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

Abstract

To provide materials and methods for efficiently delivering nucleic acids to cochlear and vestibular cells.SOLUTION: A composition for use in a method for delivering a cadherin-related 23 (CDH23) transgene to a subject comprises an adeno-associated virus (AAV) vector. The method comprises administering the AAV vector to an inner ear of the subject. The AAV vector comprises (i) an Anc80 capsid protein having a specific amino acid sequence and (ii) the CDH23 transgene. The transgene is delivered to at least 80% of inner hair cells (IHCs) and at least 80% of outer hair cells of the inner ear of the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates generally to materials and methods for delivering nucleic acids to cochlear and vestibular cells. do. [Background technology]

[0002] Genetic hearing loss is a significant problem with few treatment options other than cochlear implants. Inherited hearing problems are often due to a single gene defect. Prelingual hearing loss is one in five It is diagnosed in 100 infants, approximately 50% of which have a genetic etiology. Many different clinical subtypes can be caused by mutations in any of the following genes: Usher syndrome, which is associated with encephalopathy, accounts for 3-6% of childhood hearing loss, but is more prevalent. One of the genetic defects, estimated to account for 1-2% of hereditary hearing loss, is in the TMC1 gene. This occurs in the

[0003] The inner ear, e.g., the cochlea, in particular the inner and outer hair cells (IHCs and OHs) in the cochlea C) refers to hearing loss and hearing impairment of various etiologies, most directly related to monogenic hereditary hearing loss. It is an attractive target for gene therapy approaches to intervene in I. Efficiently translocate not only HCs and OHCs but also other inner ear cells that may be relevant for gene therapy approaches. Targeting and transducing the target gene has been challenging. Summary of the Invention [Problem to be solved by the invention]

[0004] Hearing loss is the most common sensory disorder worldwide, and half of all prelingual hearing loss is genetic. Nevertheless, the transition to clinical cochlear gene therapy is expected to be safe and clinically viable. However, progress has been hindered by the lack of a meaningful and efficient delivery modality. reported a new adeno-associated virus (AAV)-based antibody containing the Anc80 capsid protein. The novel gene delivery modalities described herein, including novel compositions and methods, are useful for IHC and The present invention provides highly efficient gene transfer into inner ear cells, including both oocytes and OHCs. As shown, Anc80 or a specific Anc80 capsid protein (e.g., Anc80-0 Adeno-associated viruses (AAVs) contain an ancestral scaffold capsid protein called 065. ) has been shown to target various cells in vivo, including IHCs and OHCs in the inner ear. It is surprisingly efficient. [Means for solving the problem]

[0005] In one embodiment, the Anc80 capsid protein and the TMC1 or TMC2 transgene In another embodiment, an AAV vector comprising the Anc80 capsid protein is provided. Protein, and MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB 2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 In one embodiment, an AAV vector is provided that comprises one or more transgenes. In some cases, the AAV vector further comprises a heterologous promoter.

[0006] In yet another embodiment, a transgene is delivered to one or more cells in the inner ear of a subject. Such methods typically involve injecting an adeno-associated virus (AAV) into the inner ear of a subject. The AAV comprises administering an AAV containing the Anc80 capsid protein and a transduction Contains genes.

[0007] In yet another embodiment, a method of treating hearing impairment (e.g., restoring hearing) or or ways to prevent hearing loss (or further hearing loss). The method typically includes administering AAV to a subject, wherein the AAV contains the Anc80 capsid. and a protein that, when expressed in one or more cells in the inner ear, provides hearing to a subject. Contains a transgene that restores

[0008] In one embodiment, the one or more cells in the inner ear are inner hair cells (IHCs) and In some embodiments, the transgene is selected from the group consisting of: outer hair cells (OHCs). 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 spiral ganglion neurons, Selected from the group consisting of vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis It is selected.

[0009] In some embodiments, the transgene is an ACTG1, ADCY1, ATOHI, AT P6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD1 64, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB 2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, CO L4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1 , COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8 L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIP C3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXC R1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1 , KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LAR S2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, ME T, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS 1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO 7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OT OG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PN PT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PT PRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17 A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMA C / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TM IE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, U SH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and and XIAP.

[0010] In some embodiments, the transgene is a neurotrophic factor (e.g., GDNF, BDNF In some embodiments, the transgene encodes an antibody. In some embodiments, the transgene encodes an immunomodulatory protein or a fragment thereof. In some embodiments, the transgene encodes an anti-oncogenic transcript. In some embodiments, the transgene is an antisense, silencing, or In some embodiments, the transgene encodes a long non-coding RNA species. From the group consisting of zinc finger nucleases, TALENs and CRISPRs Encodes a genome editing system of choice.

[0011] In some embodiments, the Anc80 capsid protein has the sequence shown in SEQ ID NO:1. In some embodiments, the Anc80 capsid protein has the sequence set forth in SEQ ID NO:2. In some embodiments, the transgene has a heterologous promoter sequence Representative heterologous promoter sequences include, but are not limited to, CMV promoters. ter, CBA promoter, CASI promoter, PGK promoter, EF-1 promoter Motor, alpha9 nicotinic receptor promoter, prestin promoter -, KCNQ4 promoter, Myo7a promoter, Myo6 promoter, Gfi l promoter, Vglut3 promoter and Atoh1 promoter .

[0012] In some embodiments, the administering step includes injecting the AncAAV through the round window. In some embodiments, the AncAAV is administered by injection through the round window. In some embodiments, the AncAAV is administered during cochleostomy or In some embodiments, the analgesic agent is administered during a canalostomy or a steroid injection. cAAVs are delivered to the middle ear and / or It is administered into the round window of the cochlea.

[0013] In some embodiments, expression of the transgene is expressed in inner hair cells (IHCs), outer hair cells ( OHC), spiral ganglion neurons, stria vascularis, vestibular hair cells and / or vestibular ganglion neurons resulting in the regeneration of neurons (e.g., Atoh1, NF2), thereby improving hearing or Restores vestibular function.

[0014] In one aspect, an article of manufacture is provided comprising the AAV vector and a pharmaceutical composition. In such products, the AAV vector contains the Anc80 capsid protein and promoter. In some embodiments, the transgene is operably linked to a target. , ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BS ND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEA CAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP , CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2 , DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, E LMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, G PR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF , HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LR TOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MIT F, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A , MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT 3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3 , PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, P OU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX1 0, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3 , TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, US H2D, VLGR1, WFS1, WHRN, and XIAP.

[0015] In another embodiment, TMC1 or TMC2 is introduced into one or more cells in the inner ear of a subject. Methods for delivering transgenes are provided. Such methods typically involve inserting an adduct into the inner ear of a subject. The method includes administering an anti-cancer agent (AAV) containing an Anc80 capsid. In another embodiment, the method comprises treating a hearing impairment in a subject. Such methods typically include the steps of administering AAV to a subject. The AAV comprises an Anc80 capsid protein and one or more cells in the inner ear. When expressed in cells, it can restore hearing to a subject or induce hearing loss (even The mice contain a TMC1 or TMC2 transgene that prevents further hearing loss (e.g., further hearing loss).

[0016] In yet another embodiment, an usher transgene is introduced into one or more cells in the inner ear of a subject. Such methods typically involve delivering adeno-associated cytotoxic T cells to the inner ear of a subject. The method includes administering an anti-cancer virus (AAV), wherein the AAV contains an Anc80 capsid protein. and a transgene. In yet another embodiment, the method comprises treating a hearing impairment in a subject. Such methods include administering to a subject an AAV, wherein the AAV comprises: Anc80 capsid protein and expressed in one or more cells in the inner ear Sometimes, an usher transgene can be included to restore hearing to the subject. Shear transgenes include, but are not limited to, MYO7A, USCH1C, CDH23, P CDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, P DZD7 is one example.

[0017] In one embodiment, the one or more cells in the inner ear are inner hair cells (IHCs) and In one embodiment, the transgene is selected from the group consisting of: outer hair cells (OHCs). It is delivered to at least 80% of the inner hair cells and at least 80% of the outer hair cells. In embodiments, the one or more cells in the inner ear include spiral ganglion neurons, vestibular hair neurons, and the like. cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis. .

[0018] In one embodiment, the Anc80 capsid protein has the sequence shown in SEQ ID NO:1. In one embodiment, the Anc80 capsid protein is set forth in SEQ ID NO:2. In one embodiment, the transgene is under the control of a heterologous promoter sequence. Representative heterologous promoter sequences include, but are not limited to, CMV promoter, CBA promoter, CASI promoter, PGK promoter, EF-1 promoter, Lufa 9 nicotinic receptor promoter, prestin promoter, KCNQ4 promoter -, Myo7a promoter, Myo6 promoter, Gfil promoter, Vglu Examples include the t3 promoter and the Atoh1 promoter.

[0019] In one embodiment, the administering step comprises injecting the AncAAV through the round window. In one embodiment, the AncAAV is administered by injection through the round window. In one embodiment, the AncAAV is administered during a cochleostomy or canalostomy. In one embodiment, the AncAAV is used in combination with one or more drug delivery vehicles. The vehicle is administered into the middle ear and / or round window.

[0020] In one embodiment, expression of the transgene is expressed in inner hair cells (IHC), outer hair cells (OH). C), spiral ganglion neurons, stria vascularis, vestibular hair cells and / or vestibular ganglion neurons regeneration of neurons (e.g., Atoh1, NF2), thereby improving hearing or hearing loss restore yard function and / or prevent hearing loss (e.g., further hearing loss).

[0021] Unless otherwise defined, all technical and scientific terms used herein are Methods and compositions of matter have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain. Methods and materials similar or equivalent to those described herein may be used in the methods and Suitable methods and materials may be used in the practice or testing of compositions of matter, including those described below. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. The document is incorporated herein by reference in its entirety. [Brief explanation of the drawings]

[0022] Part 1: Highly efficient cochlear gene transfer [Figure 1] Figures 1A-1G are representative confocal projection images of an in vitro comparison of several AAV serotypes for eGFP transgene expression in cochlear explants from C57BL / 6 mice. Figures 1A-1G show expression in the cochlear base for all serotypes, as well as the apical and basal sections for the indicated AAVs. Scale bar = 100 µM. Top: Myo7A + TuJ1; Bottom: eGFP only; Middle: overlay; S. cells = supporting cells; OHCs = outer hair cells; IHCs = inner hair cells. Figures 1H-1K are graphs showing the percentage of eGFP-positive hair cells per 100 µM after 48 h or 48 h + 5 days of incubation. N = 3 for 48 h, N = 2 for 48 h + 5 days. Error bars represent the standard error of the mean (SEM). [Figure 2]Figures 2A-2G are images showing in vivo cochlear transduction with the indicated AAV serotypes at titers indicated above each panel. Figure 2A is a confocal image of the mouse organ of Corti counterstained with Alexa-546-phalloidin (red) and imaged for eGFP (green). Scale bar = 50 µm. Figure 2B is a graph showing quantification of eGFP-positive IHCs in the basal and apical regions of AAV-eGFP-injected cochleae. Figure 2C is a graph showing quantification of eGFP-positive OHCs in the basal and apical regions of AAV-eGFP-injected cochleae. Figure 2D is an image showing sensory transduction currents recorded at P7 (left) from eGFP-negative OHCs (black) and eGFP-positive OHCs (green). The vertical scale bar indicates 200 pA; the horizontal scale bar indicates 20 ms. Currents from eGFP-negative (black) and eGFP-positive (green) P35 IHCs are shown on the right. The vertical scale bar indicates 100 pA; the horizontal scale bar indicates 20 ms. Figure 2E is a graph showing the amplitude of sensory transduction currents plotted for 103 IHCs and OHCs at the ages indicated below. Data from eGFP-negative (black) and eGFP-positive (green) IHCs are shown. The cell numbers for each group are indicated on the graph. Figure 2F is a graph showing the mean ± standard deviation (SD). ABR thresholds are plotted for four Anc80-injected ears (green) and four uninjected ears (black), along with data from one injected ear lacking eGFP fluorescence due to injection-related damage (red). Figure 2G is a graph showing the mean ± SD. DPOAE thresholds are plotted for four Anc80-injected ears (green) and four uninjected ears (black), and one negative control ear with injection damage and lacking eGFP fluorescence (red). Injection titers for data points in Figures 2B-2G are as in Figure 2A. [Figure 3]Figures 3A-3D are images showing transduction of Anc80-eGFP in the vestibular sensory epithelium. Figure 3A shows an image of a mouse utricle from a P1 mouse injected with 1 μL of Anc80-eGFP (1.7 × 10 GC / mL). The tissue was harvested, fixed, stained with Alexa546-phalloidin (red), and imaged for eGFP (green). Scale bar = 100 μm. Figure 3B shows an image of the ridge of the posterior semicircular canal from the same mouse described in Figure 3A. Scale bar = 50 μm. Figure 3C shows an image of the sensory epithelium of the human utricle. The tissue was exposed to the Anc80-eGFP vector, cultured, fixed, stained with Alexa546-phalloidin (red), and imaged for eGFP fluorescence (green). Scale bar = 100 μm. Figure 3D shows a high-magnification view of human epithelium in the utricle stained with Alexa546-phalloidin (red) and Myo7A (blue) and imaged for eGFP (green) transduced under the same conditions as in Figure 3C. White arrows in the overlay panel indicate selected eGFP-positive / Myo7A-positive cells. Scale bar = 20 μm. [Figure 4] Figures 4A-4J are representative images of an in vitro comparison of eGFP expression in cochlear explants from CBA / CaJ mice with several AAV serotypes. Figures 4A-4F are images showing results after incubation with equal doses of AAV serotypes. Scale bar = 200 μm. Error bars in Figures 4G-4J represent SEM. [Figure 5] Figures 5A-5H are images showing the eGFP qualitative expression scoring system, ranging from 0 (Figures 5D, 5H) (lowest expression) to 3 (Figures 5A, 5E) (highest level of expression), to illustrate the range of expression in terms of number and intensity of infected cells, with "0" representing no notable expression (Figures 5D, 5H), "1" representing some dimly expressing cells (Figures 5C, 5G), "2" representing low to moderate expression in a significant number of cells per microscopic field (Figures 5B, 5F), and "3" representing a high percentage of cells expressing eGFP at moderate to high levels (Figures 5A, 5E). Scale bars shown in Figure 5A (for Figures 5A-5D) and Figure 5E (for Figures 5E-5H) = 20 μm. [Figure 6] Figure 6 is a graph showing eGFP expression in limbus, supporting cells, and spiral ganglion neurons of C57BL / 6 mice using the eGFP scoring system detailed in Figure 5 above. Error bars represent SEM. Transduction of SGNs was assessed by the number of eGFP-positive cells per microscopic field. [Figure 7] Figure 7 is a graph showing eGFP expression in limbus, supporting cells, and spiral ganglion neurons of CBA / CaJ mice using the eGFP scoring system detailed in Figure 5 above. Error bars represent SEM. Transduction of SGNs was assessed by the number of eGFP-positive cells per microscopic field. [Figure 8] Figures 8A-8E are images showing widespread inner and outer hair cell transduction in mouse cochleae by Anc80. Figure 8A is an image showing a low-magnification view of the entire apical section of a mouse cochlea injected with Anc80-eGFP. The cochlea was harvested, stained with Alexa546-phalloidin (red), and imaged for eGFP (green). Scale bar = 100 μm. Figure 8B is an image showing a high-magnification view of a basal section from a different mouse cochlea injected with Anc80-eGFP. The cochlea was harvested, stained with Alexa546-phalloidin (red), and imaged for eGFP (green). Scale bar = 20 μm. Figures 8C and 8D are graphs showing a quantitative comparison of inner and outer hair cell transduction efficiency after round window injection in C57BL / 6 mice. (E) Images showing the dose-dependence of Anc80 hair cell transduction. Cochleae were exposed to two different titers of Anc80-eGFP, fixed, stained with Alexa546-phalloidin (red), and imaged for eGFP (green). Scale bar = 20 μm. [Figure 9]Figures 9A-9H show images of bilateral cochlear transduction from base to apex of mouse cochleae, assessed for eGFP transgene expression in tissue sections stained for TuJ1 (red) and Myo7A (magenta). Efficient Anc80 transduction extending to the apex was observed in the injected cochlea (Figures 9A / 9F) and also in the contralateral uninjected ear (Figures 9B-9E = apex to base). Close-up images of eGFP- and TuJ1-positive spiral ganglion neurons (Figure 9G). Reconstructed 3D images for SGN evaluation of Anc80 transduction (Figure 9H). Scale bars = 100 µm (Figures 9A-9E) and 20 µm (Figures 9F / 9G). [Figure 10] Figures 10A-10B are representations of microscopic images showing cross sections of mouse brains after unilateral cochlear injection of Anc80 (Figure 10A). Prominent expression was observed in the cerebellum, particularly in Purkinje cells (white arrowheads) (Figure 10B). Scale bars are 1 mm (Figure 10A) and 300 μm (Figure 10B). Figure 10C is an image showing anti-AAV neutralizing antibody (NAB) titers in the serum and cerebrospinal fluid (CSF) of uninjected and Anc80RWM-injected animals. Titers reflect the dilution of serum or CSF at which 50% inhibition of transduction is observed in the NAB assay. Due to sample volume limitations, the limit of sensitivity for serum NAB was 1 / 4 and for CSF was 1 / 52.5. [Figure 11] Figures 11A-11B are images and graphs, respectively, showing vestibular function after Anc80 cochlear transduction. Mice were injected with Anc80-eGFP and assessed for expression and balance function on a rotarod apparatus. Figure 11A is an image showing expression of eGFP (green) in vestibular tissue by confocal microscopy with immunofluorescent staining for Myo7A (red). Figure 11B is a graph showing the mean time ± SEM for mice to fall off the apparatus. Scale bar = 50 μm. Part 2 - Gene therapy restores function in a mouse model of Usher syndrome. [Figure 12]Figures 12A-12L show scanning electron microscopy images of the organ of Corti from Ush1cc.216G>A mutant mice. Figures 12A-12F show images of the basal, intermediate, and apical regions of the organ of Corti from c.216GA and c.216AA mutant mice. Figures 12G-12L show high-magnification images of OHCs (Figures 12G-12H) and IHCs (Figures 12I-12J). Asterisks indicate preserved hair bundles; arrowheads indicate disorganized hair bundles; and arrows indicate wavy IHC bundles. Scale bars: low magnification: 5 μm (Figures 12A-12F); high magnification: 2 μm (Figure 12G), 3 μm (Figure 12H), 2 μm (Figures 12I-12J), and 1 μm (Figures 12K, 12L). [Figure 13] Figures 13A-13H are images showing mechanotransduction in hair cells of Ush1cc.216G>A neonatal mutant mice. Figures 13A-13D are images showing FM1-43 staining to assess the presence of open transduction channels in hair cells of c.216GA and c.216AA mice. FM1-43 fluorescence in IHC appears dim when the IHC is in a different focal plane. Left: DIC, Right: FM1-43; Scale bar 10 μm; Figure 13C, Scale bar 50 μm; Figure 13D, Scale bar 10 μm. White lines in Figure 13D delineate striola (no uptake) and extrastriola (uptake). Figures 13E-13H are graphs showing mechanotransduction assessed in OHCs, IHCs, and VHCs of neonatal c.216GA and c.216AA mice. Representative transduction currents (Fig. 6E), associated current / displacement plots fitted with a second-order Boltzmann function (Fig. 6F), and mean peak transduction currents (Fig. 6G-H) were plotted. Mean peak transduction was significantly different between the two genotypes in OHCs, IHCs, and VHCs (***P<0.01, one-way ANOVA). [Figure 14]Figures 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. Figures 14A-14C show immediately dissected inner ear tissues exposed to AAV2 / 1 vectors, cultured, fixed, counterstained (Alexa Fluor phalloidin, Invitrogen), and imaged by confocal microscopy. Figure 14A scale bar: 10 μm—upper panel; 5 μm—lower panel; Figure 14B scale bar: 10 μm; Figure 14C scale bar: 3 μm; Figure 14D scale bar: 30 μm; Figure 14E scale bar: 5 μm. [Figure 15] Figures 15A-15C are images showing the restoration of mechanotransduction in hair cells of mice injected with the Anc80harmonin vector. Figures 15A-15C show mechanotransduction currents recorded in IHCs from c.216AA non-injected control mice and c.216AA mice injected with Anc80harmonin-b1 or a combination of Anc80harmonin-b1 and Anc80harmonin-a1. Organotypic cultures were prepared and recordings were performed. Corresponding I / X curves and double Boltzmann fit functions for each data set were obtained. The respective maximum mechanotransduction currents, Imax = 102.1 pA (c.216AA); 424.3 pA (c.216AA + harmonin-b1); and 341.1 pA (c.216AA + harmonin-a1 & -b1) (Figure 15B). The mean responses (mean ± SD) show significant recovery of transduction for mice injected with harmonin-b1 and harmonin-a1+-b1 relative to uninjected mice (***P<0.001). Mean transduction currents were not significantly different between mice injected with harmonin-b1 and c.216GA control mice (NS P>0.5). Recovery of mechanotransduction was not significantly improved when harmonin-a and harmonin-b were combined. Figure 15C shows a one-way ANOVA. [Figure 16]Figures 16A-16E are images showing the recovery of ABR and DPOAE thresholds in mice injected with Anc80harmonin-b1. Figure 16A shows representative ABR responses to a 16 kHz tone in c.216AA control mice and c.216AA mice injected with vectors encoding harmonin-a1, harmonin-b1, or a combination of the two. Recovered ABR thresholds around 30 dB SPL were measured in mice injected with harmonin-b1 alone or harmonin-a1 and -b1 together. Figure 16B shows average ABR responses obtained for c.216AA; c.216GA; c.216AA + harmonin-a1; c.216AA + harmonin-b1; and c.216AA + harmonin-a1 & -b1. Mean ± SE, solid lines. Dotted lines: ABR thresholds for the entire frequency range of the mouse whose 16 kHz recording is shown in Figure 16A. Figure 16C shows the average DPOAE responses obtained for c.216AA; c.216GA; c.216AA + harmonic-a1; c.216AA + harmonic-b1; and c.216AA + harmonic-a1 & -b1. Mean ± SE, solid lines. Dotted lines: DPOAE thresholds for the four mice whose recordings are illustrated in Figure 16A. Arrows indicate thresholds higher than the maximal stimulus level tested. Figures 16D-16E show ABR and DPOAE responses obtained at 6 weeks and 3 months in mice that showed initial ABR thresholds less than or equal to 45 dB. Six of the eight mice were housed for 6 months and had ABR and DPOAE assessments (dotted lines). Mean ± SE. While shifts in ABR and DPOAE thresholds were evident during the first 3 months, hearing rescue was still prominent at 6 months of age in the low-frequency range. [Figure 17]Figures 17A-17E are images showing the recovery of startle response, rotarod performance, and open-field behavior in mice injected with Anc80harmonin-a1 and Anc80harmonin-b1. Figure 17A shows startle responses to white noise stimuli recorded in control c.216GA, c.216AA, and injected c.216AA mice. Partial startle rescue was evident in mice injected with harmonin-b1, but not with harmonin-a1. Means are shown ±SE. Figure 17B shows rotarod performance in control c.216GA, c.216AA, and injected c.216AA mice. Complete recovery was observed in mice injected with harmonin-b1 and harmonin-a1 / b1; no recovery was observed with harmonin-a1 alone. Means are shown ±SE. Figures 17C-17E show open-field observations conducted for 5 minutes in control c.216GA, c.216AA, and injected c.216AA and c.216GA mice. A representative track over 2.5 minutes is shown (Figure 17B). While c.216AA mutant mice explored the entire field and repeatedly performed full-body rotations, c.216AA mice injected with harmonin-a1, harmonin-b1, or a combination of the two vectors at P1 exhibited normal behavior similar to their heterozygous c.216GA counterparts or c.216GA mice injected with truncated vectors. Figure 17C shows graphs illustrating the mean ± SE for number of rotations and distance traveled per minute. Significant recovery ***P<0.001 was observed between uninjected and injected mice. Statistical analysis by one-way ANOVA. [Figure 18]Scanning electron microscope images of the organ of Corti from mice injected with Anc80harmonin-b1. The basal, middle, and apical regions of the organ of Corti were imaged in c.216GA, c.216AA, and c.216AA mice. OHC and IHC hair bundles were preserved in c.216GA mice, whereas they appeared disorganized along the organ of Corti in c.216AA mice. Notable hair cell loss (asterisks) and hair bundle disorganization were observed in c.216AA mice, along with more pronounced degeneration in the basal end of the organ. Hair bundles in c.216AA mice lacked normal rows of stereocilia. In c.216AA mice, lower rows appeared retracted, while higher rows were preserved (arrows). While hair cell loss and bundle disorganization were still evident in rescued c.216AA mice, hair cell survival was significantly higher in the basal and middle regions of the organ. Bar graphs summarize hair cell counts. A total of 1824 cells were counted in c.216AA mice and 792 in rescued c.216AA mice. Mean ± SE. Higher magnification imaging revealed rescue of stair-like rows of cells (arrows) in many, but not all, cells (arrowheads) in injected c.216AA mice (arrows). Scale bar: low magnification: 5 μm; higher magnification: 1 μm. [Figure 19] Figures 19A-19L show SEM images of hair bundle morphology in Ush1cc.216G>A mice. Figures 19A-19C show heterozygous c.216GA mice with normal hair bundle morphology. Figures 19D-19I show disorganized hair bundles along the organ of homozygous c.216AA mutant mice. Figures 19J-19L show mildly disorganized IHC hair bundles in c.216AA mice. Distance measured from the apex: basal 3.5-4 mm; medial 1.8-2.2 mm; apical 0.6-0.8 mm. Scale bar: low magnification: 5 μm; high magnification: 1 μm. [Figure 20]Figures 20A-20J show images demonstrating mechanotransduction characteristics in c.216AA mutant mice. Figures 20A-20E show analysis of mechanotransduction in neonatal OHCs from the middle and mid-apical turns of the cochlea. To assess adaptation in c.216GA and c.216AA mutants, representative current traces from ~P = 0.5 were fitted to a double exponential decay function (Figure 20A). The fit was used to generate the degree of adaptation (Figure 20E), as well as fast (Figure 20E) and slow (Figure 20D) time constants. The 10-90% operating range did not change significantly (Figure 20B). The degree of adaptation in c.216AA mice was significantly smaller than that in heterozygous OHCs, as shown in this scatter plot (Figure 20E). Figures 20F-20J show analysis of mechanotransduction in neonatal IHCs. The values ​​for the 10-90% working range were smaller for c.216GA vs. c.216AAIHC (Figure 20G). Adaptation was always present, but was slightly slower and significantly less severe in c.216AA IHC (Figures 20H-20J). Statistical analysis is indicated for each plot: *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA. [Figure 21] Figures 21A-21C show data demonstrating the expression of fluorescently labeled harmonin-a and harmonin-b Anc80 vectors in the organ of Corti of c.216AA mice 6 weeks after dual vector injection at P1. Figures 21A-21C show confocal images of the basal turn of a 6-week-old c.216AA mouse after co-injection of AAV2 / Anc80.CMV.tdTomato::harmonin-a1 (0.5 μl; 4.11E^12 gc / ml) and AAV2 / Anc80.CMV.eGFP::harmonin-b1 (0.5 μl; 2.99E^12 gc / ml) at P1. 69% and 74% of the total cells expressed eGFP (Figure 21A) and tdTomato (Figure 21C), respectively, and 65% expressed both markers, demonstrating successful co-transduction. Scale bar: 20 μm. [Figure 22]Figures 22A-22F show data illustrating the analysis of ABR responses of control c.216GA and injected, rescued c.216AA mice. Figures 22A and 22D show example ABR responses at 8 and 16 kHz for control c.216GA and rescued c.216AA mice. Figures 22B-22C and 22E-22F show the mean Peak 1 amplitudes (Figures 22B-22D) and latencies at 8-11.3 and 16 kHz (Figures 22C-22D) 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. [Figure 23] Figures 23A-23D show that the mutant form of harmonin expressed in Ush1c.216G>A mice does not alter hair cell or auditory function. Figure 23A is a sequence alignment between the wild-type harmonin-b1 protein and the truncated form of harmonin secreted as a result of a frameshift involving cryptic splicing and the acadian G>A mutation in exon 3 of the Ush1c gene. Figure 23B shows that semiquantitative RT-PCR from the auditory organs of wild-type, c.216GA, and c.216AA mutant mice confirms 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 distortion components (DPOAE, Figure 23D) measured in injected c.216GA mice and control c.216GA and c.216AA mice. Plots are shown as mean ± SE. [Figure 24]Figures 24A-24C are images showing the restoration of accurate Ush1c splicing in the inner ear of 6-week-old mice injected with AAV2 / Anc80.CMV.harmonin-b1. Figure 24A shows that quantification of correctly spliced ​​(450 bp) and aberrant (415 bp) mRNA from the Ush1c.216A allele by semiquantitative RT-PCR demonstrates restoration of accurate Ush1c splicing in the injected (I) and contralateral ears (Figure 24C) of rescued c.216AA mice #1 and #2 (responses at 35 dB SPL at 11.3 kHz from the injected ear). Mouse #3, which had a poor ABR response (90 dB SPL at 11.3 kHz), showed moderate restoration of accurate mRNA expression, while mouse #4 (100 dB SPL at 11.3 kHz) showed no restoration. While the correct splice form is not detected in uninjected c.216AA mice (mice #5 and #6), both the correct and truncated splice forms are detected in c.216GA mice (mice #7, #8, and #9). To confirm the relative amounts of material, the corresponding mouse GAPDH was amplified, as shown in the bottom panel. Figure 24B shows an image showing that semiquantitative radiolabeled PCR analysis confirmed the presence of AAV-mUsh1c in the injected and contralateral ears of Ush1c.216AA mice. Relative levels of AAV-mUsh1c DNA were present but reduced in mice #3 and #4. Figure 24C shows that the relative amount of AAC-mUsh1c correlated with ABR thresholds. Analysis is illustrated for 11.3 and 16 kHz. Linear regression showed a high correlation between the two. [Figure 25]Figure 25 is a graph showing that long-term ABR threshold recovery correlated with OHC survival in the intermediate to apical regions of the auditory organ. Hair cell counts throughout the organ of Corti were performed postmortem on the left ears of three uninjected c.216AA mice and five injected c.216AA mice. The total number of IHC and OHC hair cells was increased in injected mice. Comparison of rescued injected mice with poorly rescued mice showed no difference in the number of IHCs, but a significant number of OHCs was noted in rescued mice. Analysis throughout the length of the organ indicated that the difference could be explained by increased survival of hair cells from the intermediate to apical regions of the organ. Inset: Two of the mice (mice #1 and #2) showed poor ABR response thresholds (≥95 dB SPL) across the entire range tested, while three (mice #3, #4, and #5) responded to sound stimuli between 5.6 and 16 kHz with thresholds ranging from 35 to 55 dB SPL. Part 3 - Gene therapy for additional mutations involved in hearing loss [Figure 26] Figures 26A-26D show representative confocal images of the cochlea of ​​a Ush1c mutant mouse injected with Anc80-Harmonin::GFP (i.e., GFP fused to the Harmonin polypeptide) through the RWM, harvested, stained for actin (red; Figure 26A), Myo7A (blue; Figure 26B), and imaged for GFP (green; Figure 26C). A merged image of Figures 26A, 26B, and 26C is shown in Figure 26D. [Figure 27] FIG. 27 is a graph showing ABR thresholds plotted as a function of tone frequency for Ush1c mutant mice (squares) and Ush1c mutant mice injected with the Anc80-Harmonin::GFP vector (circles). [Figure 28] Figures 28A-28C show representative confocal images at low magnification (Figure 28A) or high magnification (Figure 28B) of KCNQ4- / - cochleae injected with Anc80-KCNQ4 through the RWM, harvested, and stained with Alexa546-phalloidin (red) and an antibody against KCNQ4 (green), compared to high magnification of an uninjected cochlea (Figure 28C). [Figure 29]Figures 29A-29C are a series of graphs showing KCNQ4 currents in wild-type mice (Figure 29A), P10 KCNQ4- / - mice (Figure 29B), and P10 KCNQ4- / - mice injected with Anc80-KCNQ4 (Figure 29C). Cochleae were harvested 8 days after injection. [Figure 30] FIG. 30 is a series of three images showing FM1-43 uptake (FM1-43 permeates only functional Tmc1 channels) in Tmc1 − / − tissue injected with Anc80 Tmc1 vector. [Figure 31] Figure 31A shows representative images of sensory transduction currents recorded from IHCs in wild-type (left), Tmcl- / - (middle), and Anc80 Tmcl-injected Tmcl- / - mice (right). Cochleae were harvested 8 days after injection. Figure 31B shows a graphical representation of the recovery rate of the mice shown in Figure 31A. The graphs in Figure 31B show the percentage of functional cells in wild-type (left), Tmcl- / - (middle), and Anc80 Tmcl-injected Tmcl- / - mice (right). [Figure 32] FIG. 32 is a graph showing distortion product otoacoustic emission (DPOAE) thresholds as a function of stimulation frequency for wild-type, Tmc1 − / − mice, and Anc80 Tmc1- / − mice injected with Anc80 Tmc1. DETAILED DESCRIPTION OF THE INVENTION

[0023] Sensory cells in the adult mammalian cochlea lack the ability to self-repair, making it difficult to accurately measure the level and accuracy of damage. Current treatment strategies (location-dependent) involve developing auditory nerves that relay acoustic information to the brain. to compensate for permanent damage to primary sensory hair cells or spiral ganglion neurons that transmit signals to the , amplification (hearing aids), better transmission of sound (middle ear prostheses / active implants) or These approaches are transformative. However, it is the best way to restore the complex human hearing function that is so important to modern life. In particular, the main problems are limited frequency sensitivity, unnatural sound perception, , and still includes limited speech discrimination in noisy environments.

[0024] Therapeutic gene transfer into the cochlea has potential applications in treating hearing loss ranging from age-related and environmentally induced to genetic forms. It has been thought that this would be an improvement over the current standard of care. , which has been implicated in hereditary hearing loss with over 70 described causative genes (Parker & Bitner-Glindzicz, 2015, Arch. Dis. Childhood, 100:271-8). The success of this approach is due to the relevant therapeutic target cells in the organ of Corti (OC) of the cochlea. Much depends on the safe and efficient delivery of exogenous gene constructs.

[0025] The OC contains two types of sensory hair cells: those that transmit the mechanical information carried by sound to neural structures. The IHC converts the electrical signals received into the brain, a process required for complex hearing function. Other potential targets in the inner ear include OHCs, which play a role in amplifying and modulating the cochlear response. Targets include spiral ganglion neurons and columnar cells at the edge of the spiral lamina, which are protected The adjacent tectum has a protective function and can be induced to transdifferentiate into hair cells by early neonatal life. Important for maintaining membranes or supporting cells.

[0026] Injection into the cochlear duct filled with high-potassium endolymph provides direct access to hair cells. However, changes in this delicate fluid environment can disrupt the endocochlear potential and cause the injection The periphosphates surrounding the cochlea, scala tympani, and scala vestibuli may increase the risk of related toxicities. The air-filled space leaves the middle ear through either the vestibulocochlear window membrane or the round window membrane (RWM). The RWM, the only non-bony opening to the inner ear, is accessible in many animal models. It is relatively easily accessible in the United States, and administration of viral vectors using this route is well documented. In humans, cochlear implant placement involves the insertion of surgical electrodes through the RWN. Depends on it on a daily basis.

[0027] Previous studies evaluating AAV serotypes in organotypic cochlear explants and in vivo cochlear implantation The study resulted in only partial rescue of hearing in a mouse model of inherited hearing loss. In addition, adeno-associated viruses (AVVs) containing the ancestral AAV capsid proteins are highly efficient This finding supports the development of cochlear gene therapy using conventional AAV serotypes. The ancestral AAV capsid described herein overcomes the low transduction rates that have limited the success of AAV gene expression. AAV containing the protein is able to detect not only IHCs and OHCs but also hereditary hearing and hearing loss. This may be a valuable platform for inner ear gene delivery to many other inner ear cell types impaired by equilibrium disorders. In addition to providing a high transfer rate, the ancestral AAV containing AAV capsid proteins can be expressed in mice and non-human spirits upon systemic injection. It has a similar safety profile in primates and is antigenically distinct from circulating AAV. Offering potential benefits regarding pre-existing immunity that limits the effectiveness of conventional AAV vectors It was shown that:

[0028] However, cells, particularly cells in the inner ear, e.g., the cochlea (or cells of the cochlea or cochlea) Described herein are compositions and methods that allow for highly efficient delivery of nucleic acids into bovine cells. As used herein, inner ear cells include, but are not limited to, inner hair cells (IHCs), outer hair cells (EHCs), and Hair cells (OHCs), spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, Supporting cells refer to cells in the ear and cells in the stria vascularis. Supporting cells are cells in the ear that are not excitable, e.g., Refers to cells that are not hair cells or neurons. An example of a supporting cell is a Schwann cell.

[0029] Delivery of one or more nucleic acids described herein to cells of the inner ear typically results in partial hearing loss. Treat any number of inherited or acquired hearing impairments defined by hearing loss or complete deafness. The methods described herein can be used to treat, but are not limited to, recessive hearing loss, dominant hearing loss, Hearing loss due to trauma or age, as well as hearing loss due to Usher syndrome and other syndromic hearing loss The invention may be used to treat hearing impairments such as hearing loss.

[0030] Methods for producing viruses carrying specific transgenes As described herein, adeno-associated viruses containing ancestral AAV capsid proteins AAVs have been particularly useful in delivering nucleic acids (e.g., transgenes) to cells of the inner ear. One particularly effective and efficient type of ancestral AAV capsid protein is designated Anc80. The Anc80 ancestral scaffold capsid protein is represented by the ancestral scaffold capsid protein shown in SEQ ID NO: 1. One specific ancestral capsid protein belonging to the precapsid protein class is Anc 80-0065 (SEQ ID NO: 2), but the pamphlet of WO 2015 / 054653 The Anc80 ancestral capsid protein family is a family of many further ancestral capsids. Proteins are described.

[0031] The viruses described herein containing the Anc80 capsid protein can be used to encode various nucleic acids. Nucleic acid sequences delivered to cells for expression purposes can be used to deliver nucleic acid sequences to cells of the inner ear. A gene sequence is often called a transgene. Representative gene sequences that can be delivered to and expressed in inner ear cells are: Transgenes include, but are not limited to, neurotrophic factors (e.g., glial cell line-derived neurotrophic factors). GDNF, brain-derived neurotrophic factor (BDNF), neurotropin-3 (NT3), or heat shock protein (HSP)-70), immunomodulatory proteins or anti-oncogenic proteins In addition, transgenes that are delivered to and expressed in inner ear cells are also included. Representative transgenes that can be used include, but are not limited to, antibodies or fragments thereof, antisense silencing or long non-coding RNA species, or genome editing systems (e.g., Gene-modified zinc finger nucleases, transcription activator-like effector nucleases ( TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR) Additionally, transgenes that can be delivered to and expressed in inner ear cells include Typical transgenes include ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC1 4A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN1 4, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, CO L4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A 2, 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, GRXC R2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, K CNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFP L5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR18 3, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14 , MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS 2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOG L, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POL R1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX , S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC2 2A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABL O, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, T ECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM 132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, US H1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP The nomenclature used herein includes nucleic acids named , World Wide Web hereditaryhearingloss.or g / .

[0032] Expression of the transgene is regulated by the natural promoter of the transgene (i.e., a promoter (such as that found naturally with the promoter sequence) or a transgene Expression of the vector may be directed by a heterologous promoter. For example, any of the vectors described herein may be directed by a heterologous promoter. Alternatively, the transgene may be used with its native promoter. Any of the above transgenes may be used in conjunction with a heterologous promoter. When used in a gene encoding a heterologous promoter, the heterologous promoter does not naturally direct expression of that sequence (i.e., does not naturally direct expression of that sequence). Any promoter described herein is intended to refer to a promoter that is not found with that sequence in any of the sequences described herein. Exemplary heterologous promoters that can be used to direct expression of the transgene include: For example, the cytomegalovirus (CMV) promoter, the avian beta actin (CBA ) promoter, synthetic CASI promoter, phosphoglycerate kinase (PGK) promoter promoter, and elongation factor (EF)-1 promoter, alpha 9 nicotinic receptor promoter promoter, prestin promoter, growth factor-independent (GFI1) promoter, and and the vesicular glutamate transporter 3 (VGLUT3) promoter. In addition, a promoter that naturally directs expression of one of the transgenes (e.g., K CNQ4 promoter, Myo7a promoter, Myo6 promoter, or ATOH 1 promoter) is used as a heterologous promoter to direct the expression of a transgene. It is possible.

[0033] Introduction of Anc80 capsid protein into a containing virus Methods for producing genes are known in the art and involve conventional molecular biology and recombinant nucleic acid techniques. In one embodiment, a nucleic acid sequence encoding the Anc80 capsid protein is used. Constructs containing the sequence and carrying the transgene flanked by suitable inverted terminal repeats (ITRs) are used. Constructs are provided that package the transgene within the Anc80 capsid protein. This allows the device to be pinged.

[0034] The transgene is then transduced into the Anc80 capsid protein using a packaging host cell, for example. Components of the viral particle (e.g., , rep sequence, cap sequence, inverted terminal repeat (ITR) sequence) may be one or more of the sequences described herein. One or more constructs may be used to transiently or stably introduce the vector into a packaging host cell. The viruses described herein contain at least the Anc80 capsid protein. However, other components of the virus particle (e.g., rep sequences, ITR sequences) are derived from ancestral sequences. In some cases, e.g., whole virus particles, Such viruses can be purified using routine methods. It can be done.

[0035] It will be appreciated that one or more than one transgene may be delivered to the inner ear. using a single AAV vector containing the Anc80 capsid protein or Using multiple AAV vectors containing SID proteins, more than one transgene can be delivered to the inner ear. It can also be understood that it can be delivered.

[0036] Generally, as used herein, "nucleic acid" can include DNA and RNA. and may also include nucleic acids containing one or more nucleotide analogues or backbone modifications. Nucleic acids can be single-stranded or double-stranded, which is usually the case for their intended use. Nucleic acids that can be used in the methods described herein are those that are identical to known nucleic acid sequences. The nucleic acid may be a single molecule or may be used in the methods described herein. By way of example only, the nucleic acid (or encoded A polypeptide (such as a polypeptide of interest) should 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).

[0037] In calculating percent sequence identity, two sequences are aligned and the two sequences are compared. The number of perfect matches of nucleotides or amino acid residues between the strings is determined. is the length of the aligned region (i.e., the number of aligned nucleotides or amino acids The percent sequence identity is calculated by dividing the number of aligned residues by the number of aligned residues (number of aligned residues) and multiplying by 100. The length of the region ranges from a portion of one or both sequences to the full length of the shortest sequence. It is understood that a single sequence may be aligned with more than one other sequence. and thus different percent sequence identity values ​​can be obtained across each aligned region. It is also understood that the

[0038] Alignment of two or more sequences to determine percent sequence identity can be performed using computer-aided methods. This was performed using the data program ClustalW and default parameters. is performed over the entire length of a nucleic acid or polypeptide sequence. (global alignment). Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW is a method for matching a query with one or more target sequences. The best match between the two is calculated and these are compared so that identities, similarities, and differences are determined. To maximize sequence alignment, gaps of one or more residues are allowed. can be inserted into the query sequence, the subject sequence, or both. For the statement, the default parameters are used (i.e., word size: 2; Window size: 4; Scoring method: Percentage; Number of top diagonals: 4; and gap penalty: 5); for alignment of multiple nucleic acid sequences, The parameters used are: Gap opening penalty: 10.0; Gap extension penalty: Pairwise Polypeptide Sequences For alignment, the following parameters are used: word size: 1; Dough size: 5; Scoring method: Percentage; Number of top diagonals: 5 and Gap penalty: 3. For multiple alignment of polypeptide sequences, Parameters used: Weight matrix: BLOSUM (permutation matrix) lock(blocks substitution matrix)); gap opening penalty: 10.0; gap Loop extension penalty: 0.05; Hydrophilic gap: On; Hydrophobic residues: Gly, Pro , Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific Automatic gap penalty: On. ClustalW uses, for example, Baylor Col Legacy of Medicine Search Launcher website or European Bioinformatics on the World Wide Web This can be done on the Institute website.

[0039] Changes can be introduced into a nucleic acid sequence, which can be done if the nucleic acid sequence is a coding sequence. This may result in changes in the amino acid sequence of the encoded polypeptide. using natural mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis) Alternatively, a nucleic acid molecule having such an alteration can be introduced into the nucleic acid coding sequence by chemical synthesis. Such nucleic acid changes can be conservative or non-conservative in one or more amino acid residues. A "conservative amino acid substitution" may lead to conservative and / or non-conservative amino acid substitutions. those in which the acid residue is replaced with a different amino acid residue having a similar side chain (e.g., amino Dayhoff et al. (1978, Atlas of Protein S), which provides a frequency distribution table for acid substitutions. Sequence and Structure, 5(Suppl. 3):345-352) and non-conserved positions A substitution is one in which an amino acid residue is replaced with an amino acid residue that does not have a similar side chain.

[0040] The nucleic acid may be contained within a construct, which may be called a vector or a plasmid. , can be obtained commercially or produced by recombinant techniques routine in the art. Constructs containing such nucleic acids include expression elements that direct and / or control the expression of such nucleic acids. and sequences for maintaining the construct (e.g., origin of replication, selectable marker). ) Expression elements are known in the art and may include sequences such as: For example, promoters, introns, enhancer sequences, response elements or inducible Contains elements.

[0041] Methods for delivering nucleic acids to inner ear cells Methods for delivering nucleic acids into cells are generally known in the art and include transgenes. A method for delivering a virus (also called a viral particle) to inner ear cells in vivo is As described herein, about 10 8 ~about 10 12 Individual Wii The virus can be administered to a subject in a suitable volume (e.g., 10 μL, 50 μL ... μL, 100 μL, 500 μL, or 1000 μL) of artificial perilymph, e.g. It can be suspended.

[0042] Viruses containing the transgenes described herein can be expressed using any number of mechanisms. The delivery may be to cells of the inner ear (e.g., cells in the cochlea). For example, one or more of the compounds described herein may be delivered to the inner ear. a therapeutically effective amount of a composition comprising viral particles containing multiple different types of transgenes , typically in a relatively simple (e.g., outpatient) procedure through the cochlear window or vestibular window. In some embodiments, the vector may contain a transgene as described herein or A set containing a therapeutically effective number of viral particles containing one or more sets of different viral particles. Each particle in the set can contain the same type of transgene, but the number of particles can vary. Each set contains a different type of transgene from the other sets. The device can be delivered to the appropriate location in the ear during surgery (e.g., surgery or canalostomy).

[0043] In addition, delivery vehicles (e.g., acetaminophen) that facilitate drug transfer across the tympanic membrane and / or through the round window (cochlea) may be used. For example, polymers) are available, and any such delivery vehicle may be used as described herein. These can be used to deliver the viruses described in, for example, Arnold et al., 2005, Audiol. N See Eurootol., 10:53-63.

[0044] The compositions and methods described herein provide for highly efficient delivery of nucleic acids to inner ear cells, e.g., cochlear cells. For example, the compositions and methods described herein allow for the delivery of transgenes to the host. At least 80% of hair cells (e.g., at least 85, 90, 91, 92, 93, 94 , 95, 96, 97, 98 or 99%) or At least 80% of hair cells (e.g., at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the total nucleotide sequence.

[0045] As demonstrated herein, AAV containing the Anc80 capsid protein Expression of transgenes delivered using MRI has been shown to improve auditory or vestibular function for extended periods (e.g. Inner hair cells (IHCs) can be restored over a period of time (months, years, decades, even a lifetime). , outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or or vestibular ganglion neurons (e.g., Atoh1, NF2).

[0046] As described in WO 2015 / 054653, the Anc80 capsule AAV containing seroprevalence and / or the ability to differentiate from conventional AAV (i.e., i.e., it is neutralized compared to AAV that does not contain the Anc80 capsid protein In the art, seroprevalence is defined as the number of seropositive (i.e., specific is understood to refer to the proportion of subjects in a population (who have been exposed to a particular pathogen or immunogen), The number of subjects in a population who produce antibodies against another pathogen or immunogen is compared to the number of subjects in the population tested. It is calculated as the number of infected individuals divided by the total number of infected individuals. , which are routinely practiced in the art and involve samples from special populations of individuals (e.g., blood samples). Immunoassays are typically used to determine the prevalence of one or more antibodies in a population. In addition, several methods are available for determining the level of neutralizing antibodies in serum samples. For example, a neutralizing antibody assay may demonstrate a 50% or greater increase in antibody activity compared to a control sample containing no antibody. The titer is measured to determine the concentration of antibodies in the experimental sample that neutralize the infection. Fisher et al. (1999) 97, Nature Med., 3:306-12) and Manning et al. (1998, Human Gene Ther., 9:477 -85). Representative conventional AAVs include, but are not limited to, AAV8 (or AAV8 capsid protein-containing viruses) and / or AAV2 (or AAV2 viruses containing capsid proteins).

[0047] Usher Syndrome Human Usher syndrome (USH) is a rare genetic disorder that causes combined deafness and blindness It is inherited as an autosomal recessive trait and affects 16,000-20,000 people in the United States. Usher syndrome affects 100 million people and causes childhood hearing loss in 3-6% of cases. The severity of symptoms varies. It is classified into three clinical subtypes (USH-1, -2, and -3). USH-1 is the most severe. Patients affected by USH1 have congenital bilateral profound sensorineural hearing loss and vestibular apathy. and prepubertal retinitis pigmentosa (progressive bilateral symmetric alterations in retinal rod and cone function). Unless fitted with a cochlear implant, the individual will not develop the ability to speak typically. Currently, there is no biological treatment for Usher patients, but the wild-type form of the defective gene is Early reintroduction of the morphology may allow for reversal of the disease.

[0048] Six usher genes: MYO7A (myosin 7a), USH1C (harm onin), CDH23 (cadherin 23), PCDH15 (protocad herin 15), SANS (sans) and CIB2 (calcium and Integrin binding protein 2) is associated with USH1. The genes are involved in hair bundle morphogenesis in the inner ear and express proteins that are part of the interactome. encodes quality (e.g., Mathur & Yang, 2015, Biochim. Biophys. Acta, 1852:406-20 (See ). Harmonin binds to other Usher1 proteins. , which is central to the USH1 interactome. Its PDZ (PSD-59 95 / Dl ) interacting domain, harmonin acts as a scaffolding protein. In vitro binding studies have shown that it functions similarly to all other known USHs. 1 protein, two of the USH2 proteins, usherin and VLGR Similar to 1, it was shown to bind to the PDZ domain of harmonin. The proteins are divided into three distinct subclasses (a, b, and c) depending on the domain composition of the protein. They encode 10 alternative splice forms of harmin, classified into It consists of 28 exons. Three isoforms are involved in PDZ protein-protein interactions. The action domain, the coiled-coil (CC) domain, and the proline-serine-threonine ( PST) rich domains differ in number.

[0049] USH1 protein binds to hundreds of stereocilia interconnected by numerous extracellular links. The Usher gene (and its related genes) are localized at the apical end of hair cells in mechanosensory hair bundles. Cadherin 23 and protocadherin 24, which are products of USH1D and USH1E, respectively, Phosphorin 15 forms the tip-link located at the distal end of the stereocilia. In-b binds to CDH23, PCDH15, F-actin, and itself. It is found at the tip of the stereocilia near the insertion point of the sensory filament of the cell, where it transmits signals to the hair cell. Harmony-b is thought to play a functional role in early postnatal development and adaptation. Its expression is observed in both the cochlea and vestibule at postnatal day 30 (P30) Harmonin-a also binds to cadherin 23 and is found in the stereocilia. Recent reports have revealed additional roles for harmon-a at synapses. In this context, harmonized-a binds to Cav1.3 Ca2+ channels. , limiting the availability of channels through the ubiquitin-dependent pathway.

[0050] Several mouse models of Usher syndrome have been identified or engineered over the past decade. Seven of these affect harmonics. Only the Ush1cc.216G>A model exhibits the auditory defect characteristic of human Usher syndrome. Ush1cc.216G>A reproduces both the retinal defects and the retinal defects. A point mutation similar to that found in a cohort of USH1C patients from the USH1C subtype A knock-in mouse model affecting the expression of all conventional harmon isoforms was developed. The mutation is a cryptic splice site at the end of exon 3 of the Ush1c gene. The use of this cryptic splice site introduces a frameshift mutation containing a 35-bp deletion. It produces a severely truncated transcript lacking the PDZ, PST and CC domains. Homozygous C.216AA knock-in mice showed a phenotype of α-amyloid at 1 month of age. Although they suffer from severe hearing loss, heterozygous c.216GA mice do not exhibit any abnormal phenotypes. The cochlear histology in c.216AA mice is characterized by the middle and lower gyrus at P30. Disorganized hair bundles in the basal and basal turns, abnormal cell alignment, and loss of inner and outer hair cells. Indicates a loss.

[0051] In particular, the ancestral AAV capsid described herein in combination with the harmonin transgene The hair cells were successfully transduced with the harmonin splice form of the ATPase inhibitor. This promotes the expression and precise localization of harmonin, thereby reintroducing wild-type harmonin. Treating patients diagnosed with Usher syndrome-related hearing loss, e.g., USH1C-related hearing loss Furthermore, the ancestral AAV capsid described herein can be delivered to the round window membrane early after birth. Injection of AAV containing the protein resulted in hearing loss in homozygous c.216AA mice. It is demonstrated herein that the injected mammogram successfully restores splenic and vestibular function. Restoration of hearing function in mice was associated with an increase in the expression of mRNA encoding wild-type harmon This is associated not only with recovery but also with preservation of hair bundle morphology and mechanotransduction.

[0052] TMC1 / TMC2 Over 40 different mutations have been identified in TMC1 that cause hearing loss. These are further divided into 35 recessive mutations and 5 dominant mutations. Most result in severe congenital hearing loss (e.g., DFNB7 / 11), but a minority Causes late-onset moderate to severe hearing loss. All dominant mutations occur in the mid-teens. In particular, the present invention relates to a method for treating hearing loss, which causes progressive hearing loss (e.g., DFNA36). AAV vectors containing the described Anc80 capsid protein may be non-mutated (e.g., wild-type) TMC1 or TMC2 sequences, thereby preventing hearing loss (e.g., The present invention may be used to prevent hearing loss (eventually hearing loss) and / or restore hearing function.

[0053] Conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques within the skill of one of ordinary skill in the art. Techniques such as these may be used in accordance with the present disclosure. Such techniques are fully explained in the literature. The present invention is exemplified in some of the following examples. The methods and compositions are further described in the following examples, which do not limit the scope of the methods and compositions. [Example]

[0054] Part 1: Highly efficient cochlear gene transfer Example 1 Adeno-associated viruses (AAVs) containing ancestral AAV capsid proteins are safe and effective Efficient cochlear gene transfer The following methods and materials were used in Example 1.

[0055] viral vectors AAV2 / 1, 2 / 2, 2 / 6, 2 / 8, and 2 / 9 containing a CMV-driven eGFP transgene and AAV2 / Anc80L65 and woodchuck hepatitis virus post-transcriptional regulator ( WPRE) cassette was transfected as previously described (Zinn et al., 2015, Cell Reports, 12:10 56-68) Massachusetts Eye and Ear Infirmary ar) Gene Transfer Vector Core(vector.mee The AAV2 / Anc80L65 plasmid was prepared at the University of California, Berkeley, Calif. (i.harvard.edu). The reagents are available from addgene.com.

[0056] In vitro explant culture A total of 156 cochlear explant cultures from pups of both strains were cultured as previously described (Dilwali et al. The rats were then placed on day 4 of birth for evaluation as described in (Isamu Isamu, 2015, Scientific Reports, 5:18599). The temporal bones of mice were collected after decapitation, and the spiral ganglion neuron area was extracted. Cochleae were dissected for culture as organotypic explants connected to the nucleus. Two per cochlea were used. Obtain specimens, one ("apical") from the lower apical turn and one ("basal") from the upper basal turn. For each serotype, a minimum of four (CBA / CaJ, 48h), two (CBA / CaJ, 48h+5d), 3 (C57BL / 6, 48h), 2 (C57BL / 6, The basal and apical specimens were inoculated after 48 h + 5 d. The morphology of the cochlea was not maintained during the culture. If so, the sample was excluded. This provides information about the variability of transduction and allows for further analysis. A number of samples were selected to provide a basis for selection for in vivo evaluation. Rubecco's modified Eagle's medium (DMEM), 1% ampicillin, and 1% N2 supplement, plus 1% fetal bovine serum (FBS)) and 10 10 Along with AAV of GC For the 48h+5d condition, explants were incubated in 50 μl of PBS for 48h. The AAV-containing medium was replaced with fresh AAV-free medium for an additional 5 days. Human vestibular epithelium derived from the utricle was obtained from four consenting adult patients undergoing tumor resection. (Kesser et al., 2007, Gene Ther., 14:1121-1131) and cultured for 10 10 GCs were exposed to AAV for 24 hours and maintained in culture for 10 days, after which the tissues were fixed and The study was performed under the reference number 11 / LO / 0475. urrey Borders NRES Committee London (Hea This work was approved by the International Research Authority.

[0057] Animal models and general methods Wild-type C57BL / 6J and CBA / CaJ mice were obtained from Jackson Laboratories. Animals of either sex were obtained from the veterinary laboratory (Bar Harbor, ME) at an estimated 5 The ratio of 0 / 50 was used in the experiments. The group size per experiment for subsequent endpoints will depend on access to specimens and technical The reported observations for Anc80 transduction were Due to the unique and limited nature of the accessibility of various vectors (except for human vestibular tissue transduction), This was qualitatively confirmed in subsequent experiments using a single lot. and statistical analysis of transduction efficiency between serotypes due to the qualitative nature of the reported findings. Not implemented.

[0058] CSF and blood sampling Cerebrospinal fluid (CSF) sampling from the cisterna magna (Lui & Duff, 2008, J. Visualized Exp. , 21:e960) and intracardiac blood sampling via thoracotomy were performed as final procedures. The maximum amount of clear CSF per animal (no more than 5 μL) was dispensed via a pipette tube into a volume of 60 μL. PBS, this results in a slightly different starting dilution, and then The solution was standardized with additional control PBS. After obtaining blood samples in a laboratory (Tedt, Numbrecht, Germany), these were The serum was then pelleted at 1000 rpm for 8 minutes and the serum was collected along with the CSF sample (in PBS) for further use. The mixture was stored at -80°C until use.

[0059] Example 1A - Histological Analysis After a follow-up period of 5–29 days, animals were sacrificed and whole-tissue preparations of the cochlea were prepared as previously reported. The cochlea was prepared as described (Sergeyenko et al., 2013, J. Neurosci., 33:13686-94). Both whole tissue specimens and explants were stained with the corresponding secondary antibody (Alexa Fluor 555 Anti-mouse antibody and Alexa Fluor 647 anti-rabbit antibody, #A-21422 and #A-21245 Thermo Fisher Scientific, Wal tham, MA, 1:1000) together with myosin 7A (Myo7A, #25-679 0 Proteus Biosciences, Ramona, CA, 1:400) and beta-tubulin (TuJ1, #MMS-435P Biolegend, San Diego, CA, 1:200) (Dilwali et al., 2015, Scientific Reports, 5:18599). The specimens were mounted and then subjected to confocal microscopy. Each image in a given series of experiments was taken with the same settings and the strongest eG The FP signal was obtained using a laser intensity selected based on the specimen. Overview and zoom-in images for the spiral ganglion neuron (SGN) region. Z-stacks were obtained using AMIRA. 3D reconstruction was performed using SGN transfection. was used to determine more accurately.

[0060] The results in Figures 1 and 4 show that eGFP is expressed in C57BL / 6 and CBA / CaJ. The tropism of the five serotypes, monitored by expression of their respective encoding AAVs, is illustrated. Notably, eGFP expression was qualitatively brighter in cochlear cultures exposed to Anc80. , expression was evident in many cochlear cell types.

[0061] Example 1B - Quantification of eGFP Expression For in vitro data, one or two per basal and apical sample for each specimen Divide the number of eGFP-positive cells by the total number of outer or inner hair cells per 100 μm section. By doing so, we were able to identify the patterns of eGFP-positive inner hair cells (IHCs) and outer hair cells (OHCs). The percentage of sarcoidosis was quantified manually along the cochlea. All visible sarcoidosis in the cochlear explants was counted. GNs were assessed for their eGFP expression. The areas of the spiral lamina border and supporting cells were calculated as follows: A qualitative approach (adjusted for each series of experiments, as explained above) yielded 0 (expression The signal was assessed on a scale of 0 (none) to 3 (strongest signal). A control sample was used that did not contain V. Data show that in both mouse strains tested, apical and Anc80 targets IHCs and OHCs basally with between 60 and 100% efficiency. Anc80 was significantly more potent than AAV2 in IHC and OHC. showed consistently and qualitatively brighter eGFP expression (FIGS. 1 and 4).

[0062] Differences in expression between different AAVs may result in underestimation of expression at the day 2 (early) time point. To adjust for potential differences in the onset of the new cochlea, a longer-term experiment was performed. The pairs were transduced under identical conditions, but after 48 h of incubation with AAV, the explant cultures were The medium containing the cultured vector was removed and replaced with fresh medium, and the cultures were allowed to grow for an additional 5 days. The expression patterns were similar in this long-term study (referred to as 48h + 5d). In CBA / CaJ mice, AAV2 and Anc80 were observed, especially in the basal In rotation (Figures 1J, 1K and 4I, 4J), expression for AAV6, 8, and 9 Other cell types were targeted by all serotypes, including supporting cells. The rim was more permissive than the SGN, followed by the SGN (Figs. 5, 6, and 7). Anc80 transduction was consistently more efficient, as evidenced by brighter eGFP fluorescence. This resulted in higher rates and stronger expression.

[0063] Example 1C - In vivo injection A bevel-polished glass microinjection pipette was inserted into pups (P0-P2). The injection was performed through the round window membrane (RWM) using a P-2000 pipette puller (Su Capillary glass (W) was used in a thermocouple instrument (Winter Instrument, Novato, CA). Pull the pipette from the pipette holder (PI) and use a micropipette beveler (Sutter Instruments) The wafer was bevel polished (at an angle of 28°, ~20 μm) using a lithography machine (Est. Novato, CA). Tip diameter). Use a sterile swab to cover the surgical site (left mastoid). For analgesia, E MLA cream (lidocaine 2.5% and prilocaine 2.5%) was applied externally. Before surgery, body temperature was maintained on a warm pad at 38°C. Ice was applied for 2-3 minutes until the patient lost consciousness. The pups were anesthetized by inducing rapid hypothermia in water and kept cool for 5-10 minutes during surgery. The tissue was then scrubbed with Betadine and washed with 70% ethanol. The surgical site was disinfected by wiping with a cloth three times. A post-auricular incision was made to expose the transparent ear capsule. The sac is exposed and a micropipette is manually advanced through the sac and overlying fascia. The RWM was penetrated with the tip of a pipette. Approximately 1 μL of virus was injected unilaterally for over 1 minute. Among them, five (AAV1), four (AAV2), two (AAV8), and one (AAV6 ), was manually injected into the left ear of three (Anc80) C57BL / 6 animals. To adjust for these specific vector preparation factors, the invention presented herein Using different vector lots from independent preparations to confirm our qualitative findings The results of Anc80 were confirmed in a subsequent study (data not shown). Injections were performed in groups. Occasionally, the needle was inserted too deep, too shallow, or at an incorrect angle. If there was visible damage to the middle or inner ear structures, the sample was removed from further analysis. The success rate of injections ranges from ~50% to ~80%, depending on the experience level of the person giving the injection. After injection, a 6-0 black monofilament suture (Surgical Specialists) was used to secure the puncture site. The skin incision was closed using surgical instruments (Wyoming, PA). After this, the pups are placed back on a 38°C warming pad for 5-10 minutes and then placed on a 38°C warming pad for rearing. returned to her mother.

[0064] Consistent with previous reports, AAV1 transduced IHC with moderate to high efficiency (Figure 2 A, 2B). These studies demonstrated that AAV2, 6, and 8 target a small number of IHCs, and AAV 8 demonstrates roughly equivalent transduction in the apical and basal regions. Furthermore, consistent with previous reports, all of the conventional AAV serotypes tested showed However, Anc80 significantly increased OHC transduction by 20-fold ( Nearly 100% IHC and ELISA were achieved at a dose (for AAV1) to 3-fold (for AAV2) lower than the original dose. Approximately 90% of OHCs were transduced (Figures 2A-2C). As observed by imaging, 1.36 × 10 for all serotypes 12 GC equivalent Transduction in the amount of Anc80 resulted in substantial IHC and OHC transduction. However, AAV1, 2, and 8 showed minimal targeting in IHC and no activity in OHC. No significant changes were observed (Figs. 8C and 8D).

[0065] Anc80-transduced samples were then fixed, stained, and imaged by confocal microscopy. We performed phagocytosis analysis and revealed a dose-dependent effect on hair cell transduction (Figure 8E). C targeting (Fig. 2C, Fig. 8) is qualitatively distinct from other AAVs. This illustrates the biology of transduction by Anc80. The cochlea was observed throughout the entire cochlea, from the base to the apex, in all three Anc80-injected mice (Figure 1). 2A, B, C). Low-magnification photographs of the cochlear apex (Fig. 8A) show strong eGF activity far from the injection site. High magnification images of the basal tissue showed P expression in 100% of IHCs and 95% of OHCs transduced. This reveals (Figure 8B).

[0066] In some animals, strong eGFP expression was observed in the uninjected contralateral ear (Fig. 9). In mice, the cochlear aqueduct is patent, and the perilymph of the cochlea is transported to the CSF, the contralateral aqueduct, and the This provides a fluid pathway to the contralateral cochlea. We investigated whether eGFP could be transduced into neurons in the brain. We revealed strong eGFP expression in cerebral Purkinje neurons (Figures 10A and 10B). .

[0067] Some forms of hereditary hearing loss also cause vestibular dysfunction, so Anc80 may be involved in the development of vestibular dysfunction in humans. It is possible that this vector may be useful for gene delivery to organs. Human vestibular epithelium was obtained from four adult patients undergoing resection of vestibular schwannomas; The epithelium was placed in culture as previously described (Kesser et al., 2007, Gene Ther., 14:1121-3 1) For AAV-transduced samples, Figure 3C shows that both hair cells and The contrast with Myo7A in Figure 3D revealed strong eGFP fluorescence in the nuclei and supporting cells. High-magnification photographs of the stained epithelium revealed that 83% (19 / 23) of Myo7A-positive hair cells were eG They also revealed that Anc80 was expressed in both mouse and human hair cells. This suggests that efficient transduction is possible.

[0068] Example 1D - Immunological Assays Antibody titers against Anc80 in CSF and serum were determined by neutralization assay ( Zinn et al., 2015, Cell Reports, 12:1056-68) using a 96-well format. , heat-inactivated CSF or serum samples (collected as above) were serially transferred to serum-free medium ( Life Technologies, Carlsbad, CA) and then diluted with A nc80-luciferase(10 6 GC / well) for 1 hour at 37°C. The sample / Anc80-luciferase mixture was incubated with adenovirus (MOI 20) the day before. After 1 hour at 37°C, diluted serum-free medium (1 part) was added to the treated HEK293 cells. Serum medium, 2 parts serum medium) was added to each well.

[0069] After 2 days, cells were treated with lysis buffer (Promega, Madison, WI) and The mixture was frozen at -80°C for 30 minutes. Then, the substrate buffer (Tris-HCl, MgCl , ATP (Life Technologies, Carlsbad, CA), D-Lucine Ferrin (Caliper Life Sciences, Hopkinton, MA) Cells were thawed at 37°C for 15 minutes before treatment with Synergy BioTek Luminescence was measured using a plate reader (BioTek, Winooski, VT). I read the sense output.

[0070] The sensitivity of the assay and the level of sampling ensure that low-level intermediate The sum could be detected in the serum of injected mice but not in the CSF. (Figure 10C).

[0071] Example 1E - Hair Cell Electrophysiology The cochleae were excised, mounted on a coverslip, and imaged using a 63x water immersion objective and differential interference contrast optics. Axio Examiner.A1 upright microscope (Carl Zeiss, O Electrophysiological recordings were performed in MEM (m In M) 137NaCl, 5.8KCl, 10HEPES, 0.7NaH2PO4, 1.3 CaCl2, 0.9MgCl2 and 5.6D-glucose, vitamins (1:100) and and a standard solution containing amino acids (1:50) (Life Technologies, C arlsbad, CA) (pH7.4; ~310mOsm / kg) at room temperature (22℃~2 The experiment was carried out at 4°C.

[0072] Recording electrodes (3–4 MΩ) were made of R-6 glass (King Precision Glass s, Claremont, CA) and (in mM): 140 CsCl, 5 EGTA -KOH, 5 HEPES, 2.5 Na2 ATP, 3.5 MgCl2 and 0.1 CaCl The catheter was filled with intracellular solution (pH 7.4; ∼280 mOsm / kg) containing Axopa 2. tch 200B (Molecular Devices, Sunnyvale, CA) To record mechanotransduction currents, we use whole-cell sealed membranes. The tight-seal technique was used. The hair cells were maintained at -84 mV. Low-pass Bessel The current was filtered at 5 kHz by a filter and then input to a 12-bit acquisition board (Dig idata 1440A, Molecular Devices, Sunnyvale, The signals were digitized to ≥20 kHz using the pCLAMP10 software (Molecular Recordings were made using a cardiogram (Cyclotron Radiography Devices, Sunnyvale, CA).

[0073] LVPZT amplifier (E-500.00, Physik Instrumente, Ka PICMA chip type piezo actuator driven by a piezoelectric transducer (Piezoelectric transducer, prism ... Physik Instrumente, Karlsruhe, Germany ) to deflect IHC- and OHC-derived hair bundles. 8-pole Bessel filter (Model 3384 filter, Krohn H Filtered at 40 kHz by ITE Corporation, Brokton, MA For whole-bundle recording, the stereocilia row of hair cells was recorded. A rigid glass probe was designed to fit the concave side (3 mm diameter for OHC). ~4 μm diameter for IHC, 4–5 μm diameter for whole-cell electrophysiological recordings beyond P10. To do this, cochlear tissue was dissected at P5–7 and cultured in MEM (1X) + 1% FBS containing GlutaM. Incubation was performed in AXTM-1 medium at 37°C, 5% CO2 for up to 30 days.

[0074] Representative currents evoked by hair bundle bias from P7 OHCs and P35 IHCs. Is there any difference in amplitude, sensitivity or kinetics between eGFP-positive and eGFP-negative control cells? The results revealed no differences in the phenotype (Figure 2D). GFP-negative hair cells were detected after exposure to Anc80 in all regions of the cochlea and in mice aged 1 to 5 weeks. In all cases, the responses were indistinguishable from wild-type (Figure 2E). This indicates that transduction with Anc80 had no deleterious effects on sensory cell function. confirmed.

[0075] Example 1F - Hearing Test Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) data were analyzed as previously described. (Askew et al., 2015, Science Translational Med., 7:285ra108). POAE is an assay for proper cochlear amplification and entrainment, and is essential for the viability of outer hair cells. It is a sensitive measure of the potential for hearing loss (Guinan et al., 2012, Hearing Res., 293:12-20). The stimuli tested in mice were 5.6, 8, 11.3, 16, 22.6 and 32 KH. The sound pressure level varied between 10 and 90 dB at frequencies of z. four uninjected ears and one negative ear with lesions due to injection that did not contain eGFP fluorescence. Sex control ears were analyzed at P28–P30.

[0076] The minimal sound threshold required to elicit an ABR was plotted (Fig. 2F) and compared with the injected and Histological analysis revealed no difference in thresholds between injected and uninjected ears. revealed strong eGFP fluorescence in all injected ears (data not shown). In one case, there were no eGFP-positive cells, and the ABR threshold increased (Figure 2F). This suggests that the needle may have ruptured the cochlea, causing permanent damage. Despite strong outer hair cell transduction with Anc80-eGFP, no injection No difference was observed in DPOAE thresholds compared to the control ear (Fig. 2G). DPOAE data were compared with RWM injection, Anc80 transduction, and and transgene expression have all been shown to be safe for auditory function.

[0077] Example 1G - Rotarod Test Five C57BL / 6 mice were tested for balance behavior on the rotarod apparatus. Mice with impaired yard function are known to perform poorly on the rotarod apparatus. (Parker & Bitner-Glindzicz, 2015, Archives Dis. Childhood, 100:271-8). The study demonstrated the effectiveness of this rotor for detecting balance dysfunction when only one ear is affected. emphasized the effectiveness of the rod test (Fukui & Raphael, 2013, Hearing Res., 297:99-105; Gel eoc & Holt, 2014, Science, 344:1241062). Three mice were injected at P1 and P 36 and two uninjected control mice at P79. All mice were tested using the rotarod protocol. On day 1, 4 RPM Mice were trained to balance on a rotating rod for 5 min at 4°C. Mice were tested in five trials, spaced 5 minutes apart. Starting speed: 2 RPM From then on, the rod was accelerated by 1 RPM for each trial (Fukui & Raphael, 2013, Hearing Res., 297:99-105). The time (in seconds) until the mouse fell off the apparatus was recorded.

[0078] The perilymph of the cochlea is continuous with the perilymph of the vestibular labyrinth, so it is infused through the cochlear RWM. We evaluated whether the introduced Anc80-eGFP could transduce the vestibular sensory organs. Whole tissue preparations of the vestibular epithelium reveal the ovoid vestibular organ, which is sensitive to gravity and linear head movements. in both type I and type II hair cells of the capsule and sensitive to rotational head movements We found strong eGFP expression in the semicircular canals (Fig. 3A, 3B). To address safety concerns that c80 transduction may affect balance, The mice injected with the gene showed improved vestibular function in the rotarod test. The results were similar to those of the control group (Fig. 11).

[0079] Part 2 - Gene therapy restores function in a mouse model of Usher syndrome Example 2 Mouse model of Usher syndrome The following methods and materials were used in Example 2.

[0080] tissue specimen Ush1cc.216G>A heterozygous or homozygous for electrophysiological studies The utricle and organ of Corti from sex mutant mice were cultured from postnatal day 0 to 8 (P Postnatal mice were killed by rapid decapitation. The temporal bones were removed. MEM (Invitrogen, Ca) supplemented with 10 mM HEPES (pH 7.4). The organ of Corti was cut without enzymes as previously described. After treatment with 0.1 mg / ml protease for 10 minutes (Protease XXIV, Sigma), the utricle was excised. The excised organ was placed on a round cover slip. A pair of thin glass fibers, previously glued to a cover slip, was placed on the edge of the tissue and pulled downward. The tissue was used acutely or in culture in the presence of 1% fetal bovine serum. Cultures were maintained for 7 to 8 days for experiments involving in vitro viral vector infection. Therefore, the medium was changed every 2 to 3 days.

[0081] animal Ush1cc.216G>A knock-in mice were obtained from the Louisiana State University Health The strains were obtained from the Science Center of Japan. The imported strains are derived from a Cdh23 (Ahl) mutation that causes age-related hearing loss. Bred for the first time using toe clips (before P8) or ear punches (after P8). Mice were genotyped using PCR as previously described (Lentz et al., 2007, Mutat. Res., 616:139-44). For all studies, male and female mice were used. The randomization paradigm was not otherwise applied.

[0082] Generation of viral vectors Total RNA was isolated from the cochlea of ​​c.216AA mutant mice (RNAqueous micro kit, Ambion), QuantiTect Reverse Tra Reverse transcription was performed using the transcription kit (Qiagen). rmonin cDNA was purified using Platinum Taq DNA polymerase High Fidelity (Invitrogen) and primers: Trunc-harm onin.F(KpnI)GAG GTA CCA TGG ACC GGA AGG TGG CCC GAG (SEQ ID NO: 9); Trunc-harmomin.RV (Bam HI)CAG GAT CCG GAC AAT TTC ATC CCC TAC(Distribution The 387 bp PCR product was amplified using the TA clone. The clones were cloned using a sequencing kit (Invitrogen) and confirmed by sequencing. To generate the GFP fusion construct, the DNA was digested using KpnI and BamHI. The NheI-harmonin fragment was subcloned into pEGFP-C1. XbaI EGFP::trunc-harmonin cDNA was transfected into the AAV shuttle vector A conventional vector was introduced into the vector containing the AAV2 inverted terminal repeats (ITRs). The transgene cassette is packaged into a V1 capsid, in which the transgene cassette is (AAV2 / 1.CMV.EGFP::trunc-harmomin.hGH, 1.9 2 E14gc / m,BCH).

[0083] The harmonin-a1 and harmonin-b1 plasmids were kindly provided by Lily Zh EGFP-tagged, kindly provided by eng and James Bartles Labeling construct (Zheng et al., 2010, J. Neurosci., 30:7187-201)(Departmen of Cell and Molecular Biology, Northwe stern University, Feinberg School of Medicine The IgG1-containing ... Harmonin-a1 was originally isolated from mouse kidney, and harmon-b1 was isolated from mouse kidney. The harmonin-a1 construct was derived from mouse cochlear sensory epithelium. This was further modified to replace the EGFP tag with tdTomato. The untagged and untagged constructs were packaged into AAV vectors. en's Hospital's Virus Core Facility and Massachusetts E ye and Ear Infirmary's Gene Transfer Vector The r Core generated the viral vectors: AAV2 / 1.CMV. tdTomato::harmonin-a1 4.33 10^13gc / ml(BC H);AAV2 / 1.CMV.EGFP::harmonin-b1 2.73 564 10^14gc / ml(BCH);AAV2 / 1.CMV.EGFP-harmoni n-a1:2.81 10^12gc / ml(MEEI);AAV2 / 1.CMV.EG FP-trunc-harmonin;1.92 10^14gc / ml(BCH);A AV2 / Anc80.CMV.harmonin-a1:1.93 10^12gc / m l(MEEI);AAV2 / Anc80.CMV.harmonin-b1:1.74 10^12gc / ml(MEEI);AAV2 / Anc80.CMV.trunc-ha rm.WPRE:9.02 567 10^12gc / ml (MEEI) was produced. For in vitro experiments, 10 μl of concentrated vector was incubated in the presence of 1% fetal bovine serum. The dissected tissue was rapidly added to 1 ml of MEM-supplemented medium for 24 h. It was maintained for up to 10 days.

[0084] Round window membrane (RWM) injection RWM injections were performed in accordance with the Animal Care and Use Guidelines of Boston Children's Hospital. The study was conducted in accordance with the Animal Protocol #15-01-2878R approved by the Committee. 0.8 μl to 1 μl of AAV vector was injected into neonatal mice P0-P1 and P10-P12. Initially, exposure to hypothermia was used to anesthetize P0–P1 mice, but P10–P Mice were anesthetized with isoflurane. Upon anesthesia, the otic capsule was exposed and the cochlea was visualized. A post-auricular incision was made to separate the eardrum. i. Transl. Med., 7:295ra108) using a glass micropipette controlled by RW The injection was performed through the M. The volume of injected material was controlled at approximately 0.02 μl / min for 10 min. Standard postoperative care was applied. To optimize sample size and reduce variability, Subsequently, the sample size for in vivo testing was determined.

[0085] Electrophysiological recordings (in mM) 144 NaCl, 0.7 NaH2PO4, 5.8 KCl, 1.3 C aCl2, 0.9 MgCl2, 5.6 D-glucose and 10 HEPES-Na Standard artificial OH-containing solution adjusted to pH 7.4 and 320 mOsmol / kg Recordings were performed in perilymphatic fluid concentrate (Invitrogen, Carlsbad, CA). A) Vitamins (1:50) and amino acids (1:100) were added. 63X water immersion An upright Axioskop FS microscope (Z) with objective and differential interference optics eiss, Oberkochen, Germany) to extract hair cells from the apical surface. The recording pipette (3–5 MΩ) was made of borosilicate capillary glass (Garn er Glass, Claremont, CA) and (in mM): 135 KCl, 5 EGTA-KOH, 10 HEPES, 2.5 K2ATP, 3.5 MgCl2, The cells were filled with intracellular solution containing 0.1% CaCl2 at pH 7.4. Currents were recorded at a holding potential of -64 mV at room temperature. Low-pass Bessel filter Filtered at 10kHz by Axopatch Multiclamp 70 0A or Axopatch 200A (Molecular Devices, Pal Alto, CA) using a 12-bit acquisition board (Digidata 132 2) to ≥ 20 kHz, and pClamp 8.2 and 10.5 (Mole Data were collected using a vitreous optics device (Central Devices, Palo Alto, CA). Data were analyzed offline using OriginLab software and, unless otherwise stated, , expressed as mean ± standard deviation.

[0086] statistical analysis To ensure reproducibility, at least three rats per group were included at each time point. Test and control vectors were evaluated. Sample sizes are noted in the figure legends. RWM All animals that were successfully injected were included in the analysis of the study. Animals that were not successfully injected had an average Successful injection was defined as a threshold >90 dB. SPL was determined by ABR recovery. Statistical analysis was performed using Origin 2016. The experiment was carried out using the inLab Corporation. Data are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM), as indicated. One-way analysis of variance (ANOVA) was used to determine significant differences between means. .

[0087] Example 2A - Scanning Electron Microscopy (SEM) in the Mouse Usher Model Along the organ of Corti in control and mutant mice, P7, P18, and ~P42 (6 SEM was conducted at the University of Washington. n This was done in collaboration with Rubel. The inner ear was soaked in 0.1M sodium phosphate with 4% glutamate. The cells were fixed in ethanol at 4°C overnight. The next day, the cells were fixed in 0.1M sodium phosphate buffer. The specimens were rinsed three times with PB and then soaked in 1% methyl methyl tetroxide in 0.1M PB for 30 minutes in an ice bath. Specimens were then rinsed in 0.1M PB and post-fixed in a graded series of ethanol. The samples were dehydrated at 35%, 70%, 95%, and 100% (×2). It was dried, mounted on an SEM stub, and sputter coated with Au / Pd. JEOL JSM-8 SEM was performed using a 40A scanning electron microscope. Similar preparations were performed at P8 and 6-week stages. Organ of Corti explants were cultured in 0.1 M cacodylate supplemented with 2 mM CaCl. in 2% ethanol buffer (Electron Microscopy Sciences) The sections were fixed in 0.5% glutaraldehyde for 1 hour at room temperature, followed by a graded series of acetone. The specimens were dehydrated, critical point dried from liquid CO2, and coated with 4-5 nm platinum (Q150T, Qu Sputter coating by Orum Technologies, United Kingdom and observed using a field emission scanning electron microscope (S-4800, Hitachi, Japan). .

[0088] Homozygous c.216AA mutant mice are deaf and exhibit poor circulation and head shaking. This indicates vestibular dysfunction characterized by swaying behavior. Neurobiol., 70:253-67) reported that at P30, the cochlear base had distinct inner and outer hair cells. Degeneration and hair cell death were also observed in the middle turn, but not in the organ. The apical part of the inner ear was better preserved at 1 month of age. To assess hair cell survival at earlier stages, which are hypothesized to occur progressively during development SEM analysis was performed along the organ of Corti at P8 and P18. c.216GA mouse outer hair cells (OHCs) and inner hair cells (IHCs) were preserved and The bundles were properly oriented at these ages (Figures 12A-12C, 12G, 1 2I and Figures 19A-19C, 19K). However, homozygotes were not observed at both ages analyzed. Disorganized hair bundles were evident along the entire length of the organ of Corti in the syngeneic c.216AA mice. (Figs. 12D-12F, 12H, 12J-12L and Figs. 19D-19J, 19L). In 8, IHC bundles were moderately disorganized in the basal, intermediate, and apical regions ( Figure 1 2D-12F, 12J). Many IHC bundles are arranged in a wavy pattern and with light arrangement of stereocilia rows. Many OHCs in the c.216AA mutant mice showed poorly organized sequences (Fig. 12J). The organs had preserved hair bundles (Fig. 12H, 12K), while fragmented and disorganized hair bundles were present. It was clear that the sparse distribution of the spleen was along the periphery of the spleen (Figs. 12D-12F, 12L). In the sham-treated group, the destruction was more obvious, but the majority of hair cells were still present, as previously reported. (Lentz et al., 2013, Nat. Med., 19:345-50) (Figures 19D to 19F).

[0089] To evaluate hair bundle morphology in mice treated with harmonin-b1 gene therapy, To investigate the effect of 6-week-old untreated (or uninjected) and treated (or injected) mice The temporal bones of the mice were prepared for SEM analysis. The basal and intermediate regions of the brain showed severe hair cell loss (Figure 18). In the first row, OHCs were almost absent, and sparsely present in the second and third rows. In the intermediate region of the organ, the first row of OHCs was also largely absent. High-magnification SEM also revealed that the full-length organs of c.216AA mutant mice were significantly different from those of the control mice. Surprisingly, a 6-week-old c.216 In AA mice, no hair bundles were observed, and the typical staircase structure with all three rows of stereocilia was observed. Instead, hair cells from c.216AA mice were found along the first row. with retracted stereocilia, an abnormal second row, and a slightly preserved top row. In contrast, reduced hair cell loss and normal hair bundles were observed in the har The number of hair cells was observed in c.216AA mice after treatment with monin-b1. , estimated from the presence or absence of hair bundles in representative fields.

[0090] The data show that the apparent number of hair cells from base to apex of the organ in injected mice Conserved, 40-79% at the base, 68-95% at the middle, and 93% at the apex ~99% of the cells were identified (n = 1824 cells from ears of n = 4 c.216AA mice). n=792 from the ears of the cyst and n=2 rescued c.216AA. was still evident in harmonized-b1-injected mice, and most hair bundles They have three rows of stereocilia and a morphology that is nearly indistinguishable from their heterozygous controls. (Figure 18).

[0091] Example 2B - FM1-43 imaging in an Usher mouse model Five micromolar FM1-43 (Invitrogen) was diluted in extracellular recording solution. Apply to tissue for 10 seconds, then wash three times with extracellular recording solution to remove excess dye and After 5 min, the cells were immersed in water at 20x, 40x, and 63x to prevent endocytic uptake. Epifluorescence on a Zeiss Axioscope FS plus with objectives Light source, differential interference optics, and FM1-43 filter set (Chroma Te Intracellular FM1-43 was imaged using a CCD camera. Argus-20 image processor using chromatic and background fluorescence subtraction Images were captured at 16-bit using a Hamamatsu camera. Trust settings are maintained for all image acquisitions and are used in Adobe Photoshop or analyzed offline using Image-J software.

[0092] To assess early hair cell function, FM1- Uptake of FM1-43 was analyzed at P4. Upon brief application (<10 sec), FM1-43 penetrated into hair cells with functional mechanosensitive channels. The uptake of α-glucan was observed in hair cells of c.216GA mice (Fig. 13A). Bell varied among OHCs in c.216AA mice, affecting some, but not all This suggests that the cells retained functional transduction channels (Fig. 13B). The entire length of the cochlea was followed. No frequency-specific differences were observed. The expression of mitochondrial DNA was also reduced by IHC in c.216AA mice during the first postnatal week (data not shown). FM1-43 uptake was also assessed in hair cells of the utricle of mutant mice. Interestingly, in c.216AA mutant mice, uptake was significantly increased at P6 In the case of the striola, the hair cells are restricted to the extrastriola region and open during rest. This suggests the absence of mechanosensitive channels (Figs. 13C, 13D).

[0093] Example 2C - Mechanical Stimulation in the Usher Mouse Model OHC and IHC: 400mA ENV400 Amplifier (Piezoelectric One-524 dimensional drive system (Jena, Germany) PICMA chip piezo actuator (one-524 dimensional PICMA chip piezo actuator uator)(Physik Instruments,Waldbronn,German Mechanical stimulation was transmitted via a rigid glass probe mounted on the y. The tip is heat-polished (Fire polisher, H60) to fit the bundle of moving hairs. 2, World Precision Instruments Inc., Saras ota, FL) (Stauffer & Holt, 2007, J. Neurophysiol., 98:3360-9). 8-po Bessel filter (Khron-Hite, 528 Brockton, MA) By applying a voltage step at 50 kHz and filtering using The residual pipette resonance was eliminated by using a C2400 CCD camera (Hamamats The deflection of the hair bundle was monitored using a stimulator probe. Voltage steps were used to calibrate to within ±2 μm of the rest position. To ensure that the probe was not moving, a video image of the probe was recorded and the probe movement was calibrated. (Spatial resolution of ~4 nm). 10-90% of the rise time of the probe is ~20 μs. It was.

[0094] VHC: Transmits mechanical stimuli via a stiff glass probe mounted on a piezoelectric bimorph element. Coupling was performed by gentle suction of the kineolic cilia into the stimulation pipette. The voltage is applied to a piezoelectric device consisting of two bimorphs mounted on the stimulator and directly coupled to the stimulation probe. The bias was induced by applying a step. The voltage step is controlled by an 8-pole Bessel filter at 1 kHz. (Khron-Hite, Brockton, MA). The bias of the hair bundles was monitored using a meter (Hamamatsu, Japan). Previously, the movement of the stimulus probe was calibrated around its resting position (±2 μm).

[0095] During the first week after birth, the auditory and vestibular epithelium has a relatively normal morphology. In the organ of Corti, the mechanoreceptor hair cells, including the neurites, are located at P3 to P6 (Figure 12). c. Ciliary cells with normal-appearing bundles from the middle and apical turns of the cochlea of ​​a 216AA mouse. Records were obtained from those with cysts and more severely destroyed hair bundles. In the heterozygote, OHCs retained mechanosensitivity, but the response amplitude was reduced to 170 ± 80 pA. There was a significant reduction of ~63% (n=24; p<0.001, Figures 13E, 13F, 13G c. In 216AA mice, the amplitude of the response ranged from 31 to 292 pA. C. When the data were grouped according to the morphology of the hair bundle, a significant difference (p<0.01) was found. Observed: Currents evoked in mutant hair cells with severely disorganized bundles , smaller than those induced in mutant cells with more preserved hair bundles, respectively. , 120 ± 65 pA (n = 9), and 201 ± 74 pA (n = 15). Despite the reduction in β-amyloid, the response of hair cells to mechanical displacement was significantly reduced in heterozygous c.216 The stimulus response [I(X)] curves were similar to those of GA mice. The data were fitted using the Oltzmann equation (Figure 13F) and the fit was calculated using a 10-9 0% operating range (Figure 20B). No significant differences in operating range were observed between OHCs recorded from 6AA (p = 0.05 4) Similarly, hair bundles derived from IHC of c.216AA mutant mice were clearly visible under DIC microscopy. The transduction current was significantly reduced at P6 (Fig. 13E, 13F, 13G). At a holding potential of -64 mV, the I The maximum transduction current in HC (P6-P7) was 587±96 pA ( n = 21), whereas IHC of c.216AA showed 316 ± 127 pA (n = 19; p There was a significant (p<0.01) reduction in the operating range of 46% (p<0.001). , as measured by IHC in c.216AA mutant mice (Figure 20G).

[0096] Adaptation, defined as a decrease in transduction current in the presence of steady-state flux bias, also The c.216AA mutant mice showed a rapid and slow adaptation kinetics. Both components were analyzed using a double exponential fit to determine the c.216AA Although the difference was slower in IHCs and OHCs from mutant mice, The results were significant only for components (p<0.05 for OHC and p< 0.001; Figures 20C, 20D, 20H, and 20I). On the other hand, measurements at Popen = 0.5 The degree of adaptation was greater in OHCs and IHCs of hair cells in c.216A than in c.216GA. The results were significantly lower (Figures 20E, 20J; p<0.001). Mechanosensation was mildly impaired in the inner and outer hair cells of c.216AA mice. Importantly, both cell types are prerequisites for gene therapy and restoration of cell function. demonstrated survival throughout the first postnatal week.

[0097] In vestibular hair cells, mechanotransduction currents were reduced in c.216AA mice. In the extrastriola region, the current of c.216AA was 109±30 pA ( n=9, P5~P7), whereas c.216G The current in A was 231 ± 53 pA (n = 8, P6–P7) (Figures 13E, 13F, 13 H). Absence of FM1-43 uptake in the striola region (see below). Consistent with this finding (Fig. 13C, 13D), very small hair cells in the striola region Currents were recorded or no currents were recorded (6 ± 13 pA, n = 6, P5–P7 ) DIC microscopy revealed that the hair bundles of the utricle were extremely well preserved, while the transmural hair bundles were not. The induction current is generated in the hair cells in the extrastriola and striola, respectively. These findings were significantly reduced or absent, except for the striola region. The results show that the transduction apparatus assembles correctly in mutant mice. This suggests that the number of targeted but functional complexes is reduced in neonatal mice.

[0098] Next, c.216AA hairy mice were exposed to an AAV vector driving harmonin expression. The function in cells was evaluated. Possibility of functional rescue by exogenous harmon To enhance expression, we used untagged harmo The nin-a1 or harmon-b1 coding sequence was cloned into the A gene known as Anc80. It was packaged into AV capsids (Zinn et al., 2015, Cell Rep., 12:1056-68). As shown herein, Anc80 capsids are present in 100% of IHCs and 80% of OHCs. 90% of cells are transduced in vivo. Harmonin-b is expressed in both IHCs and OHCs. is required for mechanotransduction in the retina and for hearing function in both cell types AAV2 / Anc80.CMV.harmonin-b1 (0.8 μl, 1 .9×10^12gc / ml), and separately AAV2 / Anc80.CMV.harm onin-a1(1.7×10^12 gc / ml)+AAV2 / Anc80.CMV. RWM injection of a mixture of harmonin-b1 (0.5 μl + 0.5 μl) was performed. Canotransduction responses were assessed after 2 weeks of treatment.

[0099] Tissue was extracted at P5–P6, before the cochlea ossified, and maintained in culture for 10 days. HCs (>P10) do not survive ex vivo recording paradigms, but exhibit robust electrophysiological Recordings were obtained from IHC at times corresponding to P14-P16. The results are shown in Figure 15. IHCs from mice lacking IL-1 showed significantly reduced transduction currents at P16. However, the recovery of sensory transduction was significantly higher in AAV-treated mice (79±43 pA, n=8). It was clear that P1 was composed of harmonics, b1, and a1. In mice treated with α-thiamin, the values ​​were 338 ± 66 pA (n = 15) and 352 ± 28 pA, respectively. A (n = 7; Fig. 15C) showed significant recovery ( *** P We observed a significant difference in IHC scores after treatment with harmonin-b1 (<0.001). The amplitude of induction currents was not significantly different from control c.216GA mice. The level of harmonic-b1 was significantly increased by co-injection of harmonic-a1. These results suggest that early RWM injections may be beneficial for the treatment of exogenous harmonics. These results suggest that delivery of n-b1 can restore mechanotransduction in IHCs. is doing.

[0100] Example 2D - Confocal imaging in the Usher mouse model To prepare tissue for confocal imaging from postnatal mice P0-P8, Fixation was performed using 4% paraformaldehyde (PFA) for 15 minutes. Permeabilization with Triton and Alexa Fluor phalloidin (Invitro Counterstaining with 1 / 200 (L) was used to label actin filaments. Images were acquired with a SM700 Zeiss confocal microscope. In older mice (4–8 weeks), After euthanasia, the temporal bones were removed and placed in 4% PFA for 1 hour, followed by 120 mM The sensory epithelium was then excised and subjected to immunostaining. The mice were injected as described above for the purpose of the study. Mouse anti-CTBP2 (BD Bioscience #6 12044, 1 / 200) for 48 hours, followed by Alexa Fluor goat anti-mouse antibody Ribbon synapses were labeled by counterstaining with Zeiss (1 / 200) overnight at 4°C. LSM 710 laser confocal microscope (IDDRC Imaging Core Grant) Images were acquired using a Zeiss LSM Image Viewer 4. It was processed using 2.

[0101] Previous studies have identified two alternative splice forms of harmon in sensory hair cells. We demonstrated the expression of exogenous harmonin splice forms in AAV vectors. To assess transgene capacity, newborn c.216AA and wild-type (C57BL / 6J) mice were cultured in vitro. The utricle and organ of Corti from mice were cloned using a recombinant vector containing the N-terminally fused harmonin-b1 gene. GFP (eGFP::harmonin-b1) or harmonic-a1 181 tdTomato (tdTomato::harmonin-a1) fused to the N-terminus The vector was then exposed to an AAV2 / 1 vector encoding the vector P1. In vivo, the vector was administered by RWN injection (1 μl). P0-P1 tissue was incubated in the presence of ATP for 24 hours and maintained in culture for 1 week. Confocal images showed that hair cells in wild-type, c.216GA, and C.216AA mice The results showed that transduction was successful (Figures 14A-14C, 14E). VHC (Figure 14A), EGFP::har at the tips of the stereocilia in IHCs and OHCs (Figures 14B, 14C). The monin-b1 signal was clearly visible. The EGFP signal was clearly visible in the mice injected at P1. It was also detected in OHCs and IHCs in the basal part of the cochlea at P60 (Fig. 14D). mato::harmonin-a1 was detected at the base of auditory hair cells (Fig. 14E). Co-staining with ribbon synaptic marker CTBP2 frequently revealed co-localization in P7 IHCs. (Fig. 14E), but not in the P7 utricle (data not shown). .

[0102] The localization of the foreign fusion construct was observed in the distal stereocilia near the harmonin-b sensory thread insertion. This is consistent with previous studies of the localization of harmonized-a to the synaptic terminals and to the synapses. .

[0103] Example 2E - Auditory Brainstem Response (ABR) and Distortion Components (DPOAE) ABR and DPOAE were measured with xylazine (5–10 mg / kg ip) and ketamine. Recordings were made from mice anesthetized with amine (60–100 mg / kg ip). The electrodes were placed a) dorsally between the two ears (reference electrode); b) behind the left pinna (recording electrode); and c) a ground electrode (ground electrode) was inserted into the skin on the back of the animal's buttocks. The tube at the base of the ear was trimmed. For ABR recording, the ear canal and hearing device (EPL Acou) were inserted. stic system, MEEI, Boston) presented a 5 millisecond tone pip The response was amplified (10,000 times), filtered (0.1-3 kHz), and analyzed by PC Data acquisition system based on EPL, Cochlear function test The average was measured using an analog-digital board (Master's Suite, MEEI, Boston). Increase the sound level by 5-10 dB from 0-110 dB sound pressure level (decibels SPL). At each level, after "artifact removal", the polarity of the stimulus was alternated. Responses were averaged from 512 to 1024 (by measuring the number of times the threshold was determined). Thresholds were determined by visual inspection. Data were analyzed using Origin-2015 (OriginLab Corporation). The plots were made using the mean ± standard deviation of thresholds unless otherwise stated. For DPOAE, f1 and f2 primary tones (f2 / f1=1.2) f2 varies by half an octave between 5.6 and 45.2 kHz and L1-L2=1 At each f2, L2 was presented at 10 dB SPL increments. The DPOAE threshold was varied between 0 and 80 dB SPL. Defined from the average spectrum as the L2 level that elicits DPOAEs of magnitude greater than PL. The average noise floor level was below 0 dB SPL across all frequencies. SA-1 speaker driver (Tucker-Davis Technologies, Inc.) amplified 24-bit digital IO card in a PXI-1042Q enclosure Stimuli were generated by a National Instruments PXI-4461. , two electrostatic drivers (CUI CDMG150) in our conventional sound system. The signal was delivered from an electret microphone (Kno) at the end of a small probe tube. The sound pressure in the ear canal was monitored using a phonometer (Wles FG-23329-P07). Most of these experiments were not performed under blinded conditions.

[0104] To determine whether truncated harmonin interfered with normal hearing function, we analyzed the truncated protein Construction of the Anc80.CMV.trunc-harm vector for protein overexpression The vector was injected into the inner ear of c.216GA mice via the RWM. and DPOAES were measured at 4, 6, and 12 weeks with and without injection. There were no differences in thresholds between GA mice (data from 6-week-old mice shown in Figures 23C-23D). This data serves as a control for injection technique and, importantly, The vector allows the exogenous truncated harmonin to compete with the endogenous full-length harmonin. They argued that the endogenous truncated form in c.216AA hair cells was not involved in the gene therapy vector-mediated This suggests that the full-length harmonin expressed in the presence of an exogenous harmonin will not be interfered with.

[0105] Increased harmonin gene expression reduces hearing and balance function in Ush1c mice AAV2 / Anc80.CM at P0-P1 to determine which can be queued V.harmonin-a1 (0.8 μL, 1.7 × 10^12 gc / ml) or AA V2 / Anc80.CMV.harmonin-b1(0.8μl, 1.9×10^12 The RWM was injected with 1000 mg of acetaminophen (gc / ml) and the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DP) were measured. OAEs, acoustic startle reflex, open field and rotarod behavior were assessed. At 6 weeks, a stage at which 216AA mice suffer from severe hearing loss and vestibular dysfunction. Mice were evaluated at 3 and 6 months.

[0106] Twelve mice injected with AAV2 / Anc80.CMV.harmonin-a1 None of them recovered hearing function within 6 weeks (Fig. 16A-16C). This suggests that exogenous expression of -a1 is insufficient for hearing rescue. However, 25 mice injected with AAV2 / Anc80.CMV.harmonin-b1 Nineteen of the mice showed significant recovery of hearing function within six weeks. AAV2 / Anc80.CMV.harmonin-b1 was injected at 6 kHz. The best ABR thresholds for the affected ear were at 25–30 dB SPL, compared with those for wild-type mice. The threshold was surprisingly similar (Figures 16A-16B). Hz and little or no activity at 32 kHz. Consistent with the rescue of DPOAE thresholds, a rescue of DPOAE thresholds was also evident (Figure 16C). Among mice with hearing thresholds below 45 dB SPL for stimuli at ∼11.3 kHz Eight dogs were tested at a later stage to assess rescue longevity. From 6 weeks to 3 months, ~1 ABR threshold shifts at 0 dB SPL in the low frequency range and at ~30 dB SPL in the high frequency range. A similar shift was observed in the DPOAE threshold across the frequency range (Fig. 16D). After this time point, ABR thresholds and DPOAEs were observed at the slowest time points tested (Fig. 16E). The changes remained stable up to 6 months of age (Figs. 16D-16E).

[0107] For a more complete hearing rescue, especially at the high frequency end To assess whether both rmonin-a1 and harmonin-b1 are required, To achieve this, AAV2 / Anc80.CMV.tdTomato::harmonin-a1( 0.5μl;238 4.1E^12 gc / ml) and AAV2 / Anc80.CM V.eGFP::harmonin-b1(0.5μl;3.0E^12 gc / ml) 65% of hair cells were co-injected with α- and β-glucan. , both harmon-a1 and harmon-b1 were expressed (Figure 21). Fluorescently labeled harmonized-a1 was detected in AAV2 / Anc cells, possibly due to overexpression. Stereocilia of mice exposed to 80.CMV.tdTomato::harmonin-a1 Unlabeled harmonic-a1 and harmonic-b1 vectors were occasionally observed. ABR and DPOAE thresholds in mice co-injected with thiamin (Fig. 16) were significantly higher than those in mice co-injected with thiamin (Fig. 17). Similar to injection of onin-b1 alone, it did not provide any further improvement and harmonization Importantly, the data suggest that n-a1 may not be essential for auditory function. , harmonic-b1 alone is sufficient to significantly restore the hearing threshold at low frequencies. This demonstrates that (Figure 16).

[0108] To further evaluate the degree of rescue, mice with thresholds below 45 dB SPL were used. ABR waveforms from eight control c.216GA mice and eight AAV2 / Anc80 mice were analyzed. Comparison between five c.216AA mice injected with CMV.harmonin-b1 Analysis of the responses at 8–11.3 kHz and 16 kHz revealed that the amplitude of normal wave 1 (significant No difference, P>0.2, Student's t-test) and longer peak 1 latency (P>0 .001) (Figure 22), suggesting the possibility of a delay in neurotransmission at the synapse. In most animals, auditory rescue was also observed in the contralateral ear, with ABR thresholds of 11.3kJ. Hz, the SPL was as low as 20 dB (harmonic-b1: average 59.7 ± 5.3 dB) B SPL, n=15 / 25;harmonin-a1+-b1:255 average 76.2± 10.3 dB SPL, n = 4–6). Spread of AAV vectors to the contralateral ear has been previously observed. occurs via the perilymphatic duct, which is connected to the subarachnoid space in newborn mice. There is a possibility.

[0109] We investigated whether injection at a later developmental stage could result in partial hearing rescue. We also investigated AAV2 / Anc80.CMV.harmonin-b1( RWM injections of 0.8 μl were performed, and auditory thresholds were assessed at 6 weeks. None of the mice injected had detectable DPOAEs, and their ABR thresholds were higher than those of the uninjected mice. 16AA control mice (n=10; data not shown), possibly due to the older age group. This may be due to low viral transduction efficiency in the tissue or degeneration of the organ of Corti at later developmental stages. This suggests that opportunities for therapeutic intervention may be limited to the early postnatal stage.

[0110] Example 2 RT-PCR in the F-Usher Mouse Model P2-P3 wild-type heterozygous and homozygous Ush1cc.216G>A mice QUANTITECT® Reverse Trans cDNA was prepared using a Qiagen cDNA Creation Kit. cDNA encoding either the 50 bp or truncated harmonin (~35 bp) was Primers: Forward primer mUsh1c_Ex2F: 5' CTC ATT GAA AAT GAC GCA GAG AAG G 3' (SEQ ID NO: 11), liver mUsh1c_Ex5R:5' TCT CAC TTT GAT GGA CAC GGT CTT 3' (SEQ ID NO: 12). It is specific for the mouse Ush1c sequence and the target sequence is the human Ush1cc.216A allele. Because it is outside the knock-in region, both endogenous and AAV2-derived Ush1c are not detected. DNA and RNA levels were also assessed from mouse tissues taken 6 weeks after treatment. TRIzol reagent (Life Technologies) was prepared according to the manufacturer's protocol. DNA and RNA were isolated from the cochlea using a centrifuge (Giez, Carlsbad, CA). The GoScript reverse transcription system (Promega, Madison, WI) was used. RNA was reverse transcribed using GoTaq Green Master Mix (Promega). Radiolabeled PCR was performed using a PCR kit (Gas Marker, Madison, WI). For amplification of mouse Ush1c, use the following primers: mUsh1c_Ex3F (5' -GAA CCC AAC CGC CTG CCG (SEQ ID NO: 13)) and mUsh 1c_Ex4WTR(5'-TGC AGA CGG TCC AAG CGT-3'( SEQ ID NO: 14) was used.

[0111] These primers only amplify the viral Ush1 cDNA, which is homozygous. Ush1c.216AA mice were knocked in at exon 3 and exon 4, and This is because the human USH1Cc.216A gene contains a subunit of the USH1Cc.216A gene (Lentz et al. al., 2007, Mutat. Res., 616:139-44). Full-length (450 bp) and aberrantly spliced For amplification of the cDNA of the cleaved / cut harmonin (415 bp), The same primers as above were used (mUsh1c_Ex2F and mUsh1c_Ex5 R). The Gapdh primer is: mGapdh_Ex3F (5'-611 GTG AG G CCG GTG CTG AGT ATG-3' (SEQ ID NO: 15) and mGapd h_Ex4R(5'-GCC AAA GTT GTC ATG GAT GAC-3' (SEQ ID NO: 16). The products were separated on a 6% non-denaturing polyacrylamide gel and using a yphoon 9400 phosphorimager (GE Healthcare). quantified.

[0112] Previous studies have shown that truncated harmonin binds to full-length harmonin for its endogenous binding partners. This raises the possibility that the function of the truncated protein may be disrupted by competing with onin. Sustained expression of the protein was achieved by injecting an exogenous full-length harmonized vector. To address this concern, we investigated whether IFN-γ-glucan phosphate-glucosamine (IFN-γ) limited recovery in c.216AA mice (Figure 23A). To address this issue, we used RT-PCR assays to identify the c.216GA and c.216AA We examined the expression of Ush1c transcripts in mice. Consistent with previous reports, both full-length and truncated Ush1c transcripts were expressed. The Ush1c transcript encoding truncated harmon was detected in the c.216GA cochlea. In the c.216AA cochlea, only the transcript encoding the truncated harmon was expressed. was detected (Figure 23B).

[0113] The expression of AAV2 / Anc80.CMV.harmonin-b1 was confirmed, and viral expression was confirmed. To investigate the relationship between current level and ABR threshold, DN was recorded from the injected and contralateral cochleae. A and RNA were isolated and quantified by PCR and RT-PCR, respectively. c.216GA mice and AAV2 / Anc80.CMV.harmonin-b1 (0.8μl; 1.93 10^12gc / ml) injected and not injected c.21 Expression was evaluated in 6AA mice. The samples showed good ABR rescue (11.3 kHz). Two mice were injected with stimuli (thresholds ≤ 35 dB SPL) and had poor ABR rescue. (Threshold ≥ 90 dB SPL at 11.3 kHz) Two heads were included. RNA encoding the correctly spliced ​​form of n (Figure 24A) and AAV2 / Anc8 0.CMV.harmonin-b1 DNA (Figure 24B) in all injected cochleae. It was detected in the ventral cochlea and, to a lesser extent, in the contralateral cochlea of ​​all animals tested.

[0114] There was variability between animals in ABR thresholds and DNA and RNA expression levels (Fig. 2 4C). However, AAV2 / Anc80.CMV.harmonin-b1 DN A levels, the amount of RNA encoding the correct spliced ​​form of harmon and A A strong correlation was observed between the ABR threshold levels, which indicates that the variability of the ABR data is due to the AAV These results suggest that the successful recovery of ABR thresholds in mice may be a direct result of the expression of GABA. To assess long-term survival of hair cells in the sarcolemma, tissue was cultured in five 6-week-old mice. The number of IHCs and OHCs was counted (Figure 25). The number of ABRs did not change, whereas the number of ABRs increased to 5 in the three mice that showed long-term ABR rescue. OHC survival was monitored throughout the organ, except for the basal turn. I guessed (Figure 25).

[0115] Example 2G - Acoustic startle response in the Usher mouse model Using Startle Monitor (Kinder Scientific) The acoustic startle response (ASR) was measured using a piezo / plexiglass sensing assembly. A small, non-restrictive cubic Plexiglas recording chamber (27 cm) was fixed to the The mouse was placed in a room (10 cm x 10 cm x 12.5 cm) and exposed to a background white light of 60 dB SPL. Each session consisted of 35 trials, during which the subjects were allowed to habituate to the sound noise for 5 minutes. , with an intertrial interval of 30 seconds on average (range 25–35 seconds), followed by a single noise pulse. The signals were delivered over a range of 60 to 120 dB SPL in 10 dB SPL increments. A steady 60dB SPL background noise level to limit interference from external noise In the system, the pulses were arranged in a pseudo-random order. The timescale for the peak startle response (ASR amplitude) and the time from stimulus to peak startle response (ASR) was calculated. To calculate R (latency), the response to each pulse was recorded as the initial N, the maximum N, and the maximum response. The measurements were reduced to time (ms). All ASRs were performed blinded.

[0116] To assess whether ABR / DPOAE recovery resulted in behaviorally related recovery of auditory function, To achieve this, AAV2 / Anc80.CMV.harmonin-a1, AAV2 / Anc Mice injected with 80.CMV.harmonin-b1 and mice injected with both vectors Acoustic startle responses were measured in mice. Analysis of the startle response to white noise was performed in mice aged 6 weeks. AAV2 / Anc80.CMV.harmonin-b1-injected mice and both vectors Mice co-injected with lectin showed a partial rescue of the response (Figure 17A). Mice injected with only onin-a1 showed similar results to uninjected c.216AA mice. The startle response was not restored.

[0117] Example 2H - Vestibular Assessment in the Usher Mouse Model Vestibular function was assessed using the open field and rotarod balance tests. The subject was placed inside an acoustic chamber with ceiling LED lighting set at 30 lux. An open field test was conducted in a dimly lit room using a circular frame with a diameter of 42 cm. One mouse was placed in the circular open field at a time and allowed to explore for 5 minutes. The animals were tracked using Ethovision XT, allowing measurements of distance traveled and speed. All open field assessments were performed blindly. The rotarod was performed in a closed enclosure. The rotation starts at 4 rpm and continues for 0.1 rpm. -1 The mouse moves to the rod, which accelerates at a rate of On day 1, the mice were placed on the rod for 5 minutes to allow them to become accustomed to the equipment. The next day, the animals were placed on the rod for a total of five trials, with a 5-minute gap between trials. The animals were allowed to remain on the apparatus before falling to the instrumented floor of the enclosure. The length of time taken was displayed on a timer and recorded after each test run.

[0118] Because the perilymphatic space is continuous between the cochlea and the vestibular labyrinth, AAV injected through the RWM The vector can also transduce the vestibular sensory organs. Mice were tested for their performance on the rotarod. Poor rotarod performance was defined as c.2 c injected with 16AA and AAV2 / Anc80.CMV.harmonin-a1. 216AA mice (average latency to fall <22 seconds) were observed, and AAV2 / Anc80.C c.216AA mice injected with MV.harmonin-b1 and harmonin Co-injected n-a1 and -b1 vectors were tested on the rotarod for 60–120 s. , balance function was maintained, consistent with that of control c.216GA mice (Figure 17B).

[0119] The recovery of open field behavior was significantly improved by the administration of harmon-b1 and harmonin-b1. This was also observed in c.216AA mice doubly injected with n-a1 and b1. The open field exploration traces are plotted in Figure 17C. c.216AA mice explored the field edge and showed minimal whole-body rotations, whereas c.216AA mice showed significantly higher rotations / Further rotation throughout the entire chamber with increased whole-body rotation quantified as minutes Surprisingly, AAV2 / Anc80.CMV showed activity similar to that of the AAV2 / Anc80.CMV vector (Figures 17D-17E). Although we did not observe ABR rescue in mice injected with .harmonin-a1, Open field data demonstrated restoration of vestibular function to the level of control mice. V2 / Anc80.CMV.trunc-harmonic injected c.216GA ma The behavior of the mice was not different from that of control c.216GA mice, and again, the truncated and wild-type h This indicates that there is no interference between the two.

[0120] Mice injected with harmonin-a1 lost their turning behavior but showed no improvement in the rotarod test. Because the experiment failed, behavioral assays demonstrated partial pre- Page 11 ... On the other hand, mice injected with harmonized-b1 showed no significant improvement in both tests. Transduction of striola and FM1-4 restored function in the striola region (Fig. 17). The lack of uptake of 3 is a function of hair cells in the striola region and possibly type I cells. These results demonstrate that the expression of harmonin is dependent on proper expression of harmonin (Fig. 13).

[0121] While hearing rescue was predominant at low frequencies but not at high frequencies (Figure 16), Preservation of bundle morphology was observed along the entire organ (Fig. 18). The high frequency hearing loss is unlikely to be due to damage caused by the AAV vector. No changes were observed in any of the 216GA mice injected with thrombus (Figures 23C-23D). AAV targeting along the entire length of the cell may explain the lack of basal transduction efficiency. One possibility is to use other harmonics, such as a short harmonic C. nin isoforms are required for functional rescue in the basal high-frequency terminals of the cochlea Alternatively, cochlear development may begin at the basal end. Therefore, by P0, hair cells derived from the basal high-frequency terminals may have matured beyond the repair point. If so, embryonic intervention may allow for better rescue in the high-frequency region. do.

[0122] Part 3 - Gene therapy for additional mutations involved in hearing loss Example 3A - In vivo experiments An carrying the coding sequence of mouse TMC1 driven by a modified CMV promoter The c80 vector was transfected into a helper virus-free system and into a double transfection system as previously described. The triplicate was prepared using the triplicate method (Grimm et al., 2003, Mol. Ther., 7:839:50). A flag tag (FLAG) sequence was fused to the C-terminus of the TMC coding sequence to encode the expressed TMC. Iodixanol step gradient followed by iodixanol step gradient allowed visualization of proteins. Purify the Anc80.CMV.Tmc vector using ion exchange chromatography A primer set specific for human beta-globulin intron elements was used. The titer was 1 x 10 as determined by quantitative PCR. 12 From 1×10 13 gc / ml range Aliquots of virus were stored at -80°C and thawed immediately before use.

[0123] P0-P2 age mice were cultured at Boston Children's Hospital. Protocols approved by the Institutional Animal Care and Use Committee (Protocols #2659, #2146) was used for in vivo delivery of viral vectors as described below. 7BL / 6J (Jackson Laboratories) or Swiss Web A ster mouse strain (Taconic) was used as a wild-type control mouse, and TMC1 Mice carrying the mutant alleles (TMC1Δ / Δ or Tmc1− / −) were cultured as previously described. The C57BL / 6J background was used as described previously (Kawashima et al., 2011, J. Cli n. Invest., 121:4796-809).

[0124] To prepare tissue for evaluation, temporal bones were collected from mouse pups at P0–P10. Pups were euthanized by rapid decapitation and treated with 10 mM HEPES, 0.05 mg / ml anhydrous sodium. MEM (Invitrogen) supplemented with picillin and 0.01 mg / ml ciprofloxacin The temporal bones were dissected in ethanol (Progen), pH 7.40. The membranous labyrinth was isolated under a dissecting microscope and The tunica media was peeled off, and the tectorial membrane and stria vascularis were mechanically removed. The organ of Corti culture was then cultured with one end A pair of thin glass fibers bonded to an 18 mm round cover glass with Sylgard. The tissue was used for electrophysiological studies acutely. For older mice, the animals were euthanized by inhaled CO2, and then the temporal bones were removed. The tissue was collected and whole cochlear specimens were prepared.

[0125] All mean values ​​and error bars shown in the figures represent the mean ± SD. Comparisons for statistical significance between injected and uninjected ears were performed using a paired two-tailed t-test. P<0.05 was considered significant.

[0126] Example 3B - In vivo injection of viral vectors Bevel-polished glass microinjection pipette through the round window membrane (RWM) Mouse pups (P0-P2) were injected using a P-2000 pipette puller (Sutt In the ER Instruments, a pipette is drawn from the capillary glass. Beveled using a Crop Pipette Beveler (Sutter Instruments). (28° angle, ~20 μm tip diameter). Sterile needles were used to cover the surgical site (left mastoid). For pain relief using swabs, EMLA cream (lidocaine 2.5% and prilocaine) Before surgery, the patient was placed on a 37°C warm pad for 30 to 60 minutes. The temperature was maintained.

[0127] Anesthetize the pup by inducing a rapid decrease in body temperature for 2-3 min until it loses consciousness. The patient was kept on this cooling platform for 10-15 minutes during surgery. The surgical site was disinfected by scrubbing and wiping with 70% ethanol three times. A posterior incision is made to expose the transparent otic capsule, and a microinjection is performed through the capsule and overlying fascia. The pipette was placed in a micromanipulator (MP-30, Sutter Instruments) The RWM was penetrated with the tip of a micropipette.

[0128] 10 12 From 10 14Approximately 1 μL of virus with a titer between 10 9 From 1 0 11 1000 total viral particles) at 0.1 μl / min unilaterally using a pneumatic microinjector. The left ear was injected with 6-0 mono (WPI Nanoliter 2010). The skin incision was closed using filament sutures (Ethicon). The pups were then returned to the warming pad.

[0129] Example 3C - Immunofluorescence Immunostaining to determine the distribution of expression of transgenes delivered by viral vectors To this end, immunostaining was performed on freshly dissected organs of Corti and stained with PBS. The tissue was then immersion fixed in diluted 4% paraformaldehyde at room temperature for 1 hour. Rinse with PBS and permeabilize in 0.01–0.1% Triton X-100 for 30 minutes. The resulting solution was treated with Alexa Fluor 546-phalloidin (Molecular Probe s, 1:200 dilution) for 1 hour to label filamentous actin.

[0130] 2% BSA for localization of exogenously expressed TMC::FLAG fusion proteins The tissue was blocked with 5% normal goat serum for 1 hour, and the FLAG motif was removed. overnight at 4°C with antibodies against (BD Biosciences, 1:200 dilution) For hair cell counting, the tissue was blocked with normal goat serum for 1 hour. King and rabbit anti-myosin VIIa primary antibody (Proteus Bioscience s, 1:1000 dilution) overnight at 4°C, and AlexaFluor 488 (L goat conjugated to iFe Technologies, 1:200 dilution The samples were labeled with anti-rabbit antibody for 1 hour. The samples were then mounted in Vectashield mounting medium (Vector The specimen was mounted on a cover glass with the Zeiss LSM70 Imaging was performed using a confocal microscope at magnifications ranging from 10X to 63X.

[0131] Figure 26 shows homogeneous delivery of Anc80 to harmonin Ush1c mutant mice. FIG. 28 shows immunofluorescence analysis demonstrating the expression of KCNQ4 in cells of KCNQ4 mutant mice. Immunofluorescence demonstrating Anc80 delivery to the vesicles is shown. Thus, Anc80 is not responsible for the hearing loss. It is useful for treating many different genetic abnormalities (at many different loci) that result in It is an effective vector.

[0132] Example 3D - Hair Cell Electrophysiology Organotypic cochlear cultures were grown in 137 mM NaCl, 0.7 mM NaH2PO4, 5.8 mM KCl, 1.3mM CaCl2, 0.9mM MgCl2, 10mM Hepes The cells were bathed in standard artificial perilymph containing 5.6 mM D-glucose. Vitamins (1:50) and amino acids (1:100) were dissolved in 100% ethanol from Invitrogen. N was added to the solution to adjust the final pH to 7.40 (310 mosmol / kg). A recording pipette (3-5 megaohms) was prepared using R6 capillary glass ( Drawn from King Precision Glass, 135 mM CsCl, 5 mM Hepes, 5mM EGTA, 2.5mM MgCl2, 2.5mM Na2-ade Fill with intracellular solution containing phosphoinositide and 0.1 mM CaCl2, and add the final solution. CsOH was used to adjust H to 7.40 (285 mosmol / kg). Using an opatch 200B amplifier (Molecular Devices), Whole-cell voltage-clamp recordings were performed at 84 mV and room temperature (22–24°C). The sensory transduction current was filtered at 10 kHz by a Bessel filter and 16-bit Acquisition board (Digidata 1440A) and pCLAMP10 software The signals were digitized at ≥20 kHz by a digital signal processor (Molecular Devices). Data for offline analysis using OriginPro8 (OriginLab) Recorded.

[0133] Figure 29 shows the results of transfection with Anc80-KCNQ4 for mutant mice (Figure 29B). KCNQ4- / - cells (Fig. 10C) showed increased expression of potassium to near wild-type levels. The results showed that Anc80-based gene therapy was functional, demonstrating the restoration of IL-1 flow (Fig. 29A). It proves that recovery is possible.

[0134] Example 3E - Auditory Brainstem Response (ABR) ABR recordings were performed as previously described (Maison et al., 2010, J. Neurosci., 30:6751-62). Specifically, mice at P25 to P30 were given 5 ml of 0.9% saline solution. IP injection (0.1 ml / 10 g body weight) containing 0 mg ketamine and 5 mg xylazine The rats were anesthetized with acetaminophen. ABR experiments were performed in a sound-proof chamber at 32°C. Auditory function To test for this, repeatable measurements were taken at sound pressure levels between 10 and 115 dB in 5 dB increments. The ABR waveform was measured at 5.6 kHz and 10 kHz until the threshold intensity eliciting a valid ABR waveform (peaks I to IV) was found. Pure tone stimuli of 8 kHz, 11.3 kHz, 16 kHz, 22.6 kHz or 32 kHz Alternating polarity stimuli were presented to the mice. 512 to 1024 responses were collected for each sound pressure level. Waveforms with amplitudes greater than 15 μV (peak-to-peak) were considered "Arrows." The artifacts were discarded using the "Remove artifacts" function.

[0135] Before ABR testing begins, loose skin and sagging skin that typically obscure the entrance to the ear canal are removed. The cartilage was then cut with dissecting scissors and placed on each individual subject at all stimulation frequencies. The sound pressure at the entrance of the ear canal was calibrated for the primary tone. Two electrostatic earphones (CUI Miniature Dynamics) and an outer ear Knowles miniature microphone (electret capacitor) for recording sound pressure in the tunnel. A conventional probe tube speaker / microphone assembly (E The sound stimuli were delivered directly to the tested ear by a PL PXI Systems. Millisecond bursts of sound (cos 2 Delivered at 40 / sec with a 0.5 ms rise / fall at the onset ).

[0136] Subcutaneous needle electrodes were inserted into the pinna (active electrode), the vertex (reference electrode), and the buttocks (ground electrode). ABR signals were collected using a 10000x amplified ABR potential. Filtering (0.3-10kHz) was performed using conventional data acquisition software (e.g., LabVIEW). ) and digitized using a digital IO board (National Institute of The acoustic stimulus and electrode voltage were sampled at 40 μs intervals using a phono- stimulator. The waves were stored for online analysis. As the sound intensity increased, arbitrary waves (I-IV) were detected. Thresholds were visually defined as the lowest decibel level that was produced and reproduced within each experimental group. ABR thresholds were averaged and used for statistical analysis.

[0137] Figure 27 shows the delivery of Anc80 viral vectors encoding and expressing Harmonin. However, especially at low frequencies (e.g., about 5 to about 22 kHz), the auditory function is nearly complete. The figures demonstrate that recovery can be achieved.

[0138] Example 3F-Quantitative RT-PCR analysis Experiments were conducted to assess the amount of virus present in the cochlea after in vivo administration. TMC1- / - mice were injected into the left ear at P1. Cochleae were excised from both ears and then injected into the left ear at P10. The cells were maintained in culture for the corresponding 3 days. RNA was extracted and analyzed by Agilent Bioanalyzer. The quality was confirmed using a ZERO (Agilent Technologies) and described previously. As per (Kawashima et al., 2011, J. Clin. Invest., 121:4796-809) SYBR Gr TMC1-specific efficiency using eenER qPCR reagents (Invitrogen) The cDNA was reverse transcribed for quantitative RT-PCR analysis using specific primer sets.

[0139] To amplify a fragment of TMC1, use the following primers: 5'-CAT CTG CAG CCA ACT TTG GTG TGT-3' (SEQ ID NO: 17) and 5'-AGA GGT AGC CGG AAA TTC AGC CAT-3' (SEQ ID NO: 18) The expression level was measured using the 5'-TGA GCG CAA GTA CTC TGT G TG GAT-3' (SEQ ID NO: 19) and 5'-ACT CAT CGT ACT C CT GCT TGC TGA-3' (SEQ ID NO: 20) (β-actin All primers were inserted into the intron. The Actb cross-linking assay was designed and validated using melting curve analysis and negative controls. and data were collected using the ΔΔCT method in relation to the differences between injected and uninjected ears. The data was analyzed.

[0140] These results indicated that TMC1 mRNA expression was significantly higher in injected ears than in uninjected ears. demonstrated that the effect was 12 times higher.

[0141] Example 3G-Labeling of FM1-43 FM1-43 dye loading experiments were performed as previously described (Gale et al., 2001, J. Neurosci., 21:7013-25; Meyers et al., 2003, J. Neurosci., 23:4054-65; and G (Eleoc & Holt, 2003, Nat. Neurosci., 10:1019-20). Cover with adherent cochlear culture. The glass was placed under an upright microscope (Zeiss Axioscope F) on a glass-bottom chamber. S Plus). 5 μM FM1-43FX (I The tissue was washed three times with artificial perilymph and then purified with ATP. The dye was removed from the outer leaflet of the vacuole. After 5 min, intracellular FM1-43 was removed by FM1-43 filter. Imaged using an epifluorescence light source with a 63X water immersion objective. Tissues were fixed and processed for immunofluorescence as described above.

[0142] Figure 30 shows the FM1 expression by cells exposed to the Anc80 viral vector described herein. FIG. 31 is an immunostaining image showing uptake of the -43 dye. 80 TMC1 delivered by viral vectors was expressed in Tmc1-deficient hair cells in vivo. The figure demonstrates that sensory transformation was restored in the

[0143] Example 3H - Distortion Product Otoacoustic Emissions (DPOAEs) DPOAE data were collected under the same conditions and during the same recording session as ABR data. . For the generation of DPOAEs at 2f1-f2, the frequency ratio (f2 / f1) was set to 1.2. Create a secondary tone, and for each f2 / f1 pair, the f2 level is 10d lower than the f1 level. B. The sound pressure level was low. The f2 level was moved from 20 to 80 dB in 5-dB increments. To increase the signal-to-noise ratio of the recorded ear canal sound pressure, waveform and spectral flattening were performed. Equalization was used at each level. From the averaged spectra, DPOAE at 2f1-f2 The amplitude of the DPOAEs was extracted along with the noise floor at adjacent points in the spectrum. Iso-response curves were interpolated from the plots of sound level. Thresholds were calculated as DPOAE at 0 dB. is defined as the f2 level required to generate

[0144] Figure 32 shows TMC1 delivered using the Anc80 viral vector described herein. However, TMC1- / - mice exhibited a significantly higher external frequency response, especially at low frequencies (e.g., approximately 5 to approximately 16 kHz). Rescue of hair cell function is demonstrated graphically.

[0145] Other embodiments Although methods and compositions of matter have been described herein in conjunction with many different embodiments, various The above description of various embodiments is intended to be illustrative and not to limit the scope of the methods and compositions of matter. It should be understood that other aspects, advantages, and modifications are within the scope of the following claims. is within the range.

[0146] Disclosed are methods and compositions that can be used for, can be used with, can be used in preparation for, or are products of the disclosed methods and compositions. It is understood that these and other materials are described herein, and that combinations, subsets, interactions, groups, etc., of these methods and compositions are disclosed. That is, specific reference to various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, but each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed, and multiple compositions or methods are discussed, each and each combination and permutation of the compositions and methods is specifically contemplated unless specifically indicated otherwise. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Various embodiments of the present invention are described below. 1. An AVV vector comprising an Anc80 capsid protein and one or more transgenes selected from the group consisting of TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7. 2. A method for delivering a transgene to one or more cells in the inner ear of a subject, comprising: A method comprising administering an adeno-associated virus (AAV) to the inner ear of a subject, the AAV comprising an Anc80 capsid protein and a transgene. 3. The method according to claim 2, wherein the one or more cells in the inner ear are selected from the group consisting of inner hair cells (IHCs) and outer hair cells (OHCs). 4. The method according to claim 3, wherein the transgene is delivered to at least 80% of the inner hair cells and at least 80% of the outer hair cells. 5. The method of claim 2, wherein the one or more cells in the inner ear are selected from the group consisting of spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis. 6. 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, and DIAP H1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR 1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, M ET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTO F, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6 3. The method of claim 2, wherein the gene is selected from the group consisting of SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP. 7. The method according to claim 2, wherein the transgene encodes a neurotrophic factor. 8. The method according to claim 7, wherein the neurotrophic factor is selected from the group consisting of GDNF, BDNF, NT3 and HSP70. 9. The method according to claim 2, wherein the transgene encodes an antibody or a fragment thereof. 10. The method according to claim 2, wherein the transgene encodes an immunomodulatory protein. 11. The method according to claim 2, wherein the transgene encodes an anti-oncogenic transcript. 12. The method of claim 2, wherein the transgene encodes an antisense, silencing, or long non-coding RNA species. 13. The method according to claim 2, wherein the transgene encodes a genome editing system selected from the group consisting of engineered zinc finger nucleases, TALENs and CRISPRs. 14. The method described in 2 above, wherein the Anc80 capsid protein has the sequence shown in SEQ ID NO: 1. 15. The method described in 2 above, wherein the Anc80 capsid protein has the sequence shown in SEQ ID NO: 2. 16. The method of claim 3, wherein the transgene is under the control of a heterologous promoter sequence. 17. The method of claim 16, wherein the heterologous promoter sequence is selected from the group consisting of CMV promoter, CBA promoter, CASI promoter, PGK promoter, EF-1 promoter, alpha9 nicotinic receptor promoter, prestin promoter, KCNQ4 promoter, Myo7a promoter, Myo6 promoter, Gfil promoter, Vglut3 promoter and Atoh1 promoter. 18. The method described in 2 above, wherein the administering step includes injecting AncAAV through the round window. 19. The method of claim 2, wherein the AncAAV is administered by injection through the cochlear window. 20. The method of claim 2, wherein the AncAAV is administered during cochlear fenestration or canalostomy. 21. The method described in 2 above, wherein the AncAAV is administered to the middle ear and / or round window by one or more drug delivery vehicles. 22. The method described in 2 above, wherein expression of the introduced gene results in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells and / or vestibular ganglion neurons, thereby restoring hearing or vestibular function. 23. An article of manufacture comprising an AAV vector and a pharmaceutical composition, wherein the AAV vector comprises an Anc80 capsid protein and a transgene operably linked to a promoter. 24. 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, and DIA PH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR 1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, M ET, 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, 24. The article of manufacture described in claim 23, wherein the gene is selected from the group consisting of SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIAP. 25. A method for delivering a TMC1 or TMC2 transgene to one or more cells in the inner ear of a subject, comprising: A method comprising administering an adeno-associated virus (AAV) to the inner ear of a subject, the AAV comprising an Anc80 capsid protein and a transgene. 26. A method for delivering an usher transgene to one or more cells in the inner ear of a subject, comprising: A method comprising administering an adeno-associated virus (AAV) to the inner ear of a subject, the AAV comprising an Anc80 capsid protein and a transgene. 27. The method of claim 25 or 26, wherein the usher transgene is selected from the group consisting of MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, and PDZD7. 28. The method according to claim 25 or 26, wherein the one or more cells in the inner ear are selected from the group consisting of inner hair cells (IHCs) and outer hair cells (OHCs). 29. The method of claim 28, wherein the transgene is delivered to at least 80% of the inner hair cells and at least 80% of the outer hair cells. 30. The method of claim 25 or 26, wherein the one or more cells in the inner ear are selected from the group consisting of spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis. 31. The method described in 25 or 26 above, wherein the Anc80 capsid protein has the sequence shown in SEQ ID NO: 1. 32. The method described in 25 or 26 above, wherein the Anc80 capsid protein has the sequence shown in SEQ ID NO: 2. 33. A method according to claim 25 or 26, wherein the transgene is under the control of a heterologous promoter sequence. 34. The method of claim 33, wherein the heterologous promoter sequence is selected from the group consisting of a CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, an EF-1 promoter, an alpha9 nicotinic receptor promoter, a prestin promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, and an Atoh1 promoter. 35. The method described in claim 25 or 26, wherein the administering step comprises injecting AncAAV through the round window. 36. The method described in 25 or 26 above, wherein the AncAAV is administered by injection through the round window of the cochlea. 37. The method of claim 25 or 26, wherein the AncAAV is administered during cochlear fenestration or canalostomy. 38. The method described in 25 or 26 above, wherein the AncAAV is administered to the middle ear and / or round window by one or more drug delivery vehicles. 39. The method described in 25 or 26 above, wherein expression of the transgene results in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells and / or vestibular ganglion neurons, thereby restoring hearing or vestibular function.

Claims

1. 1. A composition for use in a method of delivering a cadherin-associated 23 (CDH23) transgene to a subject, the method comprising: administering an AAV vector to the inner ear of a subject; the AAV vector comprises (i) an Anc80 capsid protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) a CDH23 transgene; the transgene is delivered to at least 80% of the inner hair cells (IHCs) and at least 80% of the outer hair cells of the subject's inner ear; The method comprises injecting an AAV vector through the round window of the cochlea. composition.

2. 10. The composition of claim 1, wherein the CDH23 transgene is further delivered to one or more cells in the inner ear selected from the group consisting of spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, supporting cells, and cells in the stria vascularis.

3. 3. The composition of claim 1 or 2, wherein the CDH23 transgene is under the control of a heterologous promoter sequence, optionally selected from the group consisting of a CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, an EF-1 promoter, an alpha 9 nicotinic receptor promoter, a prestin promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, and an Atoh1 promoter.

4. The composition of any one of claims 1 to 3, wherein the method comprises delivering an AAV vector to the round window by one or more drug delivery vehicles.

5. 5. The composition of any one of claims 1 to 4, wherein the method comprises injecting an AAV vector through the round window during a cochleostomy or canalostomy.

6. 5. The composition of any one of claims 1-4, wherein expression of the CDH23 transgene results in regeneration of one or more of inner hair cells (IHC), outer hair cells (OHC), spiral ganglion neurons, stria vascularis, vestibular hair cells, and vestibular ganglion neurons.

7. 1. An article of manufacture for use in a method of delivering a cadherin-associated 23 (CDH23) transgene to the inner ear of a subject, comprising an adeno-associated virus (AAV) vector and a pharmaceutical composition, the AAV vector comprises (i) an Anc80 capsid protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) a CDH23 transgene operably linked to a promoter; the CDH23 transgene is delivered to at least 80% of the inner hair cells (IHCs) and at least 80% of the outer hair cells of the subject's inner ear; The method comprises injecting an AAV vector through the round window of the cochlea. Manufactured products.

8. The composition of any one of claims 1 to 6 or the article of manufacture of claim 7, wherein the method is a method for treating hearing impairment.

Images