaluated. The mice were evaluated at 6 weeks, when c.216aa mice developed severe hearing loss and vestibular impairment. Some mice were further tested at 3 and 6 months.

None of the 12 mice injected with AAV2/Anc80.CMV. Harmonin-a1 restored hearing function at 6 weeks (FIGS. 16A-16C), indicating that exogenous expression of harmonin-a1 was insufficient for hearing rescue. However, 19 of 25 mice injected with AAV2/anc80.Cmv. Harmonin-b1 had a clear recovery of auditory function at 6 weeks. At low frequencies (5.6 to 16 kHz), the optimal ABR threshold in AAV2/Anc80.CMV. Harmonin-B1 injected ears was 25-30dB SPL, a very similar threshold to that of wild type mice (FIGS. 16A-16B). Partial rescue was observed at 22.6kHz with little rescue at 32 kHz. Rescue of DPOAE threshold was also evident, consistent with rescue of function in OHC (figure 16C). In the late stage, 8 mice with auditory threshold <45dB SPL at 8-11.3kHz stimulation were tested to assess the duration of rescue. From 6 weeks to 3 months, -10 dB SPL ABR threshold migration was observed in the low frequency range and-30 dB SPL ABR threshold migration was observed in the high frequency range (fig. 16D). Similar migration was observed in DPOAE thresholds (fig. 16E). After this time point, ABR threshold and DPOAE still stabilized to 6 months of age (fig. 16D-16E), which is the last time point tested.

To assess whether both harmonin-a1 and harmonin-b1 are required for more complete hearing rescue (particularly at the high frequency end), AAV2/Anc80.CMV. TdTomato:: harmonin-a1 (0.5. Mu.l; 238.4.1E. Sup. 12gc/ml) and AAV2/Anc80.CMV. EGFP:: harmonin-b1 (0.5. Mu.l; 3.0E. Sup. 12gc/ml) were co-injected. As can be seen from the two fluorescence-tagged positive cells, 65% of the hair cells expressed both harmonin-a1 and harmonin-b1 (FIG. 21). Fluorescently labeled harmonin-a1 is sometimes observed in the static cilia of mice exposed to AAV2/Anc80.CMV. TdTomato:harmonin-a 1, perhaps due to overexpression. ABR and DPOAE thresholds in mice co-injected with unlabeled harmonin-a1 and harmonin-b1 vectors (fig. 16) were similar to mice injected with harmonin-b1 alone, providing no further improvement, suggesting that harmonin-a1 may be unnecessary for auditory function. Importantly, the data show that harmonin-b1 alone is sufficient to significantly restore the hearing threshold at low frequencies (FIG. 16).

To further evaluate the extent of rescue, the ABR waveforms of mice with a threshold of 45dB SPL or less were analyzed and compared between 8 control c.216GA mice and 5 c.216AA mice injected with AAV2/Anc80.CMV. Harmonin-b 1. Analysis of responses at 8-11.3kHz and 16kHz showed normal 1 st wave amplitude (no significant difference, P >0.2, student t-test) and longer 1 st peak delay (P > 0.001) (FIG. 22), suggesting that neurotransmission in the synapse may lag. In many animals, auditory rescue was also observed in the contralateral ears, which had an ABR threshold as low as 20dB SPL at 11.3kHz (harmonin-b 1: 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 into the contralateral ear has been previously observed, which may occur through the perilymphatic vessels that remain continuous with the subarachnoid space in neonatal mice.

We also examined whether injection at a late developmental stage could result in partial hearing rescue. AAV2/Anc80.CMV. Harmonin-b1 (0.8. Mu.l) RWM injections were made at P10-P12 and hearing thresholds were evaluated at 6 weeks. None of the P10-P12 injected mice had detectable DPOAE and no difference in ABR threshold from the non-injected c.216aa control mice (n =10; data not shown), suggesting that the window of opportunity for intervention may be limited to the early postnatal stage, probably due to lower efficiency of viral transduction in older tissues or degeneration of the organ of Corti in the late developmental stage.

Example 2F-RT-PCR in the Usher mouse model

Use ofReverse transcription kit (Qiagen) from P2-P3 wild type, heterozygous and pure and Ush1 c.216G>cDNA was prepared from 6 auditory organs of A mice. The 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 G3' (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 the mouse Ush1c sequence and will amplify endogenous and AAV 2-derived Ush1c, since the target sequence is located outside the region of the human knock-in Ush1c.216a allele part. DNA and RNA levels in mouse tissues collected 6 weeks after treatment were also assessed. TRIzol reagent (Life technology) was used according to the manufacturer's protocols, carlsbad, CA) isolated DNA and RNA from cochlea. The RNA was reverse transcribed using the GoScript reverse transcription system (Promega, madison, wis.). Radiolabelled PCR was performed using GoTaq Green Master Mix (Promega, madison, wis.). 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.

These primers will only amplify the viral Ush1C DNA, since homozygous Ush1c.216aa mice knock in the human Ush1c.216a gene in exons 3 and 4, which replaces the mouse sequence (Lentz et al, 2007, mutat.res., 616. For cDNA amplification of full length (450 bp) and aberrant spliced/truncated harmonin (415 bp), the same primers as described above were used ((mUsh 1c _ Ex2F and mUsh1c _ Ex 5R). Gapdh primers were mGapdh _ Ex3F (5 '-611 GTG AGG CCG GTG CTG AGT ATG-3' (SEQ ID NO: 15)) and mGapdh _ Ex4R (5-.

Since previous studies suggest the possibility that truncated harmonin might disrupt its function by competing with full-length harmonin as an endogenous binding partner, it was investigated whether sustained expression of the truncated protein might limit recovery in c.216aa mice injected with vectors expressing endogenous full-length harmonin (fig. 23A). To address this problem, the expression of Ush1c transcripts in c.216ga and c.216aa mice was examined using RT-PCR assays. Consistent with previous reports, ush1c transcripts encoding full-length and truncated harmonin were detected in c.216ga cochlea, and only transcripts encoding truncated harmonin were detected in c.216aa cochlea (fig. 23B).

To confirm AAV2/anc80.Cmv. Harmonin-b1 expression and explore the relationship between viral expression levels and ABR threshold, DNA and RNA were isolated from the injected and contralateral cochlea and quantified by PCR and RT-PCR, respectively. Expression was assessed in c.216GA 6 weeks old and in AAV2/Anc80.CMV. Harmonin-b1 (0.8. Mu.l; 1.93 10^ 12gc/ml) injected and non-injected c.216AA mice. The samples included 2 injected mice with better ABR rescue (threshold ≦ 35dB SPL at 11.3 kHz) and 2 injected mice with poor ABR rescue (threshold ≧ 90dB SPL at 11.3 kHz). RNA encoding the correctly spliced form of harmonin (fig. 24A) and AAV2/anc80.Cmv. Harmonin-B1 DNA (fig. 24B) were detected in all cochlea injected and to a lesser extent in the contralateral cochlea of all animals tested.

ABR thresholds and the amount of DNA and RNA expression varied between animals (fig. 24C). However, a strong correlation was found between AAV2/anc80.Cmv. Harmonin-b1 DNA level, the amount of RNA encoding the correct spliced form of harmonin, and the ABR threshold level, suggesting that variability in the ABR data may be a direct result of AAV expression. To assess long-term survival of hair cells in mice that have successfully restored ABR thresholds, tissue preparation and IHC and OHC counts were performed on 5 mice at 6 months of age (fig. 25). Although the number of IHCs in both cohorts did not change, 50% or more of OHCs were retained in 3 mice showing long-term ABR rescue. OHC survival was observed in all organs except baseline (fig. 25).

Example 2G Acoustic startle response in Usher mouse model

Acoustic Startle Response (ASR) was measured using a startle monitor (kidder Scientific). Mice were placed in a small-sized, non-limiting cubic plexiglass recording chamber (27 cm x10 cm x 12.5 cm) fixed on a piezo/plexiglass sensing assembly and allowed to adapt to 60dB SPL background white noise for 5 minutes. Each group consisted of 35 trials during which a single noise pulse ranging from 60-120dB SPL at 10dB SPL intensity was provided at an average 30s (ranging from 25-35 s) inter-trial interval. The pulses are arranged in a pseudo-random order under a constant 60dB SPL background noise to limit external noise interference. The startle monitor reduces the response to each pulse to the longest time (ms) to measure the first N, max N, and response to calculate the peak startle response (ASR amplitude) and the time from stimulus to peak startle response (ASR latency). ASR was performed in the blind state.

To assess whether ABR/DPOAE recovery produced behavior-related recovery of auditory function, acoustic startle responses were measured in mice injected with AAV2/anc80.Cmv. Harmonin-a1, AAV2/anc80.Cmv. Harmonin-b1, and both vectors. Analysis of the startle response 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 (fig. 17A). Mice receiving only harmonin-a1 were similar to c.216AA non-injected mice and showed no recovery of the startle response.

Example 2H vestibular assessment in Usher mouse model

Vestibular function was assessed using the open field and rotarod balance test. Open field testing was performed using a circular frame 42cm in diameter, placed in a sound room with LED illumination on top, set to 30lux in the center, and in a dark room. One mouse was placed in a round open field at a time and allowed to explore for 5min. Ethovision XT was used to record and track behavior so that distance traveled and speed could be measured. Open field evaluations were performed blindly. Rotarod performance experiments involved placing mice on rods in a closed housing, which were initially spun at 4rpm and 0.1rpm s ^-1 Is accelerated. Mice were familiarized with the apparatus by placing them on the bar for 5min on day 1. The following day, animals were placed on the rods for a total of 5 trials. A 5min rest period was set between trials. The timer shows the length of time that the animal can stand on the device before falling onto the instrument housing plate and is recorded after each test batch.

Since the perilymphatic space between the cochlea and the vestibular labyrinth is continuous, it is also possible to transduce the vestibular sensory organs by RWM injection of AAV vectors. To assess vestibular behavior, mice were tested for their performance on a rotarod. Although the rotarod performance observed in c.216aa and c.216aa mice injected with AAV2/anc80.Cmv. Harmonin-a1 was poor (< 22sec on average to the latency of a fall), c.216aa mice injected with AAV2/anc80.Cmv. Harmonin-B1 and co-injected with harmonin-a1 and-B1 vectors maintained a balanced function on the rotarod for 60-120 seconds, consistent with control c.216aa mice (fig. 17B).

Recovery of open field behavior was also observed in harmonin-b1 and harmonin a1 and b1 double-injected c.216AA mice. A representative open field exploration trajectory is shown in fig. 17C. c.216ga mice explored the borders of this 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 as rotations/min (fig. 17D-17E). Surprisingly, while ABR rescue was not observed in mice injected with AAV2/anc80.Cmv. Harmonin-a1, open field data indicated that their vestibular function returned to the levels 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 indicating the absence of interference between truncated and wild-type harmonin (fig. 17C-17E).

Behavioral assays indicated that partial vestibular rescue was produced using harmonin-a1 because of elimination of rotational behavior, but mice injected with harmonin-a1 failed the rotarod test. On the other hand, mice injected with harmonin-b1 had functional recovery in both tests (FIG. 17). The absence of transduction and FM1-43 uptake in the groove region suggests that hair cells and possibly type I cell function in the groove region may be dependent on correct harmonin expression (FIG. 13).

Although the auditory rescue was significant at low frequencies rather than high frequencies (fig. 16), preservation of hair bundle morphology was observed at 6 weeks in the whole organ (fig. 18). The absence of rescue at high frequencies is unlikely to be due to injection injury. High frequency hearing loss was observed in any of the c.216ga injected with AAV vector (fig. 23C-23D). By way of explanation, AAV targeting the entire length of the cochlea disproves the lack of transduction efficiency at the base. One possibility is the presence of other harmonin isoforms, such as brehmonin-c, which may be necessary for rescue function at the high frequency end of the cochlea base. Alternatively, as cochlear development begins at the basal end, it is likely that at P0 the hair cells at the high frequency end of the basal portion have matured beyond their point of repair. If this is the case, performing embryo intervention may result in better rescue of the high frequency zone.

Part 3 Gene therapy for other mutations involved in hearing loss

Example 3A-bodyInternal experiment

An Anc80 vector carrying the mouse TMC1 coding sequence driven by the modified CMV promoter was generated using a helper virusless system and double transfection method as described previously (Grimm et al, 2003, mol. Ther.,7 839. The triple FLAG-tag (FLAG) sequence was fused to the C-terminus of the TMC coding sequence to enable visualization of the expressed protein. The Anc80-CMV-Tmc vector was purified using a discontinuous gradient of iodixanol followed by ion exchange chromatography. Titers ranged from 1 × 10 by quantitative PCR using primer pairs specific for the human β -globin intron element ¹² To 1X 10 ¹³ gc/ml. The virus aliquots were stored at-80 ℃ and thawed prior to use.

Mice aged P0-P2 were used for in vivo delivery of viral vectors according to the protocol approved by the institutional animal care and use committee of boston children hospital (protocol #2659, # 2146) as follows. C57BL/6J (Jackson Laboratories) or the Swiss Webster mouse strain (Taconnic) were used as wild type control mice, as well as mice carrying the TMC1 mutant allele (TMC 1. Delta./. Or Tmc 1-/-) as described previously (Kawashima et al, 2011, J. Clin. Invest.,121 4796-809) were C57BL/6J background.

To prepare the tissues for evaluation, temporal bones were collected from mouse pups at P0-P10. Pups were sacrificed by rapid decapitation and temporal bones were dissected in pH 7.40 MEM (Invitrogen) supplemented with 10mM HEPES, 0.05mg/ml ampicillin, and 0.01mg/ml ciprofloxacin. The membranous labyrinth was isolated in the dissected field, reissner's membrane was peeled off, and the tectorial membrane and vascular striations were mechanically removed. The organ cultures of Corti were laid flat under a pair of thin glass fibers, one end of which was adhered to an 18mm circular glass cover glass with Sylgard. The tissues were immediately used for electrophysiological studies. For mice older than P10, CO inhalation ₂ The temporal bone was collected after the animal was sacrificed and cochlear whole tissue specimen inclusion was prepared.

All mean and error bars in the figure represent the mean ± SD. Statistical significance was compared between injected and non-injected ears using a two-sided paired t-test. P <0.05 was considered significant.

EXAMPLE 3B in vivo injection of viral vectors

Mouse pups (P0-P2) were injected through a Round Window Membrane (RWM) using a beveled glass microinjection pipette. The pipette was drawn out on a P-2000 pipette drawing Instrument (Sutter Instrument) using a glass capillary tube and obliquely cut (tip diameter 20 μm,28 ℃ angle) using a micropipette oblique cutter (Sutter Instrument). The surgical site (left mastoid) was covered with EMLA cream (lidocaine 2.5% and prilocaine 2.5%) using a sterile swab for analgesia. The body temperature was maintained by placing the pad on a 37 ℃ warming pad for 30-60 minutes prior to surgery.

The pups were anesthetized by rapid induction of hypothermia for 2-3 minutes until loss of consciousness, and held in this state on a cold plate for 10-15 minutes during surgery. The surgical site was sterilized by scrubbing with biot iodine and rubbing three times with 70% ethanol. A retroauricular incision was made to expose the clear bleb, and a micropipette (MP-30, sutter Instrument Company) was advanced through the bleb and overlying fascia, and passed through the RWM with the micropipette tip.

Titers were adjusted to 10. Mu.l/min using a pneumatic microsyringe (WPI Nanoliter 2010) ¹² To 10 ¹⁴ gc/mL (10 total virus particles) ⁹ To 10 ¹¹ ) Approximately 1. Mu.l of virus was injected unilaterally into the left ear. The skin incision was closed using 6-0 monofilament suture (Ethicon). The pup is then returned to the warm mattress for recovery.

Example 3C-immunofluorescence

Immunostaining was performed to determine the distribution of transgene expression delivered by the viral vector. For this purpose, immunostaining was performed on freshly dissected organ of Corti, which was fixed by immersion at room temperature for 1h using 4% paraformaldehyde diluted in PBS. Tissues were then washed in PBS, permeabilized in 0.01-0.1% triton X-100 for 30min, and counterstained for 1h with AlexaFluor 546-phalloidin (Molecular Probes,1, 200 dilution) to label actin filaments.

For localization of exogenously expressed TMC:FLAGfusion protein, tissues were blocked using 2% BSA and 5% normal goat serum for 1 hour and incubated overnight at 4 ℃ using antibodies against the FLAG motif (BD Biosciences,1 dilution 200. For hair cell counts, tissues were blocked in normal goat serum for 1 hour, stained overnight at 4 ℃ with rabbit anti-myosin VIIa primary antibody (Proteus Biosciences,1, 1000 dilution), and labeled with goat anti-rabbit antibody conjugated to AlexaFluor488 (Life Technologies,1, 200 dilution) for 1 hour. Samples were mounted on glass coverslips with Vectashield mounting (Vector Laboratories) and imaged at 10X-63X magnification using a Zeiss LSM700 confocal microscope.

Fig. 26 shows immunofluorescence demonstrating uniform Anc80 delivery of Harmonin to Ush1c mutant mice, and fig. 28 shows immunofluorescence demonstrating delivery of KCNQ4 to cellular Anc80 in KCNQ4 mutant mice. Thus, anc80 is an effective vector for the treatment of a variety of different genetic defects (at a variety of different loci) that lead to hearing loss.

Example 3D-Hair cell electrophysiology

Soaking the organ type cochlear culture in a solution containing 137mM NaCl and 0.7mM NaH ₂ PO ₄ 、5.8mM KCl、1.3mM CaCl ₂ 、0.9mM MgCl ₂ 10mM Hepes and 5.6mM D-glucose. To the solution was added vitamin (1 (50) and amino acid (1. Recording pipettes (3 to 5 megaohms) were drawn using R6 capillary Glass (King Precision Glass) and filled with an intracellular solution containing: 135mM CsCl, 5mM Hepes, 5mM EGTA, 2.5mM MgCl ₂ 、2.5mM Na ₂ Adenosine triphosphate and 0.1mM CaCl ₂ Wherein the final pH was adjusted to 7.40 (285 mosmol/kg) using CsOH. Whole cell, tight seal voltage clamp recordings were made at room temperature (22 ℃ to 24 ℃) and-84 mV using Axomatch 200B (Molecular Devices). Sensory conduction currents were filtered at 10kHz using a low pass Bessel filter, digitized at ≧ 20kHz using a 16-bit acquisition plate (Digidata 1440A), and recorded using pCLAMP 10 software (Molecular Devices). Data were stored and analyzed offline using OriginPro 8 (OriginLab).

Fig. 29 shows that potassium current was restored to near wild-type levels in KCNQ 4-/-cells transfected with Anc80-KCNQ4 (fig. 10C) compared to mutant mice (fig. 29B), thereby indicating that gene therapy with Anc80 was able to restore function.

Example 3E Auditory Brainstem Response (ABR)

ABR recordings were performed as described previously (Maison et al, 2010, j. Neurosci, 30. Briefly, P25-P30 mice were anesthetized by IP injection (0.1 ml/10 g-body weight) with 50mg ketamine and 5mg xylazine diluted into 5ml 0.9% saline. The ABR experiment was performed in a sound proof booth at 32 ℃. To test hearing function, mice were provided with pure tone stimulation at 5.6kHz, 8kHz, 11.3kHz, 16kHz, 22.6kHz, or 32kHz with sound pressure levels between 10 and 115dB in 5dB steps until reaching a threshold intensity causing a repeatable ABR waveform (peaks I-IV) was detected. Using alternating polarity stimulation, 512 to 1024 responses were collected and averaged for each sound pressure level. Waveforms with amplitudes greater than 15 μ V (peak to valley) are rejected by the "exclude artifact" function.

Prior to starting the ABR test, the skin and cartilage that normally cover the entrance of the external auditory canal were trimmed with dissecting scissors and the sound pressure at the entrance of the ear canal was corrected for each test subject at all stimulation frequencies. Acoustic stimulation was delivered directly to the ear under study by a custom probe tube speaker/microphone assembly (EPL PXI system) consisting of two electrostatic earphones (CUI Miniature Dynamics) for producing the primary tone and a Knowles Miniature microphone (electric transducer) for recording the ear canal sound pressure. The acoustic stimulus consists of 5-ms audio pulses (in cos) ² First 0.5ms up-down at 40/s transmission).

ABR signals were acquired using a hypodermic needle electrode inserted into the pinna (active electrode), apex (reference electrode) and buttocks (ground electrode). The ABR potentials were amplified (10,000x), passed through filtering (0.3-10 kHz) and digitized using custom data acquisition software (LabVIEW). The acoustic stimulus and electrode voltage were sampled at 40-mus intervals using digital I-O plates (National Instruments) and stored for off-line analysis. The threshold is defined as the lowest decibel level visually at which any wave (I-IV) can be detected and reproduced as the sound intensity increases. ABR thresholds in each experimental group were averaged and used for statistical analysis.

Fig. 27 graphically demonstrates that delivery of Anc80 viral vectors encoding and expressing Harmonin can provide nearly complete restoration of auditory function, particularly at lower frequencies (e.g., about 5 to about 22 kHz).

Example 3F-quantitative RT-PCR analysis

This experiment was performed to assess the amount of virus present in the cochlea after in vivo administration. Left ear injection into 2 TMC 1-/-mice at P1. The cochlea was isolated from the left and right ears and kept in the medium for 3 days (equivalent to P10). RNA was extracted and its quality confirmed using an Agilent bioanalyzer (Agilent Technologies), reverse transcribed into cDNA for quantitative RT-PCR analysis using an effective primer pair specific for TMC1 and SYBR GreenER qPCR reagent (Invitrogen) as described previously (Kawashima et al, 2011, j.clin.invest., 121.

For amplification of TMC1 fragments, the following primers were used: 5. 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 the intron and verified using melting point curve analysis and negative control analysis of the data using the Δ Δ Δ CT method to determine changes relative to Actb and differences between injected and non-injected ears.

These results indicate that TMC1 mRNA was expressed 12-fold in the injected ear compared to the non-injected ear.

Example 3G-FM 1-43 labelling

FM1-43 dye loading experiments were performed as described previously (Gale et al, 2001, J.Neurosci.,21, 7013-25, meyers et al, 2003, J.Neurosci., 23. Coverslips with cochlear cultures adhered thereto were placed on the glass bottom chamber of an upright microscope (Zeiss Axioscope FS Plus). 5- μ M FM1-43FX (Invitrogen) diluted with artificial perilymph fluid was applied for 10sec and the tissue was washed 3 times in artificial perilymph fluid to remove the dye from the outer leaves of the cell membrane. After 5 minutes, intracellular FM1-43 was imaged using an FM1-43 filter set and an epifluorescent light source with a 63X water immersion objective. Tissues were fixed and treated for immunofluorescence detection as described above.

Figure 30 is an immunostaining image showing FM1-43 dye uptake by cells exposed to an Anc80 viral vector as used herein, and figure 31 illustrates that TMC1 delivered by an Anc80 viral vector as described herein is able to restore the sensory conductance of TMC 1-deficient hair cells in vivo.

Example 3H distortion product otoacoustic emission (DPOAE)

DPOAE data were collected under the same conditions and in the same record groups as the ABR data. To produce DPOAEs at 2f1-f2, the consonants are produced at a frequency ratio of 1.2 (f 2/f 1), where for each f2/f1 pair, the f2 level is 10dB and the sound pressure level is below the f1 level. The f2 level is scanned from 20 to 80dB in 5-dB steps. Waveform and spectral averaging is used at each level to increase the signal-to-noise ratio of the recorded ear canal sound pressure. The amplitude of DPOAE at 2f1-f2 is extracted from the average spectrum, as well as the noise floor at nearby points in the spectrum. Equal response curves are inserted into the DPOAE amplitude versus level curves. The threshold is defined as the f2 level required to produce DPOAE at 0 dB.

Figure 32 illustrates that TMC1 delivered using the Anc80 your viral vector as described herein is able to rescue the function of outer hair cells in TMC 1-/-mice, particularly at lower frequencies (e.g., about 5 to about 16 kHz).

Other embodiments

It should be understood that while the application has been described in conjunction with many different aspects of the methods and compositions of matter, the foregoing description of various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

Methods and compositions are disclosed that can be used, can be used in combination, can be used to make the disclosed methods and compositions, or are products thereof. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, although specific reference to each various individual and collective combinations and permutations of such compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a plurality of compositions or methods are discussed, each and every combination and permutation of the compositions and methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Sequence listing

Ant 80 capsid protein (SEQ ID NO: 1)

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAX1KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMX2AGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGX3STNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKX4LNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX5FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRX6LQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTX7NQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITX8EEEIKTTNPVATEX9YGTVATNLQSX10NTAPATGTVNSQGALPGMVWQX11RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL

where X1 = K/R; X2 = A/S; X3 = A/G; X4 = R/K; X5 = E/Q; X6 = T/E; X7 = A/T; X8 = S/N; X9 = Q/E; X10 = S/A; X11 = N/D

Ant 80-L0065 capsid protein (SEQ ID NO: 2)

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITNEEEIKTTNPVATEEYGTVATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL

pAAV-TMC2 (SEQ ID NO:3)

Left inverted terminal repeat (L-ITR) 1-130 nt

Cytomegalovirus (CMV) promoter 206-799 nt

Simian Virus 40 (SV 40) misc intron 831-963 nt

Transmembrane channel 1 (TMC 1ex 1): 982-3,267 nt

Post-transcriptional regulatory element (WPRE) from woodchuck hepatitis virus 3,268-3,821 nt

Bovine growth hormone (bGH) polyA signal 3,822-4,086 nt

Right reverse terminal repeat (R-ITR) 4,124-4,253 nt

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgcggATGCCACCCAAAAAAGTGCAAATCCAAGTGGAGGAGAAAGAAGAGGATACAGAGGAAAGCTCAAGTGAAGAAGAAGAAGATAAGCTACCCAGAAGAGAGAGCTTGAGACCAAAGAGGAAACGGACCAGAGATGTCATCAATGAGGATGACCCAGAACCGGAGCCGGAGGATGAAGAAACAAGAAAGGCAAGAGAAAAAGAAAGGCGGAGGAGGCTGCGGAGAGGAGCGGAAGAAGAAGAAGAAATTGATGAAGAGGAATTAGAACGGTTAAAAGCACTGCTCGATGAGAATAGACAAATGATCGCTACTGTCAAATGTAAACCTTGGAAAATGGAGAAGAAAATTGAAGTTCTCAAGGAAGCAAAGAAATTTGTGAGTGAGAATGAAGGCGCTCTTGGGAAAGGAAAGGGAAAGAAGTGGTTTGCATTTAAGATGATGATGGCCAAGAAATGGGCAAAATTCCTCCGAGATTTTGAGAACTTCAAAGCGGCTTGCGTCCCATGGGAAAACAAAATCAAGGCAATTGAAAGTCAGTTTGGTTCCTCAGTGGCCTCGTACTTCCTGTTCCTCAGGTGGATGTACGGCGTCAACATGGTTCTCTTTGTGTTGACCTTCAGCCTCATCATGTTACCGGAGTACCTCTGGGGTTTACCGTACGGCAGCTTACCTAGGAAAACAGTCCCAAGAGCTGAAGAAGCATCTGCAGCCAACTTTGGTGTGTTGTATGACTTCAATGGCCTGGCGCAGTACTCTGTCCTCTTTTATGGCTATTACGACAATAAACGCACGATCGGATGGCTGAATTTCCGGCTACCTCTTTCCTACTTCCTGGTGGGGATTATGTGCATTGGATACAGCTTCCTGGTTGTCCTCAAAGCGATGACCAAAAATATTGGTGACGATGGTGGTGGCGATGACAACACTTTCAACTTCAGCTGGAAGGTGTTCTGTAGCTGGGACTATCTGATTGGTAACCCTGAAACAGCCGACAACAAGTTTAACTCTATCACGATGAACTTTAAGGAAGCCATCATAGAAGAGAGAGCCGCACAGGTGGAGGAGAACATCCACCTCATCAGATTTCTGAGGTTTCTCGCTAACTTCTTCGTGTTCCTCACACTTGGTGCAAGTGGATACCTCATCTTTTGGGCTGTGAAGCGATCCCAGGAGTTCGCCCAGCAAGATCCTGACACCCTTGGGTGGTGGGAAAAAAATGAAATGAACATGGTAATGTCCCTCCTGGGGATGTTCTGTCCCACCCTGTTTGACTTATTTGCTGAACTGGAAGATTACCATCCTCTCATTGCTCTGAAGTGGCTCCTGGGGCGCATTTTTGCTCTTCTTCTAGGCAACTTGTATGTATTCATTCTCGCCTTGATGGATGAGATTAACAACAAGATTGAAGAGGAGAAGCTTGTGAAGGCCAATATTACCCTGTGGGAAGCCAACATGATTAAGGCTTACAATGAATCTCTCTCTGGGCTCTCTGGGAACACCACAGGAGCACCCTTTTTCGTTCATCCTGCAGATGTCCCTCGCGGTCCCTGCTGGGAAACAATGGTGGGGCAGGAATTCGTGCGTCTCACCGTTTCTGACGTCCTGACCACTTACGTCACGATCCTCATTGGCGACTTCCTCAGAGCATGTTTCGTGAGGTTCTGCAATTACTGCTGGTGCTGGGACTTAGAATATGGATATCCTTCATACACAGAATTCGACATCAGTGGCAACGTCCTCGCTCTGATCTTCAACCAAGGCATGATCTGGATGGGCTCCTTCTTCGCTCCTAGCCTCCCGGGCATCAACATCCTCCGTCTCCACACATCCATGTATTTCCAGTGCTGGGCTGTGATGTGCTGCAATGTTCCCGAGGCCAGGGTGTTCAAAGCTTCCAGATCCAACAACTTCTACCTCGGCATGCTGCTACTCATCCTCTTCCTGTCCACCATGCCGGTCCTGTACATGATCGTCTCCCTCCCGCCATCTTTTGATTGTGGGCCCTTCAGTGGTAAAAACAGGATGTTTGAAGTCATCGGTGAGACCCTGGAACATGACTTCCCAAGCTGGATGGCGAAGATCCTGAGGCAGCTTTCTAACCCCGGCCTTGTCATTGCTGTCATTCTGGTGATGGTTCTGACCATCTATTATCTCAATGCTACTGCCAAGGGCCAGAAAGCAGCGAATCTGGACCTCAAAAAGAAGATGAAACAGCAAGCTTTGGAGAACAAAATGCGAAACAAGAAAATGGCAGCGGCTCGAGCAGCTGCAGCTGCTGGTGGCCAGTAAggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaatagtggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgcggttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaagg

pAAV-TMC1ex2 (SEQ ID NO:4)

L-ITR: 1-130

CMV promoter 206-799

831-963 intron of SV40 misc

TMC1ex2: 982-3,255

WPRE: 3,256-3,809

bGH polyA signals 3,810-4,074

R-ITR: 4,112-4,241

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgcggATGTTGCAAATCCAAGTGGAGGAGAAAGAAGAGGATACAGAGGAAAGCTCAAGTGAAGAAGAAGAAGATAAGCTACCCAGAAGAGAGAGCTTGAGACCAAAGAGGAAACGGACCAGAGATGTCATCAATGAGGATGACCCAGAACCGGAGCCGGAGGATGAAGAAACAAGAAAGGCAAGAGAAAAAGAAAGGCGGAGGAGGCTGCGGAGAGGAGCGGAAGAAGAAGAAGAAATTGATGAAGAGGAATTAGAACGGTTAAAAGCACTGCTCGATGAGAATAGACAAATGATCGCTACTGTCAAATGTAAACCTTGGAAAATGGAGAAGAAAATTGAAGTTCTCAAGGAAGCAAAGAAATTTGTGAGTGAGAATGAAGGCGCTCTTGGGAAAGGAAAGGGAAAGAAGTGGTTTGCATTTAAGATGATGATGGCCAAGAAATGGGCAAAATTCCTCCGAGATTTTGAGAACTTCAAAGCGGCTTGCGTCCCATGGGAAAACAAAATCAAGGCAATTGAAAGTCAGTTTGGTTCCTCAGTGGCCTCGTACTTCCTGTTCCTCAGGTGGATGTACGGCGTCAACATGGTTCTCTTTGTGTTGACCTTCAGCCTCATCATGTTACCGGAGTACCTCTGGGGTTTACCGTACGGCAGCTTACCTAGGAAAACAGTCCCAAGAGCTGAAGAAGCATCTGCAGCCAACTTTGGTGTGTTGTATGACTTCAATGGCCTGGCGCAGTACTCTGTCCTCTTTTATGGCTATTACGACAATAAACGCACGATCGGATGGCTGAATTTCCGGCTACCTCTTTCCTACTTCCTGGTGGGGATTATGTGCATTGGATACAGCTTCCTGGTTGTCCTCAAAGCGATGACCAAAAATATTGGTGACGATGGTGGTGGCGATGACAACACTTTCAACTTCAGCTGGAAGGTGTTCTGTAGCTGGGACTATCTGATTGGTAACCCTGAAACAGCCGACAACAAGTTTAACTCTATCACGATGAACTTTAAGGAAGCCATCATAGAAGAGAGAGCCGCACAGGTGGAGGAGAACATCCACCTCATCAGATTTCTGAGGTTTCTCGCTAACTTCTTCGTGTTCCTCACACTTGGTGCAAGTGGATACCTCATCTTTTGGGCTGTGAAGCGATCCCAGGAGTTCGCCCAGCAAGATCCTGACACCCTTGGGTGGTGGGAAAAAAATGAAATGAACATGGTAATGTCCCTCCTGGGGATGTTCTGTCCCACCCTGTTTGACTTATTTGCTGAACTGGAAGATTACCATCCTCTCATTGCTCTGAAGTGGCTCCTGGGGCGCATTTTTGCTCTTCTTCTAGGCAACTTGTATGTATTCATTCTCGCCTTGATGGATGAGATTAACAACAAGATTGAAGAGGAGAAGCTTGTGAAGGCCAATATTACCCTGTGGGAAGCCAACATGATTAAGGCTTACAATGAATCTCTCTCTGGGCTCTCTGGGAACACCACAGGAGCACCCTTTTTCGTTCATCCTGCAGATGTCCCTCGCGGTCCCTGCTGGGAAACAATGGTGGGGCAGGAATTCGTGCGTCTCACCGTTTCTGACGTCCTGACCACTTACGTCACGATCCTCATTGGCGACTTCCTCAGAGCATGTTTCGTGAGGTTCTGCAATTACTGCTGGTGCTGGGACTTAGAATATGGATATCCTTCATACACAGAATTCGACATCAGTGGCAACGTCCTCGCTCTGATCTTCAACCAAGGCATGATCTGGATGGGCTCCTTCTTCGCTCCTAGCCTCCCGGGCATCAACATCCTCCGTCTCCACACATCCATGTATTTCCAGTGCTGGGCTGTGATGTGCTGCAATGTTCCCGAGGCCAGGGTGTTCAAAGCTTCCAGATCCAACAACTTCTACCTCGGCATGCTGCTACTCATCCTCTTCCTGTCCACCATGCCGGTCCTGTACATGATCGTCTCCCTCCCGCCATCTTTTGATTGTGGGCCCTTCAGTGGTAAAAACAGGATGTTTGAAGTCATCGGTGAGACCCTGGAACATGACTTCCCAAGCTGGATGGCGAAGATCCTGAGGCAGCTTTCTAACCCCGGCCTTGTCATTGCTGTCATTCTGGTGATGGTTCTGACCATCTATTATCTCAATGCTACTGCCAAGGGCCAGAAAGCAGCGAATCTGGACCTCAAAAAGAAGATGAAACAGCAAGCTTTGGAGAACAAAATGCGAAACAAGAAAATGGCAGCGGCTCGAGCAGCTGCAGCTGCTGGTGGCCAGTAAggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaatagtggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgcggttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaagg

pAAV-TMC2 (SEQ ID NO:5)

L-ITR: 1-130

CMV promoter 206-799

831-963 intron of SV40 misc

TMC2: 981-3,647

WPRE: 3,655-4,208

bGH polyA Signal 4,209-4,473

R-ITR: 4,511-4,640

ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagctagctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcctgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccaggcggccgccATGAGCCCCCAGTTAAAGAGCTTGGACGAGGAAGGTGACAAGTCAGCAAGAAGACCCACAAGGAAACAAACCTCCAGAGCTGCATGTCCCCAAGACGGGCACCGAGCCCAATCTAGCCGGAAGGATCCTGCTAAGGGTAGCCCAAGACCAGGGTCTTCCCGGAAGAAACAGATGGAACATGGAAGCTATCACAAGGGGTTGCAGGGACAGAAACCACGAAAGGTGGAGAGGTCTCTACAAGGGAGGAAGAAGGATCGGAGAACTTCCCTTAAGGAGCAGAGAGCATCTCCAAAGAAGGAGAGGGAGGCTCTGAGGAAGGAGGCAGGCAAGCAGCTGAGAAAACCCAGGTCCACTTCCTTGGGCTCCAGTGTCTCTACTGGAGACTCCCTGTCTGAGGAGGAGCTGGCTCAGATCCTGGAACAGGTAGAAGAAAAAAAGAAGCTCATCACTACCGTGAGGAACAAACCCTGGCCCATGGCAAAGAAGCTGAGGGAACTCAGGGAAGCCCAAGCCTTTGTGGAGAAGTATGAAGGAGCCTTGGGGAAAGGCAAGGGCAAACACCTCTACGCCTACAGGATGATGATGGCTAAGAAATGGGTCAAGTTTAAGAGGGACTTTGATAATTTCAAGACTCAATGTATTCCCTGGGAAATGAAGATCAAGGACATTGAAAGTCACTTCGGTTCTTCTGTGGCATCTTACTTCATCTTTCTCCGATGGATGTATGGAGTTAACCTTGTCCTTTTTGGCTTaATATTTGGTCTAGTCATCATCCCAGAGGTGCTGATGGGCATGCCCTATGGAAGTATACCCAGAAAGACGGTGCCTCGGGCTGAGGAAGAGCGAGCCATGGACTTCTCTGTCCTTTGGGATTTTGAGGGCTACATCAAATATTCTGCTCTCTTCTATGGCTACTACAACAACCAGCGGACCATTGGATGGCTGAGGTACAGGCTGCCCATGGCTTACTTTATGGTGGGGGTCAGCGTGTTTGGCTACAGCTTGATGATCGTCATTAGGTCGATGGCCAGCAATACCCAGGGTAGCACCAGTGAGGGGGACAGTGACAGCTTCACGTTCAGCTTCAAGATGTTCACCAGCTGGGACTACCTCATCGGGAATTCAGAGACAGCAGACAACAAATATGTCTCCATCACTACCAGCTTCAAGGAGTCTATAGTGGACGAACAAGAGAGTAACAAAGAAGGGAATATCCACCTGACAAGATTCCTCCGCGTCCTGGCCAACTTTCTCATTCTCTGCTGTCTGTGTGGAAGCGGGTACCTCATTTACTTTGTGGTGAAACGGTCCCAGGAGTTCTCCAAAATGCAAAATGTCAGCTGGTATGAAAGGAATGAGGTGGAGATCGTGATGTCTCTGCTAGGGATGTTTTGTCCCCCTCTGTTTGAAACCATCGCTGCCTTGGAGAATTATCACCCACGAACTGGGCTGAAGTGGCAGCTGGGCCGCATCTTTGCCCTTTTCCTGGGAAACCTCTACACGTTTCTCCTGGCCCTCATGGACGATGTCCACCTTAAGCTTTCTAATGAGGAAAAAATCAAGAACATCACTCACTGGACCCTGTTTAACTATTACAATTCCTCAGGTGGGAATGAGAGTGTGCCCCGGCCACCACCACACCCTGCAGATGTGCCCAGAGGTTCTTGCTGGGAGACAGCTGTGGGCATTGAGTTTATGAGGCTCACCGTGTCTGACATGCTGGTAACATACCTCACCATCTTGGTCGGAGATTTCCTCCGAGCTTGTTTTGTCCGGTTCATGAATCACTGCTGGTGTTGGGACCTCGAGGCTGGTTTTCCCTCATATGCCGAGTTTGATATTAGTGGAAATGTGTTGGGTTTGATCTTCAACCAAGGAATGATCTGGATGGGCTCCTTCTATGCTCCAGGACTGGTGGGCATCAATGTCCTGCGCCTGTTGACCTCCATGTACTTCCAGTGCTGGGCAGTGATGAGCAGCAACGTTCCCCATGAGCGTGTGTTTAAAGCCTCCCGATCCAACAACTTCTACATGGGCCTGCTGCTGTTGGTGCTCTTCCTCAGCCTCCTGCCTGTGGCCTACACTGTCATGTCTCTCCCACCCTCGTTTGACTGTGGCCCCTTCAGTGGGAAAAACAGAATGTACGATGTCCTCCATGAGACCATCGAGAACGATTTCCCTAAGTTCCTGGGCAAGATCTTTGCGTTCCTTGCCAACCCAGGCCTGATCATTCCAGCCATCCTGCTAATGTTTCTGGCCATTTACTACCTGAACTCAGTTTCAAAAAGTCTTTCCAGAGCTAATGCCCAGCTGCGAAAGAAGATCCAAGCGCTCCGTGAAGTTGAGAAGAACCATAAATCCATCAAGGGAAAAGCCATAGTCACATATTCAGAGGACACAATCAAGAACAGCTCCAAAAATGCCACCCAGATACATCTTACTAAAGAAGAGCCCACATCTCACTCTTCCAGCCAAATCCAGACCCTGGACAAGAAAGCGCAGGGCCCCCACACCTCCAGTACTGAGGGTGGGGCCTCGCCGTCTACCTCCTGGCACCATGTTGGGTCTCAACCACCGAGAGGCAGACGAGATTCTGGCCAACCCCAGTCTCAGACTTATACAGGCAGGTCACCTTCTGGAAAGAGAACCCAGAGGCCTCACAACTGAtaagcttggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttataatttcaggtggcatctttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaatagtggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagtaatggtaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgcggttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaagg

pAAV-Pmyo6-TMC1ex1 (SEQ ID NO:6)

L-ITR: 1-141

Myosin 6 (myo 6) promoter 155-1,396

TMC1ex1: 1,425-3,710

hGH polyA Signal 3,745-4,225

R-ITR: 4,262-4,402

cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcgTGCAAGAACCCTCACTGGCTGAACTATCTTGCCAGCCCCTTATTTTGTTTTCATATTAACCTCTTTTTTCTAGTAAAGGAGATGTTTGCTCTCAAATTTGCATAGGAATGTAATATTTAATTTAAAAAGATGACCCACATATGACCTTATAAGGACAGTAAAATTAAACAACCGGAAAGATAAAGCGGGCCAGTTGGCTCAGTTCTATAAAACCAGCCCACAAGGATTGTCACTATTCTTAGGCTTGCGCGGGCTACATGATGAGTTCCAGGACTGCCTGGTTACAGACCGAGACTCTCTCAAGAGTCCAGATAAACAACAACAAAGGGGGCGAGGTGGAAATACAGGGGCTGTAAGAAGTAAATATGATATCTGCATGGGAGGCTAGCCAGAGAAGAAAAAATTTTCTTCCGTGGTTCAATCCTCCAAGGGCTGAACAGGAAGTTGACGCAGGCAGGTGAGGAGCACGAGCCTAGATGGGCTGCGGTGCCACCCTTAATCCCCACAAGCGAGTTCCTCCGCAATTCGCCTGTCCCACTCTCAACTTTTCTTCAACTGACTCTTTGCTGTGGTCCCTCGCTGTGGCAGTGGAAACAACTACCACTGCGAGGTAGGGAATGTCATGAGGGGCTACCTGCAGCCCTTGGCTTGCAGGGATGCAGGGATGCGGTCGGAACCTGAGGCCCCGCCCTTCTCTTGCCCCACGCCATTAGGCCACGCCCCTACCCAGCACTCCTTCAACCACCCCCTTCCCCGGCGCCTCATGAGGTCCCGCCCCTCTCAACCCTAGCTCTTGAGGCCTCCCCTTCACAGCCGCCCCGGCGTTCCTTGACTTGAGGCCACGTCCCTCTGCTCCTTCATTCCCAAGACCCTACGCTTTGCGAGTCCTCCCTGTCCTGCTGCCTAGGACCCCGCCCCTCTCAGCCCTTCTGCCCCAAGACCCCGCCCCTTAGGCTGTTCCCGCCCACTGGCCAATGAAGACCCGCCCTTTCTTTAGCCGCCCCGCCCCGGTCCCACAAAATCCCGCCTCCGGCCCCGCCTCCCGCCCCCTTGGGCGCTCCGTAGCAGTGACGTGCGCAGGCTGGGCACTCTGCAGGGCTCTCTGGCCGGCGGGTGGAGACCGATCCGGGATCTGTCCCAGCAGGAAGCGTATCCCGGCCGCCGTCGTGCTGTCGTCTCCGGTGCTCGCTCTCGGCCGCGGTGTCGCGCTTGCCCTTCGCGCCCGCAGCCCGGCAGCCTCTCgagCTCAAGCTTCGAATTCgtcgacaggATGCCACCCAAAAAAGTGCAAATCCAAGTGGAGGAGAAAGAAGAGGATACAGAGGAAAGCTCAAGTGAAGAAGAAGAAGATAAGCTACCCAGAAGAGAGAGCTTGAGACCAAAGAGGAAACGGACCAGAGATGTCATCAATGAGGATGACCCAGAACCGGAGCCGGAGGATGAAGAAACAAGAAAGGCAAGAGAAAAAGAAAGGCGGAGGAGGCTGCGGAGAGGAGCGGAAGAAGAAGAAGAAATTGATGAAGAGGAATTAGAACGGTTAAAAGCACTGCTCGATGAGAATAGACAAATGATCGCTACTGTCAAATGTAAACCTTGGAAAATGGAGAAGAAAATTGAAGTTCTCAAGGAAGCAAAGAAATTTGTGAGTGAGAATGAAGGCGCTCTTGGGAAAGGAAAGGGAAAGAAGTGGTTTGCATTTAAGATGATGATGGCCAAGAAATGGGCAAAATTCCTCCGAGATTTTGAGAACTTCAAAGCGGCTTGCGTCCCATGGGAAAACAAAATCAAGGCAATTGAAAGTCAGTTTGGTTCCTCAGTGGCCTCGTACTTCCTGTTCCTCAGGTGGATGTACGGCGTCAACATGGTTCTCTTTGTGTTGACCTTCAGCCTCATCATGTTACCGGAGTACCTCTGGGGTTTACCGTACGGCAGCTTACCTAGGAAAACAGTCCCAAGAGCTGAAGAAGCATCTGCAGCCAACTTTGGTGTGTTGTATGACTTCAATGGCCTGGCGCAGTACTCTGTCCTCTTTTATGGCTATTACGACAATAAACGCACGATCGGATGGCTGAATTTCCGGCTACCTCTTTCCTACTTCCTGGTGGGGATTATGTGCATTGGATACAGCTTCCTGGTTGTCCTCAAAGCGATGACCAAAAATATTGGTGACGATGGTGGTGGCGATGACAACACTTTCAACTTCAGCTGGAAGGTGTTCTGTAGCTGGGACTATCTGATTGGTAACCCTGAAACAGCCGACAACAAGTTTAACTCTATCACGATGAACTTTAAGGAAGCCATCATAGAAGAGAGAGCCGCACAGGTGGAGGAGAACATCCACCTCATCAGATTTCTGAGGTTTCTCGCTAACTTCTTCGTGTTCCTCACACTTGGTGCAAGTGGATACCTCATCTTTTGGGCTGTGAAGCGATCCCAGGAGTTCGCCCAGCAAGATCCTGACACCCTTGGGTGGTGGGAAAAAAATGAAATGAACATGGTAATGTCCCTCCTGGGGATGTTCTGTCCCACCCTGTTTGACTTATTTGCTGAACTGGAAGATTACCATCCTCTCATTGCTCTGAAGTGGCTCCTGGGGCGCATTTTTGCTCTTCTTCTAGGCAACTTGTATGTATTCATTCTCGCCTTGATGGATGAGATTAACAACAAGATTGAAGAGGAGAAGCTTGTGAAGGCCAATATTACCCTGTGGGAAGCCAACATGATTAAGGCTTACAATGAATCTCTCTCTGGGCTCTCTGGGAACACCACAGGAGCACCCTTTTTCGTTCATCCTGCAGATGTCCCTCGCGGTCCCTGCTGGGAAACAATGGTGGGGCAGGAATTCGTGCGTCTCACCGTTTCTGACGTCCTGACCACTTACGTCACGATCCTCATTGGCGACTTCCTCAGAGCATGTTTCGTGAGGTTCTGCAATTACTGCTGGTGCTGGGACTTAGAATATGGATATCCTTCATACACAGAATTCGACATCAGTGGCAACGTCCTCGCTCTGATCTTCAACCAAGGCATGATCTGGATGGGCTCCTTCTTCGCTCCTAGCCTCCCGGGCATCAACATCCTCCGTCTCCACACATCCATGTATTTCCAGTGCTGGGCTGTGATGTGCTGCAATGTTCCCGAGGCCAGGGTGTTCAAAGCTTCCAGATCCAACAACTTCTACCTCGGCATGCTGCTACTCATCCTCTTCCTGTCCACCATGCCGGTCCTGTACATGATCGTCTCCCTCCCGCCATCTTTTGATTGTGGGCCCTTCAGTGGTAAAAACAGGATGTTTGAAGTCATCGGTGAGACCCTGGAACATGACTTCCCAAGCTGGATGGCGAAGATCCTGAGGCAGCTTTCTAACCCCGGCCTTGTCATTGCTGTCATTCTGGTGATGGTTCTGACCATCTATTATCTCAATGCTACTGCCAAGGGCCAGAAAGCAGCGAATCTGGACCTCAAAAAGAAGATGAAACAGCAAGCTTTGGAGAACAAAATGCGAAACAAGAAAATGGCAGCGGCTCGAGCAGCTGCAGCTGCTGGTGGCCAGTAAGCGGCCGCTCGAGCCTAAGCTTCTAGAagatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccacgtgcggaccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

pAAV-Pmyo6-TMC1ex1 (SEQ ID NO:7)

L-ITR: 1-141

Myo6 promoter 155-1,396

TMC1ex2: 1,425-4,439

hGH polyA signal 4,474-4,954

R-ITR: 4,991-5,131

cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcgTGCAAGAACCCTCACTGGCTGAACTATCTTGCCAGCCCCTTATTTTGTTTTCATATTAACCTCTTTTTTCTAGTAAAGGAGATGTTTGCTCTCAAATTTGCATAGGAATGTAATATTTAATTTAAAAAGATGACCCACATATGACCTTATAAGGACAGTAAAATTAAACAACCGGAAAGATAAAGCGGGCCAGTTGGCTCAGTTCTATAAAACCAGCCCACAAGGATTGTCACTATTCTTAGGCTTGCGCGGGCTACATGATGAGTTCCAGGACTGCCTGGTTACAGACCGAGACTCTCTCAAGAGTCCAGATAAACAACAACAAAGGGGGCGAGGTGGAAATACAGGGGCTGTAAGAAGTAAATATGATATCTGCATGGGAGGCTAGCCAGAGAAGAAAAAATTTTCTTCCGTGGTTCAATCCTCCAAGGGCTGAACAGGAAGTTGACGCAGGCAGGTGAGGAGCACGAGCCTAGATGGGCTGCGGTGCCACCCTTAATCCCCACAAGCGAGTTCCTCCGCAATTCGCCTGTCCCACTCTCAACTTTTCTTCAACTGACTCTTTGCTGTGGTCCCTCGCTGTGGCAGTGGAAACAACTACCACTGCGAGGTAGGGAATGTCATGAGGGGCTACCTGCAGCCCTTGGCTTGCAGGGATGCAGGGATGCGGTCGGAACCTGAGGCCCCGCCCTTCTCTTGCCCCACGCCATTAGGCCACGCCCCTACCCAGCACTCCTTCAACCACCCCCTTCCCCGGCGCCTCATGAGGTCCCGCCCCTCTCAACCCTAGCTCTTGAGGCCTCCCCTTCACAGCCGCCCCGGCGTTCCTTGACTTGAGGCCACGTCCCTCTGCTCCTTCATTCCCAAGACCCTACGCTTTGCGAGTCCTCCCTGTCCTGCTGCCTAGGACCCCGCCCCTCTCAGCCCTTCTGCCCCAAGACCCCGCCCCTTAGGCTGTTCCCGCCCACTGGCCAATGAAGACCCGCCCTTTCTTTAGCCGCCCCGCCCCGGTCCCACAAAATCCCGCCTCCGGCCCCGCCTCCCGCCCCCTTGGGCGCTCCGTAGCAGTGACGTGCGCAGGCTGGGCACTCTGCAGGGCTCTCTGGCCGGCGGGTGGAGACCGATCCGGGATCTGTCCCAGCAGGAAGCGTATCCCGGCCGCCGTCGTGCTGTCGTCTCCGGTGCTCGCTCTCGGCCGCGGTGTCGCGCTTGCCCTTCGCGCCCGCAGCCCGGCAGCCTCTCgagCTCAAGCTTCGAATTCgtcgacaggATGTTGCAAATCCAAGTGGAGGAGAAAGAAGAGGATACAGAGGAAAGCTCAAGTGAAGAAGAAGAAGATAAGCTACCCAGAAGAGAGAGCTTGAGACCAAAGAGGAAACGGACCAGAGATGTCATCAATGAGGATGACCCAGAACCGGAGCCGGAGGATGAAGAAACAAGAAAGGCAAGAGAAAAAGAAAGGCGGAGGAGGCTGCGGAGAGGAGCGGAAGAAGAAGAAGAAATTGATGAAGAGGAATTAGAACGGTTAAAAGCACTGCTCGATGAGAATAGACAAATGATCGCTACTGTCAAATGTAAACCTTGGAAAATGGAGAAGAAAATTGAAGTTCTCAAGGAAGCAAAGAAATTTGTGAGTGAGAATGAAGGCGCTCTTGGGAAAGGAAAGGGAAAGAAGTGGTTTGCATTTAAGATGATGATGGCCAAGAAATGGGCAAAATTCCTCCGAGATTTTGAGAACTTCAAAGCGGCTTGCGTCCCATGGGAAAACAAAATCAAGGCAATTGAAAGTCAGTTTGGTTCCTCAGTGGCCTCGTACTTCCTGTTCCTCAGGTGGATGTACGGCGTCAACATGGTTCTCTTTGTGTTGACCTTCAGCCTCATCATGTTACCGGAGTACCTCTGGGGTTTACCGTACGGCAGCTTACCTAGGAAAACAGTCCCAAGAGCTGAAGAAGCATCTGCAGCCAACTTTGGTGTGTTGTATGACTTCAATGGCCTGGCGCAGTACTCTGTCCTCTTTTATGGCTATTACGACAATAAACGCACGATCGGATGGCTGAATTTCCGGCTACCTCTTTCCTACTTCCTGGTGGGGATTATGTGCATTGGATACAGCTTCCTGGTTGTCCTCAAAGCGATGACCAAAAATATTGGTGACGATGGTGGTGGCGATGACAACACTTTCAACTTCAGCTGGAAGGTGTTCTGTAGCTGGGACTATCTGATTGGTAACCCTGAAACAGCCGACAACAAGTTTAACTCTATCACGATGAACTTTAAGGAAGCCATCATAGAAGAGAGAGCCGCACAGGTGGAGGAGAACATCCACCTCATCAGATTTCTGAGGTTTCTCGCTAACTTCTTCGTGTTCCTCACACTTGGTGCAAGTGGATACCTCATCTTTTGGGCTGTGAAGCGATCCCAGGAGTTCGCCCAGCAAGATCCTGACACCCTTGGGTGGTGGGAAAAAAATGAAATGAACATGGTAATGTCCCTCCTGGGGATGTTCTGTCCCACCCTGTTTGACTTATTTGCTGAACTGGAAGATTACCATCCTCTCATTGCTCTGAAGTGGCTCCTGGGGCGCATTTTTGCTCTTCTTCTAGGCAACTTGTATGTATTCATTCTCGCCTTGATGGATGAGATTAACAACAAGATTGAAGAGGAGAAGCTTGTGAAGGCCAATATTACCCTGTGGGAAGCCAACATGATTAAGGCTTACAATGAATCTCTCTCTGGGCTCTCTGGGAACACCACAGGAGCACCCTTTTTCGTTCATCCTGCAGATGTCCCTCGCGGTCCCTGCTGGGAAACAATGGTGGGGCAGGAATTCGTGCGTCTCACCGTTTCTGACGTCCTGACCACTTACGTCACGATCCTCATTGGCGACTTCCTCAGAGCATGTTTCGTGAGGTTCTGCAATTACTGCTGGTGCTGGGACTTAGAATATGGATATCCTTCATACACAGAATTCGACATCAGTGGCAACGTCCTCGCTCTGATCTTCAACCAAGGCATGATCTGGATGGGCTCCTTCTTCGCTCCTAGCCTCCCGGGCATCAACATCCTCCGTCTCCACACATCCATGTATTTCCAGTGCTGGGCTGTGATGTGCTGCAATGTTCCCGAGGCCAGGGTGTTCAAAGCTTCCAGATCCAACAACTTCTACCTCGGCATGCTGCTACTCATCCTCTTCCTGTCCACCATGCCGGTCCTGTACATGATCGTCTCCCTCCCGCCATCTTTTGATTGTGGGCCCTTCAGTGGTAAAAACAGGATGTTTGAAGTCATCGGTGAGACCCTGGAACATGACTTCCCAAGCTGGATGGCGAAGATCCTGAGGCAGCTTTCTAACCCCGGCCTTGTCATTGCTGTCATTCTGGTGATGGTTCTGACCATCTATTATCTCAATGCTACTGCCAAGGGCCAGAAAGCAGCGAATCTGGACCTCAAAAAGAAGATGAAACAGCAAGCTTTGGAGAACAAAATGCGAAACAAGAAAATGGCAGCGGCTCGAGCAGCTGCAGCTGCTGGTGGCCAGTGGATCCACCGGCCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGCGGCCGCTCGAGCCTAAGCTTCTAGAagatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccacgtgcggaccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

pAAV-Pmyo6-TMC2 (SEQ ID NO:8)

L-ITR: 1-141

Myo6 promoter 155-1,396

TMC2: 1,425-4,091

hGH polyA Signal 4,126-4,606

R-ITR: 4,643-4,783

cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcgTGCAAGAACCCTCACTGGCTGAACTATCTTGCCAGCCCCTTATTTTGTTTTCATATTAACCTCTTTTTTCTAGTAAAGGAGATGTTTGCTCTCAAATTTGCATAGGAATGTAATATTTAATTTAAAAAGATGACCCACATATGACCTTATAAGGACAGTAAAATTAAACAACCGGAAAGATAAAGCGGGCCAGTTGGCTCAGTTCTATAAAACCAGCCCACAAGGATTGTCACTATTCTTAGGCTTGCGCGGGCTACATGATGAGTTCCAGGACTGCCTGGTTACAGACCGAGACTCTCTCAAGAGTCCAGATAAACAACAACAAAGGGGGCGAGGTGGAAATACAGGGGCTGTAAGAAGTAAATATGATATCTGCATGGGAGGCTAGCCAGAGAAGAAAAAATTTTCTTCCGTGGTTCAATCCTCCAAGGGCTGAACAGGAAGTTGACGCAGGCAGGTGAGGAGCACGAGCCTAGATGGGCTGCGGTGCCACCCTTAATCCCCACAAGCGAGTTCCTCCGCAATTCGCCTGTCCCACTCTCAACTTTTCTTCAACTGACTCTTTGCTGTGGTCCCTCGCTGTGGCAGTGGAAACAACTACCACTGCGAGGTAGGGAATGTCATGAGGGGCTACCTGCAGCCCTTGGCTTGCAGGGATGCAGGGATGCGGTCGGAACCTGAGGCCCCGCCCTTCTCTTGCCCCACGCCATTAGGCCACGCCCCTACCCAGCACTCCTTCAACCACCCCCTTCCCCGGCGCCTCATGAGGTCCCGCCCCTCTCAACCCTAGCTCTTGAGGCCTCCCCTTCACAGCCGCCCCGGCGTTCCTTGACTTGAGGCCACGTCCCTCTGCTCCTTCATTCCCAAGACCCTACGCTTTGCGAGTCCTCCCTGTCCTGCTGCCTAGGACCCCGCCCCTCTCAGCCCTTCTGCCCCAAGACCCCGCCCCTTAGGCTGTTCCCGCCCACTGGCCAATGAAGACCCGCCCTTTCTTTAGCCGCCCCGCCCCGGTCCCACAAAATCCCGCCTCCGGCCCCGCCTCCCGCCCCCTTGGGCGCTCCGTAGCAGTGACGTGCGCAGGCTGGGCACTCTGCAGGGCTCTCTGGCCGGCGGGTGGAGACCGATCCGGGATCTGTCCCAGCAGGAAGCGTATCCCGGCCGCCGTCGTGCTGTCGTCTCCGGTGCTCGCTCTCGGCCGCGGTGTCGCGCTTGCCCTTCGCGCCCGCAGCCCGGCAGCCTCTCgagCTCAAGCTTCGAATTCgtcgacaggATGAGCCCCCAGTTAAAGAGCTTGGACGAGGAAGGTGACAAGTCAGCAAGAAGACCCACAAGGAAACAAACCTCCAGAGCTGCATGTCCCCAAGACGGGCACCGAGCCCAATCTAGCCGGAAGGATCCTGCTAAGGGTAGCCCAAGACCAGGGTCTTCCCGGAAGAAACAGATGGAACATGGAAGCTATCACAAGGGGTTGCAGGGACAGAAACCACGAAAGGTGGAGAGGTCTCTACAAGGGAGGAAGAAGGATCGGAGAACTTCCCTTAAGGAGCAGAGAGCATCTCCAAAGAAGGAGAGGGAGGCTCTGAGGAAGGAGGCAGGCAAGCAGCTGAGAAAACCCAGGTCCACTTCCTTGGGCTCCAGTGTCTCTACTGGAGACTCCCTGTCTGAGGAGGAGCTGGCTCAGATCCTGGAACAGGTAGAAGAAAAAAAGAAGCTCATCACTACCGTGAGGAACAAACCCTGGCCCATGGCAAAGAAGCTGAGGGAACTCAGGGAAGCCCAAGCCTTTGTGGAGAAGTATGAAGGAGCCTTGGGGAAAGGCAAGGGCAAACACCTCTACGCCTACAGGATGATGATGGCTAAGAAATGGGTCAAGTTTAAGAGGGACTTTGATAATTTCAAGACTCAATGTATTCCCTGGGAAATGAAGATCAAGGACATTGAAAGTCACTTCGGTTCTTCTGTGGCATCTTACTTCATCTTTCTCCGATGGATGTATGGAGTTAACCTTGTCCTTTTTGGCTTaATATTTGGTCTAGTCATCATCCCAGAGGTGCTGATGGGCATGCCCTATGGAAGTATACCCAGAAAGACGGTGCCTCGGGCTGAGGAAGAGCGAGCCATGGACTTCTCTGTCCTTTGGGATTTTGAGGGCTACATCAAATATTCTGCTCTCTTCTATGGCTACTACAACAACCAGCGGACCATTGGATGGCTGAGGTACAGGCTGCCCATGGCTTACTTTATGGTGGGGGTCAGCGTGTTTGGCTACAGCTTGATGATCGTCATTAGGTCGATGGCCAGCAATACCCAGGGTAGCACCAGTGAGGGGGACAGTGACAGCTTCACGTTCAGCTTCAAGATGTTCACCAGCTGGGACTACCTCATCGGGAATTCAGAGACAGCAGACAACAAATATGTCTCCATCACTACCAGCTTCAAGGAGTCTATAGTGGACGAACAAGAGAGTAACAAAGAAGGGAATATCCACCTGACAAGATTCCTCCGCGTCCTGGCCAACTTTCTCATTCTCTGCTGTCTGTGTGGAAGCGGGTACCTCATTTACTTTGTGGTGAAACGGTCCCAGGAGTTCTCCAAAATGCAAAATGTCAGCTGGTATGAAAGGAATGAGGTGGAGATCGTGATGTCTCTGCTAGGGATGTTTTGTCCCCCTCTGTTTGAAACCATCGCTGCCTTGGAGAATTATCACCCACGAACTGGGCTGAAGTGGCAGCTGGGCCGCATCTTTGCCCTTTTCCTGGGAAACCTCTACACGTTTCTCCTGGCCCTCATGGACGATGTCCACCTTAAGCTTTCTAATGAGGAAAAAATCAAGAACATCACTCACTGGACCCTGTTTAACTATTACAATTCCTCAGGTGGGAATGAGAGTGTGCCCCGGCCACCACCACACCCTGCAGATGTGCCCAGAGGTTCTTGCTGGGAGACAGCTGTGGGCATTGAGTTTATGAGGCTCACCGTGTCTGACATGCTGGTAACATACCTCACCATCTTGGTCGGAGATTTCCTCCGAGCTTGTTTTGTCCGGTTCATGAATCACTGCTGGTGTTGGGACCTCGAGGCTGGTTTTCCCTCATATGCCGAGTTTGATATTAGTGGAAATGTGTTGGGTTTGATCTTCAACCAAGGAATGATCTGGATGGGCTCCTTCTATGCTCCAGGACTGGTGGGCATCAATGTCCTGCGCCTGTTGACCTCCATGTACTTCCAGTGCTGGGCAGTGATGAGCAGCAACGTTCCCCATGAGCGTGTGTTTAAAGCCTCCCGATCCAACAACTTCTACATGGGCCTGCTGCTGTTGGTGCTCTTCCTCAGCCTCCTGCCTGTGGCCTACACTGTCATGTCTCTCCCACCCTCGTTTGACTGTGGCCCCTTCAGTGGGAAAAACAGAATGTACGATGTCCTCCATGAGACCATCGAGAACGATTTCCCTAAGTTCCTGGGCAAGATCTTTGCGTTCCTTGCCAACCCAGGCCTGATCATTCCAGCCATCCTGCTAATGTTTCTGGCCATTTACTACCTGAACTCAGTTTCAAAAAGTCTTTCCAGAGCTAATGCCCAGCTGCGAAAGAAGATCCAAGCGCTCCGTGAAGTTGAGAAGAACCATAAATCCATCAAGGGAAAAGCCATAGTCACATATTCAGAGGACACAATCAAGAACAGCTCCAAAAATGCCACCCAGATACATCTTACTAAAGAAGAGCCCACATCTCACTCTTCCAGCCAAATCCAGACCCTGGACAAGAAAGCGCAGGGCCCCCACACCTCCAGTACTGAGGGTGGGGCCTCGCCGTCTACCTCCTGGCACCATGTTGGGTCTCAACCACCGAGAGGCAGACGAGATTCTGGCCAACCCCAGTCTCAGACTTATACAGGCAGGTCACCTTCTGGAAAGAGAACCCAGAGGCCTCACAACTGAGCGGCCGCTCGAGCCTAAGCTTCTAGAagatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccacgtgcggaccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

Claims (26)

1. Use of an adeno-associated virus (AAV) in the preparation of a kit for delivering a transgene to at least 80% of Inner Hair Cells (IHC) and at least 80% of Outer Hair Cells (OHC) in the inner ear of a subject for treating a hearing disorder, wherein the delivering comprises:

administering an adeno-associated virus (AAV) to the inner ear of the subject, wherein the AAV comprises (i) a nucleic acid sequence consisting of SEQ ID NO: 2and (ii) a transgene, and (ii) an Anc80 capsid protein.

2. The use of claim 1, wherein the transgene is further delivered to spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, support cells, and cells in the blood vessel veins in the inner ear of the subject.

3. The use of claim 1, wherein the transgene is selected from the group consisting of: <xnotran> 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 XIAP. </xnotran>

4. The use of claim 1, wherein the transgene encodes a neurotrophic factor.

5. The use of claim 4, wherein the neurotrophic factor is selected from the group consisting of: GDNF, BDNF, NT3 and HSP70.

6. The use of claim 1, wherein the transgene encodes an antibody or fragment thereof.

7. The use of claim 1, wherein the transgene encodes an immunomodulatory protein.

8. The use of claim 1, wherein the transgene encodes an anti-oncogenic transcript.

9. The use of claim 1, wherein the transgene encodes an antisense, silent or long non-coding RNA species.

10. The use of claim 1, wherein the transgene encodes a genome editing system selected from the group consisting of: zinc finger nucleases, TALENs and CRISPR which are modified by genetic engineering.

11. The use of claim 1, wherein the transgene is under the control of a heterologous promoter sequence.

12. The use of claim 11, wherein the heterologous promoter sequence is selected from the group consisting of: CMV promoter, CBA promoter, CASI promoter, PGK promoter, EF-1 promoter, alpha 9 nicotine receptor promoter, dynein promoter, KCNQ4 promoter, myo7a promoter, myo6 promoter, gfi1 promoter, vglut3 promoter and Atoh1 promoter.

13. The use of claim 1, wherein the step of administering comprises injecting the AAV through a round window.

14. The use of claim 1, wherein the AAV is administered during a cochlear ostomy or during a tracheostomy (canalostomy).

15. The use of claim 1, wherein the AAV is administered to the middle ear and/or round window via one or more drug delivery vehicles.

16. The use of claim 1, wherein expression of the transgene results in regeneration of Inner Hair Cells (IHC), outer Hair Cells (OHC), spiral ganglion neurons, blood vessels veins, vestibular hair cells, and/or vestibular ganglion neurons, thereby restoring hearing or vestibular function.

17. Use of an adeno-associated virus (AAV) in the preparation of a kit for delivering a TMC1 or TMC2 transgene to at least 80% of Inner Hair Cells (IHC) and at least 80% of Outer Hair Cells (OHC) in the inner ear of a subject for the treatment of a hearing disorder, wherein said delivery comprises:

administering an adeno-associated virus (AAV) to the inner ear of the subject, wherein the AAV comprises (i) a nucleic acid sequence consisting of SEQ ID NO: 2and (ii) the TMC1 or TMC2 transgene.

18. Use of an adeno-associated virus (AAV) in the preparation of a kit for delivering a Usher transgene 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 for treating a hearing disorder, wherein the delivering comprises:

administering an adeno-associated virus (AAV) to the inner ear of the subject, wherein the AAV comprises (i) a nucleic acid sequence consisting of SEQ ID NO: 2and (ii) the Usher transgene, and (ii) the Anc80 capsid protein.

19. The use of claim 18, wherein the Usher transgene is selected from the group consisting of: MYO7A, USH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7.

20. The use of claim 17 or 18, wherein the transgene is further delivered to one or more of spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, support cells, and cells in the blood vessel veins in the inner ear of the subject.

21. The use of claim 18, wherein the transgene is under the control of a heterologous promoter sequence.

22. The use of claim 21, wherein the heterologous promoter sequence is selected from the group consisting of: CMV promoter, CBA promoter, CASI promoter, PGK promoter, EF-1 promoter, alpha 9 nicotinic receptor promoter, dynein promoter, KCNQ4 promoter, myo7a promoter, myo6 promoter, gfi1 promoter, vglut3 promoter and Atoh1 promoter.

23. The use of claim 17 or 18, wherein the step of administering comprises injecting the AAV through a round window.

24. The use of claim 17 or 18, wherein the AAV is administered during a cochlear ostomy or during a tubostomy.

25. The use of claim 17 or 18, wherein the AAV is administered to the middle ear and/or round window via one or more drug delivery vehicles.

26. The use of claim 17 or 18, wherein expression of the transgene results in regeneration of Inner Hair Cells (IHC), outer Hair Cells (OHC), spiral ganglion neurons, blood vessels veins, vestibular hair cells and/or vestibular ganglion neurons, thereby restoring hearing or vestibular function.