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

Abstract

Provided herein are materials and methods for efficient delivery of nucleic acids to cochlear and vestibular cells, and methods of treating sensory transduction disorders associated with genetic defects.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application 62 / 638,697, filed March 5, 2018, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Hereditary hearing loss is a significant problem with few treatment options other than cochlear implantation. Congenital hearing problems are often caused by single-gene defects. Prelingual deafness is diagnosed in approximately 1 in 500 infants, with about 50% having a genetic cause. Usher syndrome is associated with a large number of different clinical subtypes, each caused by mutations in any of a large number of different genes; Usher syndrome is the cause of early childhood deafness in 3% to 6% of cases. A more common gene defect occurs in the TMC1 gene, estimated to account for 1% to 2% of all hereditary deafness. The most severe form of Usher syndrome, USH1, is associated with defects in the following six genes: USH1, MYO7A (myosin 7a), USH1C (harmonin), CDH23 (cadherin 23), PCDH15 (protocadherin 15), SANS (sans; also known as USH1G), and CIB2 (calcin-binding protein 2).

[0004] The inner ear, such as the cochlea, especially the inner and outer hair cells (IHC and OHC) within the cochlea, is an attractive target for polynucleotide therapy pathways aimed at intervening in hearing loss and deafness of various etiologies, most directly in the single-gene form of congenital deafness. However, effectively targeting and transducing IHC and OHC, and other inner ear cells that may be associated with gene therapy pathways, is a challenge. Summary of the Invention

[0005] The present disclosure provides AAV9-php.b vectors comprising a transgene encoding a polypeptide of interest (e.g., TMC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCNQ4, TMPRSS3, STRC, EYA4, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c), OTOF, GPR98, MY06, MY015A, LOXHD1, POU3F4, EYA1, WFS1, ACTG1, TMIE, PJVK, SYNE4, and FAM65B), and methods of administering the vectors to the inner ear of a subject having a genetic deficiency in hearing and / or vestibular mechanosensation to treat the subject.

[0006] The present disclosure is based, at least in part, on the discovery that AAV9-php.b-CMV-GFP (also referred to as AAV-php.b-CMV-GFP) effectively and specifically targets sensory cells of the inner ear, including inner and outer hair cells, in vivo.

[0007] In one aspect, the present disclosure provides AAV9-php.b vectors, wherein the vectors contain a polynucleotide encoding myosin 7a, harmonin (e.g., harmonin-a, harmonin-b, or harmonin-c), cadherin 23, protocadherin 15, USH2A, ADGRV1 / VLGR1 / GPR98, WHRN, CLRN1, HARS, SANS, and calcium modulating binding protein 2, or any other polypeptide described herein.

[0008] In another aspect, the present disclosure provides AAV9-php.b vectors, wherein the vectors encode a capsid having at least about 85% sequence identity to AAV9-php.b and contain a promoter that directs expression of a human TMC1 polynucleotide.

[0009] In another aspect, the present disclosure provides AAV9-php.b vectors, wherein the vectors contain a promoter that directs expression of a downstream polynucleotide, the promoter being an Espin promoter, a PCDH15 promoter, a PTPRQ promoter, a Myo6 promoter, a KCNQ4 promoter, a Myo7a promoter, a Synapsin promoter, a GFAP promoter, a CMV promoter, a CAG promoter, a CBH promoter, a CBA promoter, a U6 promoter, or a TMHS (LHFPL5) promoter.

[0010] In another aspect, the present disclosure provides cells containing the AAV9-php.b vectors of the preceding aspects. In another aspect, the present disclosure provides cells containing the AAV9-php.b vectors of the preceding aspects.

[0011] In another aspect, the application provides a method of expressing a polypeptide in the inner ear of a subject, the method involving contacting an inner ear cell with an AAV9-php.b vector encoding the polypeptide of interest, wherein the AAV9-php.b vector transfects at least about 85%, 90%, 95%, or more inner hair cells or outer hair cells.

[0012] In another aspect, the application provides a method of expressing a polypeptide in the inner ear of a subject, the method involving contacting an inner ear cell with an AAV9-php.b vector encoding the polypeptide of interest, wherein the AAV9-php.b vector transfects at least about 85%, 90%, 95%, or more inner hair cells or outer hair cells.

[0013] In another aspect, the application provides a method of treating an inner ear disorder associated with a genetic defect in a subject, the method involving contacting a cell of the subject with an AAV9-php.b vector, wherein the vector contains a polynucleotide encoding any one or more of myosin 7a, harmonin, cadherin 23, protocadherin 15, USH2A, ADGRV1 / VLGR1 / GPR98, WHRN, CLRN1, HARS, SANS, and calcium modulin binding protein 2.

[0014] In another aspect, the application provides a method of treating an inner ear disorder associated with a genetic defect in a subject, the method involving contacting a cell of the subject with an AAV9-php.b vector, wherein the vector contains a promoter that is any one of the Espin promoter, the PCDH15 promoter, the PTPRQ promoter, the Myo6 promoter, the KCNQ4 promoter, the Myo7a promoter, the Synapsin promoter, the GFAP promoter, the CMV promoter, the CAG promoter, the CBH promoter, the CBA promoter, the U6 promoter, and the TMHS (LHFPL5) promoter.

[0015] In another aspect, the application provides a method of treating an inner ear disorder associated with a genetic defect in a subject, the method involving contacting a cell of the subject with an AAV9-php.b vector, wherein the vector encodes a capsid having at least about 85% sequence identity to AAV9-php.b and contains a promoter operably linked to a polynucleotide encoding a UDH1 polypeptide that is myosin 7a, harmonin, cadherin 23, protocadherin 15, USH2A, ADGRV1 / VLGR1 / GPR98, WHRN, CLRN1, HARS, SANS, or calcium modulin binding protein 2.

[0016] In various embodiments of the above aspects of the application described herein, or any other aspect, the inner ear defect is a genetic disorder associated with a genetic alteration in a polypeptide expressed in the inner ear. In other embodiments, the genetic defect is associated with partial hearing loss, complete deafness, or partial or complete vestibular dysfunction. In other embodiments of the above aspects, the promoter is any one or more of the following: Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNQ4 promoter, Myo7a promoter, Synapsin promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and TMHS (LHFPL5) promoter. In other embodiments of the above aspects, the vector transduces inner hair cells, outer hair cells, vestibular hair cells, spiral ganglion, or vestibular ganglion with an efficiency of at least about 70% or higher. In other embodiments of the above aspects, the harmonin polypeptide is harmonin-a, harmonin-b, or harmonin-c. In other embodiments of the above aspects, the cell is an outer hair cell, an inner hair cell, a vestibular hair cell, a spiral ganglion, or a vestibular ganglion. In other embodiments of the above aspects, the vector contains a promoter that directs expression of a downstream polynucleotide, the promoter being any one or more of the following: Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNQ4 promoter, Myo7a promoter, Synapsin promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, or TMHS (LHFPL5) promoter. In other embodiments of the above aspects, the downstream polynucleotide is a TMC1, TMC2, or USH1 polypeptide, and the USH1 polypeptide is Myosin 7a, harmonin, Cadherin 23, Protocadherin 15, USH2A, ADGRV1 / VLGR1 / GPR98, WHRN, CLRN1, HARS, SANS, and CIB2. In particular embodiments of the above aspects, the harmonin polypeptide is harmonin-a, harmonin-b, or harmonin-c. In other embodiments of the above aspects, the AAV9-php.b vector targets inner hair cells and outer hair cells with a targeting efficiency of at least about 70%, 80%, 90%, 95%, or higher, even up to 100%. In other embodiments of the above aspects, the human polypeptide is TMC2, harmonin-a, harmonin-b, or harmonin-c. In other embodiments of the above aspects, the inner ear defect is a hearing disorder or a vestibular disorder. In other embodiments, administration of the vector increases, improves, or maintains hearing and / or vestibular function in the subject.In other embodiments, the hearing and / or vestibular function that is improved or maintained is related to the preservation of stereociliary bundle morphology and / or recovery of mechanotransduction. In other embodiments of the above aspects, the inner ear disorder is Usher syndrome.

[0017] Definitions

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following references provide one of ordinary skill with a general definition of many of the terms used in this application: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker et al., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0019] “AAV9-php.b vector” means a viral vector comprising an AAV9-php.b polynucleotide or fragment thereof that transfects inner ear cells. In one embodiment, the AAV9-php.b vector transfects at least 70% of inner hair cells and 70% of outer hair cells upon administration to the inner ear of a subject or in vitro contact with cells derived from the inner ear. In other embodiments, at least 85%, 90%, 95%, or nearly 100% of inner hair cells and / or 85%, 90%, 95%, or nearly 100% of outer hair cells are transfected. Transfection efficiency can be assessed in mouse models using a gene encoding GFP. The sequence of an exemplary AAV9-php.b vector is provided below.

[0020]

[0021] “Mechanosensation” means a response to a mechanical stimulus. Touch, hearing, and balance are examples of mechanosensation inputs being converted into neuronal signals. Mechanosensation inputs are converted into a response to a mechanical stimulus by a process called “mechanotransduction.”

[0022] “Myosin 6 (Myo6) promoter” means a regulatory polynucleotide sequence comprising or consisting of a nucleic acid sequence sufficient to direct expression of a downstream polynucleotide in an outer hair cell, an inner hair cell, a vestibular cell, a spiral ganglion cell, or a vestibular ganglion cell and having at least about 85% sequence identity to the following nucleotide sequence:

[0023]

[0024] By "myosin 1 A (Myo7A) promoter" is meant a regulatory polynucleotide sequence comprising or consisting of a nucleic acid sequence sufficient to direct expression of a downstream polynucleotide in outer hair cells, inner hair cells, vestibular cells, spiral ganglion cells, or vestibular ganglion cells and having at least about 85% sequence identity to the following nucleotide sequence:

[0025]

[0026] “TMC1 polypeptide” means a polypeptide or fragment thereof having mechanotransduction channel activity having at least about 85% or greater amino acid sequence identity to NCBI Reference Sequence: NP_619636.2. An exemplary TMC1 amino acid sequence is provided below:

[0027]

[0028]

[0029] “TMC1 polynucleotide” means a polynucleotide encoding a TMC1 polypeptide. An exemplary TMC1 polynucleotide sequence is provided as NCBI Reference Sequence: NM_138691.2, which is reproduced below:

[0030]

[0031]

[0032] “TMC2 polypeptide” means a polypeptide or fragment thereof that plays a role in mechanotransduction having at least about 85% or greater amino acid sequence identity to NCBI Reference Sequence: NP_542789. An exemplary TMC2 amino acid sequence is provided below:

[0033]

[0034]

[0035] “TMC2 polynucleotide” means a polynucleotide encoding a TMC2 polypeptide. An exemplary polynucleotide sequence is provided below:

[0036]

[0037]

[0038] “harmonin” polypeptide means a polypeptide or fragment thereof that plays a role in mechanosensation or interacts with any one or more of USH1C, USH1G, CDH23, and MY07A having at least about 85% amino acid sequence identity to Q9Y6N9-1 (isoform 1), Q9Y6N9-2, Q9Y6N9-3, Q9Y6N9-4, Q9Y6N9-5. An exemplary harmonin-a polypeptide (isoform 1) sequence is provided below:

[0039] >sp|Q9Y6N9|USH1C_HUMAN Harmonin OS=Homo sapiens GN=USH1C PE=1 SV=3

[0040] MDRKVAREFRHKVDFLIENDAEKDYLYDVLRMYHQTMDVAVLVGDLKLVINEPSRLPLFDAIRPLIPLKHQVEYDQLTPRRSRKLKEVRLDRLHPEGLGLSVRGGLEFGCGLFISHLIKGGQADSVGLQVGDEIVRIN GYSISSCTHEEVINLIRTKKTVSIKVRHIGLIPVKSSPDEPLTWQYVDQFVSESGGVRGSLGSPGNRENKEKKVFISLVGSRGLGCSISSGPIQKPGIFISHVKPGSLSAEVGLEIGDQIVEVNGVDFSNLDHKEAVN VLKSSRSLTISIVAAAGRELFMTDRERLAEARQRELQRQELLMQKRLAMESNKILQEQQEMERQRRKEIAQKAAEENERYRKEMEQIVEEEEKFKKQWEEDWGSKEQLLLPKTITAEVHPVPLRKPKYDQGVEPELEP ADDLDGGTEEQGEQDFRKYEEGFDPYSMFTPEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKVVVSAVYERGAAERHGGIVKGDEIMAINGKIVTDYTLAEAEAALQKAWNQGGDWIDLVVAVCPPKEYDDELTFF

[0041] “Ush1C polynucleotide” means nucleic acid molecule encoding a harmonin polypeptide. An illustrative sequence of Ush1C polynucleotide NM_005709 is provided below:

[0042]

[0043]

[0044] Other exemplary harmonin sequences are provided below:

[0045] Harmonin-B

[0046] Predicted >XM_011519832.2: Harmonin (USH1C), a component of the Homo sapiens USH1 protein network, transcriptional variant X3, mRNA

[0047]

[0048] Harmonin-B polypeptide

[0049] MDRKVAREFRHKVDFLIENDAEKDYLYDVLRMYHQTMDVAVLVGDLKLVINEPSRLPLFDAIRPLIPLKHQVEYDQLTPRRSRKLKEVRLDRLHPEGLGLSVRGGLEFGCGLFISHLIKGGQADSVGLQVGDEIVRINGYSISSCTHEEVINLIRTKKTVSIKVRHIGLIPVKSSPDEPLTWQYVDQFVSESGGVRGSLGSPGNRENKEKKVFISLVGSRGLGCSISSGPIQKPGIFISHVKPGSLSAEVGLEIGDQIVEVNGVDFSNLDHKEAVNVLKSSRSLTISIVAAAGRELFMTDRERLAEARQRELQRQELLMQKRLAMESNKILQEQQEMERQRRKEIAQKAAEENERYRKEMEQIVEEEEKFKKQWEEDWGSKEQLLLPKTITAEVHPVPLRKPKSFGWFYRYDGKFPTIRKKGKDKKKAKYGSLQDLRKNKKELEFEQKLYKEKEEMLEKEKQLKINRLAQEVSETEREDLEESEKIQYWVERLCQTRLEQISSADNEISEMTTGPPPPPPSVSPLAPPLRRFAGGLHLHTTDLDDIPLDMFYYPPKTPSALPVMPHPPPSNPPHKVPAPPVLPLSGHVSASSSPWVQRTPPPIPIPPPPSVPTQDLTPTRPLPSALEEALSNHPFRTGDTGNPVEDWEAKNHSGKPTNSPVPEQSFPPTPKTFCPSPQPPRGPGVSTISKPVMVHQEPNFIYRPAVKSEVLPQEMLKRMVVYQTAFRQDFRKYEEGFDPYSMFTPEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKVVVSAVYERGAAERHGGIVKGDEIMAINGKIVTDYTLAEAEAALQKAWNQGGDWIDLVVAVCPPKEYDDELASLPSSVAESPQPVRKLLEDRAAVHRHGFLLQLEPTDLLLKSKRGNQIHR

[0050] Harmonin-C

[0051] NM_001297764.1 Homo sapiens USH1 protein network component harmonin (USH1C), transcript variant 3, mRNA

[0052]

[0053] Harmonin-C polypeptide

[0054] MDRKVAREFRHKVDFLIENDAEKDYLYDVLRMYHQTMDVAVLVGDLKLVINEPSRLPLFDAIRPLIPLKHQVEYDQLTPRRSRKLKEVRLDRLHPEGLGLSVRGGLEFGCGLFISHLIKGGQADSVGLQVGDEIVRINGYSISSCTHEEVINLIRTKKTVSIKVRHIGLIPVKSSPDEPLTWQYVDQFVSESGGVRGSLGSPGNRENKEKKVFISLVGSRGLGCSISSGPIQKPGIFISHVKPGSLSAEVGLEIGDQIVEVNGVDFSNLDHKEGRELFMTDRERLAEARQRELQRQELLMQKRLAMESNKILQEQQEMERQRRKEIAQKAAEENERYRKEMEQIVEEEEKFKKQWEEDWGSKEQLLLPKTITAEVHPVPLRKPKYDQGVEPELEPADDLDGGTEEQGEQDFRKYEEGFDPYSMFTPEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKVVVSAVYERGAAERHGGIVKGDEIMAINGKIVTDYTLAEAEAALQKAWNQGGDWIDLVVAVCPPKEYDDELTFF

[0055] "KCNQ4 polypeptide" means a polypeptide that has at least about 85% identity to NP_004691.2 or a fragment thereof and has potassium voltage-gated channel activity. An exemplary amino acid sequence is provided at NP_004691.2, the sequence of which is reproduced below.

[0056]

[0057] "KCNQ4 polynucleotide" means a polynucleotide that encodes a KCNQ4 polypeptide. An exemplary KCNQ4 polypeptide sequence is provided at NM_004700, which is reproduced below.

[0058]

[0059]

[0060]

[0061] A "KCNQ4 promoter" means a regulatory polynucleotide sequence comprising or consisting of a nucleic acid sequence sufficient to direct expression of a downstream polynucleotide in outer hair cells, inner hair cells, vestibular cells, spiral ganglion cells, or vestibular ganglion cells and having at least about 85% sequence identity to the following nucleotide sequence:

[0062] ACGCGTCCGGCTTCCCGGCCCCGCGCGCTGCCCCCGCCACGCGGTTCGGCCCAGGCACCAACTCGGCCGCCCGTGCGCCCTGCCCCGCCGCCTGCTCCGCGCGTTCCCTCCCTCCGCCTCGCCTCGCTTGCTCGCTCGCTCCCTCCCGATTTGGGAAGGCGGCCGCGGGGCGGGCGGGGGAGGGGCGGGGCGGGGGAGGGTGACATGTGAGCGGCGCGCGCCGGTGGCAGGTGGAAAGGCGAGCGGCATGGAGCGCGTAATAAGAGAGTTGGAGTCGGAAAGAGCAGCCCCAGTCGCCGGGGAAGCGGGAGGTCAGTGCGGGCTCCGGCGGCCCCCAGGCTCCGAGCGCCCGCCCGCGGCCCCGGCCCGGCCCCTAGCCCCCGCCGCCCGCGCCCGCCCCGGGTCGCCCCTCTGGCCCCGGGTCCGAGCCATGCGTCTCTGAGCGCCCCGAGCGCGCCCCCGCCCCGGACCGTGCCCGGGCCCCGGCGCCCCCAGCCCGGCGCCGCCc

[0063] A "TMPRSS3 polypeptide" means a protein having at least about 85% amino acid sequence identity to NP_001243246 or a fragment thereof having protease activity. An exemplary TMPRSS3 sequence is as follows:

[0064] Transmembrane protease, serine 3 isoform 4 [Homo sapiens]

[0065] NCBI Reference Sequence: NP_001243246.1

[0066] >NP_001243246.1 Transmembrane protease, serine 3 isoform 4 [Homo sapiens]

[0067] MGENDPPAVEAPFSFRSLFGLDDLKISPVAPDADAVAAQILSLLPLKFFPIIVIGIIALILALAIGLGIHFDCSGKYRCRSSFKCIELIARCDGVSDCKDGEDEYRCVRVGGQNAVLQVFTAASWKTMCSDDWKGHYANVACAQLGFPSYVSSDNLRVSSLEGQFREEFVSIDHLLPDDKVTALHHSVYVREGCASGHVVTLQCTACGHRRGYSSRIVGGNMSLLSQWPWQASLQFQGYHLCGGSVITPLWIITAAHCVYDLYLPKSWTIQVGLVSLLDNPAPSHLVEKIVYHSKYKPKRLGNDIALMKLAGPLTFNEMIQPVCLPNSEENFPDGKVCWTSGWGATEDGGDASPVLNHAAVPLISNKICNHRDVYGGIISPSMLCAGYLTGGVDSCQGDSGGPLVCQERRLWKLVGATSFGIGCAEVNKPGVYTRVTSFLDWIHEQMERDLKT

[0068] A "TMPRSS3 polynucleotide" means a polynucleotide encoding a TMPRSS3 polypeptide. An exemplary TMPRSS3 polypeptide sequence is provided at NCBI NM_001256317, which is incorporated by reference below:

[0069] > NM_001256317.1 Homo sapiens transmembrane serine protease 3 (TMPRSS3), transcript variant F, mRNA

[0070]

[0071]

[0072] "STRC polynucleotide" means a nucleic acid molecule encoding a STRC polypeptide. Exemplary STRC polynucleotide sequences are as follows:

[0073]

[0074] "EYA4 polypeptide" means a protein having at least about 85% amino acid sequence identity to NP_001287941.1 or a fragment thereof having transcriptional regulatory activity.

[0075] >NP_001287941.1 Eye-shape homolog 4 isoform e [Homo sapiens]

[0076] MEDSQDLNEQSVKKTCTESDVSQSQNSRSMEMQDLASPHTLVGGGDTPGSSKLEKSNLSSTSVTTNGTGVITSSGYSPRSAHQYSPQLYPSKPYPHILSTPAAQTMSAYAGQTQYSGMQQPAVYTAYSQTGQPYSLPTYDLGVMLPAIKTESGLSQTQSPLQSGCLSYSPGFSTPQPGQTPYSYQMPGSSFAPSSTIYANNSVSNSTNFSGSQQDYPSYTAFGQNQYAQYYSASTYGAYMTSNNTADGTPSSTSTYQLQESLPGLTNQPGEFDTMQSPSTPIKDLDERTCRSSGSKSRGRGRKNNPSPPPDSDLERVFVWDLDETIIVFHSLLTGSYAQKYGKDPPMAVTLGLRMEEMIFNLADTHLFFNDLEECDQVHIDDVSSDDNGQDLSTYSFATDGFHAAASSANLCLPTGVRGGVDWMRKLAFRYRRVKELYNTYKNNVGGLLGPAKRDAWLQLRAEIEGLTDSWLTNALKSLSIISTRSNCINVLVTTTQLIPALAKVLLYSLGGAFPIENIYSATKIGKESCFERIVSRFGTNITYVVIGDGRDEEHAANQHNMPFWRISSHSDLLALHQALELEYL

[0077] "EYA4 polynucleotide" means a nucleic acid molecule encoding an EYA4 polypeptide. An exemplary EYA4 polynucleotide sequence is provided at NCBI Reference: NM_001301012.1, which is reproduced below:

[0078]

[0079] By "Espin promoter" is meant a sequence derived from NCBI Reference Sequence: NG_015866.1 that is sufficient to direct expression of a downstream polynucleotide in outer or inner hair cells, vestibular hair cells, spiral ganglion or vestibular ganglion. In an embodiment, the Espin promoter comprises or consists of at least about 350, 500, 1000, 2000, 3000, 4000, 5000 or more base pairs upstream of the Espin coding sequence.

[0080] By "Protocadherin-related 15 (PCDH15) promoter" is meant a sequence derived from NCBI Reference Sequence: NG_009191 that is sufficient to direct expression of a downstream polynucleotide in outer or inner hair cells, vestibular hair cells, spiral ganglion or vestibular ganglion. In an embodiment, the PCDH15 promoter comprises at least about 350, 500, 1000, 2000, 3000, 4000, 5000 or more base pairs upstream of the PCDH15 coding sequence. In some embodiments, the PCDH15 promoter comprises or consists of a nucleic acid sequence having at least about 85% sequence identity to the following nucleotide sequence:

[0081] TCTTCACCTGTCATTTTCAACCAGCCTCAGCCTATCTGCTCTGTCACAATCACTACTAAAATATGTTCCTAAATTGCTTGTTTCTAGATCCTTCCTTCTCATATGCTCAGGTGAACACATGGGTGAAATTTAATATGGAATTGAAATATGTACTATGCAAGATAGATTCCTTAAGAAATGTTTCTCTGATTTATATGACATAATTGTATTTTACTAGTTTACCTGTCCATCTGTAAAACTTTGTTTTGGAGATTTCATATATTACAATGTTTAAGAAATATGCTATAATGTTTTGTATAGTATATTTCTTCGTGATAACCTTATATACTACCAGTCACACGTGTTTGTAAAAATCTAAAGAGTACTTTTGGCTCCTACAGAATGTGTGAAGTTGTGAAATTGTTTTTTTGTTTTGTTTTGTTTTGTTTTTATGCCCCAAAGATGTGGAGGGCTTCATATAAGAGGGTAGATTTAATGAGAGAGAGAGGGAGAGACAGAGAGAATGATAAAAGAAGCTTAAGAGATTATTTTATCTTGTCAACGACATTGTTATTGAATGTAAGCTGCTAAACTTCTTAGATAAAGTAAAACAGTAAAAACAAACACACAAAACAGAACAGAGAATCATCAGACAGGCTGACGAACACAGTACAATAAAGCAGCCAGTACCGATGATCAGTGGACATCAATTTGTCTTTTGGGCTGTAGCACCTGCTACTAATTGGTGCAAAGCGCTCACCAGTCAGTGCGTGGTTTAGCGCACTCAGCTGTCTCCTGTATGTGCTGCGAGAAGCAAGATAGCTAATTGCTGTTGCTTCAGTGCCAGTGAAATCAACGTGCTGAGCTAATAGCGACAGATAGAGGGCAGACAGATTCCTGCTAGCAGCTTAGTGTTAGTTGCTTGTGGTAACTAAGGCAGGTGGCATACATCTCAGAACGTGGAGAATGATGGTATGCTTTCTGA

[0082]

[0083] “Lipoma HMGIC Fusion Partner-Like 5 (LHFPL5) Promoter” also referred to as “TMHS” promoter, means a promoter derived from NCBI Reference Sequence: GeneID: 222662 that is sufficient to direct expression of a downstream polynucleotide in outer or inner hair cells, vestibular hair cells, spiral ganglion or vestibular ganglion. In one embodiment, the TMHS promoter comprises at least about 350, 500, 1000, 2000, 3000, 4000, 5000, or more base pairs upstream of the PCDH15 coding sequence. In some embodiments, the TMHS promoter comprises or consists of a nucleic acid sequence having at least about 85% sequence identity to the following nucleotide sequence:

[0084] GCCCAGTGGAATTTTCCTAGTTCTTTACACTAGCCATGTATTTACCTATAAAATCAGGAGAAATATGTATATATATAATATATTAAAACATATATATATTTAAATGGGGAAATATGTAACAAACAAATAGAAACAAGGGGAGAAAGGCATTGTATTTGACAAAACACATATGTTCAGGTCTGAGAAGGCTCATAAAGAATGTTGTCTGCTATACTTTGTAGTTGCTTCTGTTATCACACAATCAGTCTGCATATACAGGCGTTTTATATATATATTTATATAGACTACATATATACGTATATTATATATGTAAATATTTCACTGTCTTTGAGGACGGGGGCCCTGTCTTTTTTATCTGTGGTTTTGCTTAGATGTCCTCCAACATAATCTTAACACATAGTATGCTTTTAGAAATCGTTGACTGAATGCTAAGGACGAAAAACCGGTGACCAGAAGGCAACCAGGAAAGGCTTTGCTGACCTCCGGAGTGGTGGAGTTGGAGGTTCTGGGAAGGCGACTAGGGAGCCAGGCAGGGGCGGGGTGGGATGGGATGTGGACAGCGCTTTTGCGGGGGGAAAGCGTTTTTGCTGCTGGAATTGAGCAGTAGGAATGTGTCAGTCACATCCCCACCTTCCCAATTCTTGTCATCTCGGTTCAGGAAGGTGAACGGTGTTCCGATTCCCCGCGGCGGGGGCCTGTAGTGGGAGCTCTGCCCCTTCCCCGCCTCTGCTGCAGGCCCCGCCCCTCGCCCGGAACCCCGGGGCGCTGGCCGCGGTGCTGAAACGGCGCCCTCCGCGGACGGAGGAGGGGGCGGGGCTCTCGGGAGCCGTGAGCCGGGAAGAGGGAGACGGGCAGGGCGGCGCCAGCAGGCCCTGGTGGGCTTGGGAGGAGGCAGGAGACTGGAGACAGCCTCGGCTAGAGCGGACACAGGCACCTGGCAAGCTTTCCTTGACCAAATCAAGGT

[0085] A "synapsin promoter" also referred to as "Syn promoter" means a regulatory polynucleotide sequence comprising or consisting of a nucleic acid sequence sufficient to direct expression of a downstream polynucleotide in an outer hair cell, an inner hair cell, a vestibular cell, a spiral ganglion cell, or a vestibular ganglion cell and having at least about 85% sequence identity to the nucleotide sequence of:

[0086] tctagactgcagagggccctgcgtatgagtgcaagtgggttttaggaccaggatgaggcggggtgggggtgcctacctgacgaccgaccccgacccactggacaagcacccaacccccattccccaaattgcgcatcccctatcagagagggggaggggaaacaggatgcggcgaggcgcgtgcgcactgccagcttcagcaccgcggacagtgccttcgcccccgcctggcggcgcgcgccaccgccgcctcagcactgaaggcgcgctgacgtcactcgccggtcccccgcaaactccccttcccggccaccttggtcgcgtccgcgccgccgccggcccagccggaccgcaccacgcgaggcgcgagatagggggGcacgggcgcgaccatctgcgctgcggcgccggcgactcagcgctgcctcagtctgcggtgggcagcggaggagtcgtgtcgtgcctgagagcgcagtc

[0087] A "agent" means a polypeptide, a polynucleotide, or a small compound.

[0088] "Alleviate" means to reduce, suppress, attenuate, diminish, retard, or stabilize the development or progression of a disease or pathology.

[0089] "Alter" means a change (increase or decrease) in the level of expression or activity of a gene or polypeptide, as detected by methods known in the art such as those described herein. As used herein, an alteration includes a change in expression level of at least 10%, preferably a change of 25%, more preferably a change of 40%, and most preferably a change in expression level of 50% or greater.

[0090] In the present disclosure, "comprise," "contain," and "have" and the like can have the meaning ascribed to them in U.S. patent law and can mean "include," "comprehend," and the like; "consisting essentially of' and the like have the meaning ascribed to them in U.S. patent law and the term is open-ended, allowing for the presence of more than the recited components, as long as the basic or novel characteristics of the recited components are not changed by the presence of more than the recited components, but does not include prior art embodiments.

[0091] "Detecting" refers to establishing the presence, absence, or amount of an analyte to be detected.

[0092] "Disease" means any condition or pathology that impairs or interferes with the normal functioning of a cell, tissue, or organ. Examples of diseases include genetic disorders that are characterized by a loss of function of a protein expressed in, for example, the inner ear of a subject and that plays a role in mechanosensory transduction. In another embodiment, the disease is Usher syndrome (e.g., USH1) or presbycusis. In one embodiment, the disease is a hearing disorder associated with a defect in a gene such as TMC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCNQ4, TMPRSS3, STRC, EYA4, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c), OTOF, GPR98, MY06, MY015A, LOXHD1, POU3F4, EYA1, WFS1, ACTG1, TMIE, PJVK, SYNE4, and FAM65B.

[0093] "Effective amount" means the amount of an agent required to alleviate the symptoms of a disease relative to an untreated patient. The effective amount of active compound utilized in practicing the present application for therapeutic treatment of a disease is dependent on the subject and disease state, and thus can vary, depending on the mode of administration and the age, body weight, and general health condition of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. This amount is referred to as the "effective" amount.

[0094] "Fragment" means a portion of a polypeptide or nucleic acid molecule. The portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. A fragment can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0095] "Hybridization" means hydrogen bonding between complementary nucleic acid bases, which can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleic acid bases that pair by forming hydrogen bonds.

[0096] The terms "isolated," "purified," or "biologically pure" denote a material that is not present in its natural state or in a naturally occurring mixture. "Isolated" indicates a degree of separation from original sources or surrounding material. "Purified" indicates a higher degree of separation from surrounding material than "isolated." A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein. In other words, a nucleic acid or peptide of the application is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques or chemical synthesis, respectively. Purity is typically determined by analytical chemistry techniques, e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein gives essentially one band on an electrophoretic gel. For proteins that can be modified, e.g., by phosphorylation or glycosylation, different modifications can give rise to different isolated proteins, which can be purified independently.

[0097] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that is not found in the natural occurring genome of the organism from which the nucleic acid molecule of the application is derived flanked by the genes that naturally flank the gene in the genome. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction analysis) independent of other sequences. Moreover, the term includes an RNA that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0098] "Isolated polypeptide" means a polypeptide of the application that has been separated from a substantial portion of the proteins and naturally occurring organic molecules with which it would naturally be associated. Typically, a polypeptide is isolated for at least 60% by weight when the polypeptide is free from its naturally associated proteins and naturally occurring organic molecules. Preferably, the preparation contains at least 75% by weight, more preferably at least 90% by weight, and most preferably 99% by weight of a polypeptide of the application. An isolated polypeptide of the application can be obtained, for example, by extraction from natural sources, expression of a recombinant nucleic acid encoding the polypeptide, or chemical synthesis of the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0099] "Marker" means any protein or polynucleotide that has an altered level of expression or activity that is associated with a disease or disorder.

[0100] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

[0101] "Promoter" means a polynucleotide sufficient to direct transcription of a downstream polynucleotide.

[0102] "Decrease" or "increase" means at least a 10%, 25%, 50%, 75%, or 100% negative or positive change.

[0103] "Reference" means a standard or control condition.

[0104] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of or all of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will typically be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will typically be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

[0105] Nucleic acid molecules useful in the methods of the application include any nucleic acid molecule that encodes a polypeptide of the application or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the application include any nucleic acid molecule that encodes a polypeptide of the application or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule.

[0106] "Hybridize" means to pair with a complementary polynucleotide sequence, such as a gene described herein, or a portion thereof, under a variety of stringent conditions to form a double-stranded molecule. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0107] For example, stringent salt concentration would generally be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridizations can be obtained in the absence of organic solvents such as formamide, while high stringency hybridizations can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will generally include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Variable additional factors, such as hybridization time, concentration of detergent such as sodium dodecyl sulfate (SDS), and the presence or absence of denaturing carrier DNA, are well known to those skilled in the art. Various levels of stringency are achieved by combining these various conditions as needed. In a preferred embodiment, hybridization will occur in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30°C. In a more preferred embodiment, hybridization will occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37°C. In a most preferred embodiment, hybridization will occur in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42°C. Useful variations on these conditions will be apparent to those skilled in the art.

[0108] For most applications, the stringency of the wash step following hybridization will also vary. Wash stringency conditions can be defined by salt concentration and temperature. As above, wash stringency can be increased by either lowering the salt concentration or increasing the temperature. For example, the salt concentration for a stringent wash is preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. The temperature for a stringent wash will generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the wash step will occur in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS at 25°C. In a more preferred embodiment, the wash step will occur in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 42°C. In a more preferred embodiment, the wash step will occur in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68°C. Additional variations on these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are disclosed, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0109] By "substantially identical" is meant that a polypeptide or nucleic acid molecule exhibits at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, the sequence will be at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, or even 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0110] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705) BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). The software matches identical or similar sequences by setting a degree of homology for various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within each of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, wherein a probability score between e -3 and e -100 indicate closely related sequences.

[0111] "Subject" means a mammal, including, but not limited to, humans and non-human mammals, such as a bovine, equine, canine, ovine, or feline.

[0112] "Transgene" means a gene that has been artificially inserted into a cell and becomes part of the genome of an organism that develops from that cell. Such a transgene can include a gene that is heterologous (i.e., foreign) to some or all of the transgenic organism, or can represent a gene that is homologous to an endogenous gene of the organism.

[0113] Ranges provided herein are understood to be shorthand for all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0114] As used herein, "treat" and the like refer to reducing or alleviating a disorder and / or symptoms associated therewith. It will be appreciated that, while not precluded, treatment of a disorder or symptoms does not require that the disorder, symptoms, or conditions associated with the disorder or symptoms be completely eliminated.

[0115] As used herein, the term "or" is understood to encompass both exclusive and inclusive examples. As used herein, the terms "a" and "the" are understood to encompass both singular and plural alternatives unless otherwise indicated or otherwise evident from the context.

[0116] As used herein, the term "about" is understood to be within the normal tolerance range of the art, for example, within 2 standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term "about."

[0117] As used herein, the description of a series of chemical groups for any variable in a definition includes the definition of that variable as any single group or as combinations of the listed groups.

[0118] Any composition or method provided herein can be combined with any other composition or method provided herein.

[0119] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this method and composition of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the method and composition of matter, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. BRIEF DESCRIPTION OF DRAWINGS

[0120] Figure 1 Sensory hair cells showing transduction with AAV9-PHP.B CMV-GFP. Left panel is an image of sensory hair cells transduced with AAV9-PHP.B CMV-GFP and stained with Alexa Fluor® 647 conjugated phalloidin. Right panel is an image of sensory hair cells transduced with AAV9-PHP.B CMV-GFP showing 100% inner hair cell uptake of the vector and positivity for green fluorescent protein (GFP).

[0121] Figure 2Images from different mice and injections using AAV9-PHP.B CMV-GFP transduction of sensory hair cells. 100% of inner hair cells and 100% of outer hair cells were transduced and were GFP positive. Very few other cells expressed GFP. We injected the inner ear of four mice. Three of the four mice showed similar expression to that described below, with 100% of sensory hair cells transduced. The fourth showed in Figure 1

[0122] Figure 3 High magnification images from different mice are shown. In this case, the tissue was stained with Myo7a to illuminate the cell bodies of hair cells (blue), and the bundle of this cilium was stained with phalloidin (red). The top panel shows the merge of all three color channels. The middle panel shows GFP (green) expression with 100% of transduced inner hair cells and 100% of transduced outer hair cells. The bottom panel shows Myo7a and phalloidin.

[0123] Figure 4 An illustration of the inner ear is shown, which shows the injection sites used to transduce mice. "P0-P5" and ">P5" refer to injections at post-hearing stages P0 to P4 and at post-hearing stages later than P5, respectively.

[0124] Figures 5A to 5D Comparison of oval window injection and round window membrane (RWM) injection of Anc80-Cmv-eGFP. Figure 5A Confocal images of the cochlear apex transduced with AAV2 / Anc80L65-GFP at P1 via oval window injection. Figure 5B Confocal images of the cochlear apex transduced with AAV2 / Anc80L65-GFP at P1 via RWM injection. Figure 5A The scale bar applies to Figure 5B and represents 0.2 μιη. Figure 5C Series of high magnification (630x) confocal images from the apical (top row), mid- region (middle row) and basal region (bottom row) of the cochlea transduced with Anc80-GFP via oval window injection and RWM injection at P1. The scale bar shown in the top row represents 20 μιη. Figure 5D Graphical comparison of the percentage of eGFP positive inner hair cells (IHC) and outer hair cells (OHC) from image sections following oval window injection (bottom) and RWM injection (top).

[0125] Figures 6A to 6D Comparison of oval window injection and RWM injection of PHP.B-Cmv-eGFP. Figure 6A Confocal images of the cochlear apex transduced with AAV9-PHP.B-CMV-GFP at P1 using the AAV production platform at EPFL Lausanne ​Confocal images of the cochlear apex after oval window injection of AAV9.PHP.B-Cmv-eGFP generated at EPFL. Figure 6A The scale bar applies to Figures 6A to 6C and represents 0.2 μιη. Figure 6B Confocal images of the cochlear apex after oval window injection of AAV9.PHP.B-Cmv-eGFP generated at EPFL. Figure 6C Confocal images of the cochlear apex after RWM injection of AAV9.PHP.B-Cmv-eGFP at P1. Figure 6D Series of 100 μιη confocal images (63x) of the apical, mid and basal regions of the cochlea after RWM or oval window injection of PHP.B-Cmv-eGFP produced at EPFL at P1. Figure 6E Graphical comparison of the percentage of eGFP positive inner (IHC) and outer hair cells (OHC) after oval window and RWM injection with AAV9.PHP.B-CMV-eGFP.

[0126] Figures 7A to 7C Comparison of the efficiency to transduce hair cells with PHP.B and Anc80 at the same titer (3.5E+12 viral genomes / mL). Figure 7A Confocal images of the cochlear apex after oval window injection with PHP.B adeno-associated viral vector (AAV). The scale bar applies to Figure 7A and Figure 7B and represents 0.2 μιη. Figure 7B Confocal images of the cochlear apex after oval window injection with Anc80 AAV. Figure 7C Graphical comparison of the percentage of eGFP positive inner (IHC) and outer hair cells (OHC) after oval window injection with PHP.B AAV (bottom) and Anc80 AAV (top).

[0127] Figure 8A and Figure 8B show that AAV9.PHP.B has a higher specificity than Anc80. Figure 8A Series of confocal images of a mouse cochlea after oval window injection of Anc80-Cmv-eGFP-WPRE at P1 and harvesting at P15. Figure 8A The scale bar applies to Figure 8B and represents 100 μιη. Figure 8BSeries of confocal images of a P1 cochlea injected with PHP.B-Cmv-eGFP via the oval window and harvested at P15.

[0128] Figure 9A and Figure 9B Example of PHP.B-Cmv-eGFP targeting inner and outer hair cells at postnatal and mature stages. Figure 9A Series of 100 pm confocal images (63x) of the apical, mid and basal regions of the cochlea after oval window injection with PHP.B-GFP at P7 and P16. The scale bar present in the uppermost image applies to all images in Figure 9A Figure 9A and represents 20 pm. Figure 9B Graphical comparison of the percentage of eGFP positive inner and outer hair cells counted from Figure 9A images.

[0129] Figures 10A to 10C Example of hair cell transduction in vivo in wild type mice is not affected by injection of AAV9-Php.b-CMV-GFP at P1. Figure 10A Series of graphs illustrating representative current families of sensory transduction currents elicited by mechanical deflections of the stereociliary bundle from a GFP positive outer hair cell at P7 (lower graph), a GFP positive inner hair cell at P7 (middle graph) and a GFP positive inner hair cell at P29 (upper graph). Figure 10B Current-displacement plot of transduction currents from P7 outer hair cells (OHCs) of GFP negative and GFP positive cells. Figure 10C Graph showing peak transduction currents from GFP positive cells with amplitudes similar to WT cells (Landegger et al., Nat. Biotech, 2017).

[0130] Figures 11A to 11C Example of auditory brainstem recording (ABR) thresholds and distortion product otoacoustic emission (DPOAE) thresholds are not affected by injection of PHP.B-Cmv-eGFP at P1, P7 and P16. Figure 11 shows several panels of graphs illustrating ABR and DPOAE thresholds measured at about P30 in un-injected WT mice (black dashed lines) and mice injected with Anc80-Cmv-eGFP (light grey) or AAV9-Php.b-Cmv-eGFP (dark grey) at P1 Figure 11A ), P7 Figure 11B ) or P16 Figure 11C .

[0131] Figure 12A and Figure 12B ​For example, compared with Anc80-Cmv-eGFP, PHP.B-Cmv-eGFP injected at P1, P7, and P16 showed a higher transduction rate in vestibular hair cells. Figure 12A A series of confocal images of the utricle, horizontal crest, and anterior crest after injection of PHP.B-Cmv-eGFP (top row) and Anc80-Cmv-eGFP (bottom row) via the utricle at P1, P7, and P16. Figure 12B A series of confocal images of the bladders after injection of PHP.B-Cmv-eGFP (top row) and Anc80-Cmv-eGFP (bottom row) into the utricle at P1, P7, and P16. Figure 12A The scale bar is applied to all images and represents 100 μm.

[0132] Figure 13 For a series of images, compare vestibular transduction after injection of PHP.B-Cmv-eGFP (top row) and Anc80-Cmv-eGFP (bottom row) via the utricle at P1, P7, and P16. The scale bar for the first group is applied to all images and is expressed in 100 μm.

[0133] Figure 14 A series of confocal images illustrating frozen sections of mouse vestibular tissue expressing a vector driven by utricle injection. The scale bar for the first set is applied to all images and indicates 200 μm.

[0134] Figure 15 A series of confocal images of cryosections of the cochlea (top row, scale bar 100 μm), cryosections of the vestibule (middle row, scale bar 200 μm), and fully sealed dissections (bottom row, scale bar 100 μm) from C57 mice injected via the utricle at P1 with PHP.B-Syn-eGFP.

[0135] Figure 16A and Figure 16B The results showed that AAV9-PHP.B-Cmv-TMC1 restored the auditory function of TMC1 mutant mice. Figure 16A The following graph illustrates an auditory brainstem response (ABR) threshold that is superior to that of AAV1-CMV-TMC1 or Anc80-CMV-TMC1. Single channels (without error bars) are transduced using PHP.B-CMV-TMC1 and have thresholds similar to wild-type (WT). Figure 16B The figure shows the aberration product otoacoustic emission (DPOAE) thresholds of five mice injected with AAV9-PHP.B-Cmv-Tmc1.

[0136] Figures 17A to 17D Examples of the ability of different promoters to drive transgene expression in the inner ear.Figure 17A A series of confocal images illustrating the ability of the Pcdh15 promoter to drive transgene expression in inner and outer hair cells. Figure 17B A confocal image showing the ability of the Myo6 promoter to drive transgene expression in inner and outer hair cells. Figure 17C An image showing the ability of the Myo7a promoter to drive transgene expression in inner and outer hair cells. Figure 17D A series of confocal images showing the ability of the KCNQ4 promoter to specifically drive transgene expression in outer hair cells. DETAILED DESCRIPTION

[0137] The present disclosure provides compositions and methods for delivering and expressing in a subject's inner ear cells, such as cochlear cells (e.g., inner hair cells or outer hair cells), a protein required for mechanosensation, including hearing and / or vestibular function (e.g., MC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCNQ4, TMPRSS3, STRC, EYA4, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c), OTOF, GPR98, MY06, MY015A, LOXHD1, POU3F4, EYA1, WFS1, ACTG1, TMIE, PJVK, SYNE4, and FAM65B), wherein the level of activity of the protein in the subject is lost or decreased.

[0138] The present disclosure is based, at least in part, on the discovery that an adeno-associated viral vector, AAV-PHP.B, which encodes a capsid comprising the 7-mer sequence TLAVPFK, is extremely efficient and specific for expressing a protein of interest in inner and outer hair cells of the inner ear.

[0139] AAV-PHP.B

[0140] AAV-PHP.B vectors were generated using a Cre recombination-dependent approach to selectively restore the capsid to a predefined Cre-expressing target cell population, also known as CREATE. This approach and vectors useful in the methods of the present application are described by Deverman et al. in Nat Biotechnol. 2016 February; 34(2): 204-209, entitled "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain" and U.S. Patent Publication No. 20170166926, each of which is incorporated by reference herein in its entirety. A library of AAV variants was generated by inserting a 7-amino acid random sequence (7-mer) between AA588 and 589 of the AAV9 capsid (VP1 position). AAV-PHP.B encodes the 7-mer sequence TLAVPFK and was tested for its ability to effectively deliver a transgene to the cochlea, and was effective if it showed clear specificity and robust expression in inner and outer hair cells.

[0141] Usher Syndrome

[0142] Human Usher Syndrome (USH) is a rare genetic condition that causes both deafness and blindness. As an autosomal recessive trait, it affects 16,000 to 20,000 people in the United States and is the cause of 3% to 6% of early childhood deafness. Usher syndrome is divided into three clinical subtypes (USH-1, USH-2, and USH-3) depending on the severity of symptoms. USH1 is the most severe form. Patients affected by USH1 suffer from congenital bilateral profound sensorineural hearing loss, vestibular reflex loss, and prepubertal retinitis pigmentosa (progressive, bilateral, symmetric degeneration of retinal rod-cone cell function). Unless equipped with a cochlear implant, individuals typically do not develop the ability to speak. Although there is currently no biological treatment for Usher Syndrome patients, early reintroduction of a wild-type version of the defective gene can potentially reverse the disease.

[0143] Six Usher genes are associated with USH1 : MY07A (myosin 7a), USH1C (harmonin), CDH23 (cadherin 23), PCDH15 (pre-cadherin 15), SANS (sans), and CIB2 (calcium- integrin binding protein 2). These genes encode proteins involved in morphogenesis of the stereociliary bundle in the inner ear and as part of an interacting group (see, e.g., Mathur & Yang, 2015, Biochim. Biophys. Acta, 1852:406-20). Harmonin resides at the center of the USH1 interacting group, where it binds to other Usher1 proteins. Because of its PDZ (PSD-59 95 / Dlg / ZO-1) interaction domain, harmonin has been proposed to function as a scaffold protein. In vitro binding studies have shown that all other known USH1 proteins bind to the PDZ domain of harmonin, as do two USH2 proteins, usherin and VLGR1. The USH1C gene consists of 28 exons, which encode 10 alternatively spliced forms of harmonin, and is divided into three different subclasses (a, b, and c) depending on the composition of the protein's domains. The three isoforms differ in the number of PDZ protein-protein interaction domains, coiled-coil (CC) domains, and proline-serine-threonine (PST)-rich domains.

[0144] The USH1 proteins are also localized in the mechanosensory stereociliary bundles at the apical end of the hair cells, which are composed of hundreds of stereocilia that are interconnected by a large number of extracellular links. Cadherin 23 and pre-cadherin 15 are the products of the Usher genes USH1D and USH1E, respectively, and form the tip links located at the distal end of the stereocilia. Harmonin-b binds to CDH23, PCDH15, F-actin, and itself. It is found at the tips of the stereocilia close to the insertion point of the inner tip links in the hair cells and is thought to play a functional role in the transduction and adaptation of the hair cells. Harmonin-b is expressed in the early postnatal stage, but its expression in both the cochlea and the vestibule decreases around postnatal day 30 (P30). Harmonin-a also binds to cadherin 23 and is found in the stereocilia. A recent report revealed an additional role for harmonin-a at the synapse, where harmonin-a associates with Cav1.3 Ca 2+ The channel is associated with a ubiquitin-dependent pathway that limits the availability of the channel.

[0145] Over the past 10 years, several mouse models for Usher syndrome have been identified and engineered, seven of which affect harmonin. Of these models, only one, the Ush1c c.216G>A model, recapitulates both the auditory and retinal defects that are characteristic of human Usher syndrome. Ush1c c.216G>A is a knock-in mouse model that affects expression of all traditional harmonin isoforms due to a point mutation analogous to that seen in a French-Acadian USH1C patient cohort. This mutation introduces a cryptic splice site at the end of exon 3 of the Ush1c gene. Use of this cryptic splice site generates a frameshift transcript with a 35 bp deletion and results in translation of a severely truncated protein that lacks the PDZ, PST, and CC domains. Homozygous c.216AA knock-in mice suffer from severe hearing loss by 1 month of age, while heterozygous c.216GA mice do not have any abnormal phenotypes. Cochlear histology of c.216AA mice shows disorganized hair bundle, abnormal cell rows, and loss of both inner and outer hair cells in the mid- and basal turns at P30.

[0146] It is shown herein that the AAV9-PHP.B vector successfully transduces hair cells and drives expression of a protein of interest (i.e., GFP) within the hair cells. As such, this vector can be used to deliver other proteins of interest to hair cells for the treatment of Usher syndrome and other hearing impairments.

[0147] TMC1 / TMC2

[0148] Over 40 distinct mutations have been identified in TMC1 that cause deafness. They are classified as 35 recessive mutations and 5 dominant mutations. Most recessive mutations cause severe congenital hearing loss (e.g., DFNB7 / 11), but some also cause moderate to severe hearing loss that is late-onset. All dominant mutations cause progressive hearing loss (e.g., DFNA36) and onset in the middle of the teenage years. In particular, a non-mutant (e.g., wild-type) TMC1 sequence or a TMC2 sequence can be delivered using the AAV9-PHP.B vector as described herein to prevent hearing loss (e.g., further hearing loss) and / or restore hearing function.

[0149] Therapeutic strategies for treating hearing loss

[0150] Due to the lack of self-repairing ability of sensory cells in the adult mammalian cochlea, current therapeutic strategies (depending on the level and exact location of the lesion) rely on amplification (hearing aids), better sound propagation (middle ear prostheses / active implants), or direct neural stimulation (cochlear implants) to compensate for permanent damage of primary sensory hair cells or spiral ganglion neurons, which form the auditory nerve and convey acoustic information to the brain. Although these approaches have been revolutionary, they are far from optimal in restoring the complex human hearing function that is so important for modern life. In particular, major problems still include limited frequency selectivity, sound not sounding normal, and limited speech recognition in noisy environments.

[0151] Therapeutic genes delivered to the cochlea have been considered as a further improvement over current standards of care ranging from age- and environmentally induced hearing loss to genetic forms of deafness. Over 300 loci have been linked to genetic hearing loss, and over 70 disease-causing genes have been described (Parker & Bitner-Glindzicz, 2015, Arch. Dis. Childhood, 100:271-8). Success of these approaches in therapy significantly depends on safe and efficient delivery of exogenous gene constructs to the therapeutic cell targets in the organ of Corti in the cochlea.

[0152] The organ of Corti comprises two types of sensory hair cells: inner hair cells, which transduce mechanical information carried by sound into electrical signals that are propagated to neuronal structures; and outer hair cells, which serve to amplify and tune the cochlear response, a process required for complex hearing function. Other potential targets in the inner ear include spiral ganglion neurons, pillar cells of the spiral limbus, which are important for maintaining adjacent tectorial membrane or supporting cells, which have a protective function and can be triggered to trans-differentiate into hair cells until the early neonatal stage.

[0153] Injection into the cochlear duct, which is filled with high-potassium endolymph, provides direct access to hair cells. However, this change in the delicate fluid environment can disturb the endocochlear potential, increasing the risk of injection-related toxicity. The space surrounding the cochlear duct, scala tympani, and scala vestibuli, which is filled with perilymph, can be reached from the middle ear or through the round window membrane round window membrane is the only non-bony opening into the inner ear, can be reached relatively early in various animal models, and viral vector administration using this route is well tolerated. In humans, cochlear implant placement typically relies on insertion of a surgical electrode through the RWM.

[0154] Previous studies evaluating AAV phenotypes in organotypic cochlear explants as well as in in vivo inner ear injections have shown only partial repair in congenital deaf mouse models. Surprisingly, the AAV9-PHP.B vector transduces hair cells with high efficiency. This finding overcomes the low transduction rate that has limited the successful development of cochlear gene therapy using traditional AAV serotypes. The AAV9-PHP.B vector as described herein provides a valuable platform for delivering inner ear genes to inner and outer hair cells and other array of inner ear cell types that are affected by genetic hearing and balance disorders.

[0155] The AAV9-PHP.B vector provides high efficiency delivery of nucleic acids encoding proteins of interest. In particular, the present disclosure provides AAV9-PHP.B vectors comprising one of the following promoters: Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNQ4 promoter, Myo7a promoter, Synapsin promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and TMHS (LHFPL5) promoter. In particular embodiments, the promoter directs expression of a polynucleotide encoding one or more of TMC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCNQ4, TMPRSS3, STRC, EYA4, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c), OTOF, GPR98, MY06, MY015A, LOXHD1, POU3F4, EYA1, WFS1, ACTG1, TMIE, PJVK, SYNE4, and FAM65B in cells, particularly inner ear cells, e.g., cells of the cochlea (or cells of the cochlea or cochlear cells). As used herein, inner ear cells refer to, without limitation, inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, vestibular ganglion neurons, and supporting cells. Supporting cells refer to non-excitable cells within the ear, e.g., cells other than hair cells or neurons. An example of a supporting cell is a Schwann cell.

[0156] Delivery of one or more nucleic acids described herein to inner ear cells can be used to treat any congenital or acquired hearing disorder, which are typically defined by partial hearing loss or complete deafness. The methods described herein can be used to treat hearing disorders such as, without limitation, recessive deafness, dominant deafness, Usher syndrome, and other symptomatic deafness, as well as hearing loss due to trauma or aging.

[0157] Methods of making viruses carrying specific transgenes

[0158] As described herein, AAV-PHP.B is particularly effective in delivering nucleic acids (e.g., a polynucleotide encoding one or more of TMC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCN04, TMPRSS3, STRC, EYA4, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c)) into cells of the inner ear. AAV-PHP.B vectors advantageously transduce more than about 60%, 70%, 80%, 90%, 95%, or even 100% of inner or outer hair cells.

[0159] In particular embodiments, AAV-PHP.B vectors have native or engineered hair cell tropism. In some embodiments, AAV9-php.b delivers a transgene (e.g., a polynucleotide encoding one or more of TMC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCN04, TMPRSS3, STRC, EYA4, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c), OTOF, GPR98, MY06, MY015A, LOXHD1, POU3F4, EYA1, WFS1, ACTG1, TMIE, PJVK, SYNE4, and FAM65B) to the inner ear of a subject.

[0160] In one embodiment, AAV-PHP.B vectors using promoters (e.g., Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNQ4 promoter, Myo7a promoter, Synapsin promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and TMHS (LHFPL5) promoter) comprising a polynucleotide expressing one or more of TMC1, TMC2, MY07A, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCNQ4, TMPRSS3, STRC, EYA4, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c), OTOF, GPR98, MY06, MY015A, LOXHD1, POU3F4, EYA1, WFS1, ACTG1, TMIE, PJVK, SYNE4, and FAM65B are used to treat hearing disorders. Nucleic acid sequences delivered to a cell for expression purposes are often referred to as transgenes. Representative transgenes that can be delivered to and expressed in cells of the inner ear include, without limitation, transgenes encoding polypeptides that function in auditory and / or vestibular mechanosensation (e.g., TMC1, TMC2, MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 (e.g., harmonin-a, harmonin-b, or harmonin-c), OTOF, GPR98, MY06, MY015A, LOXHD1, POU3F4, EYA1, WFS1, ACTG1, TMIE, PJVK, SYNE4, and FAM65B), KCNQ4, TMPRSS3, STRC, EYA4, transgenes encoding neurotrophic factors (e.g., GDNV, BDNF, or HSP70).

[0161] Expression of the transgene can be directed by the native promoter of the transgene (i.e., the promoter found naturally with the transgene coding sequence) or expression of the transgene can be directed by a heterologous promoter (e.g., Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNQ4 promoter, Myo7a promoter, synapsin promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and TMHS (LHFPL5) promoter). For example, any of the transgenes described herein can be used with its native promoter. Alternatively, any of the transgenes described herein can be used with a heterologous promoter. As used herein, a heterologous promoter refers to a promoter that does not naturally direct expression of the sequence (i.e., is not found naturally in the sequence). Representative heterologous promoters that can be used to direct expression of any of the transgenes referred to herein include, for example, a CMV promoter, a CBA promoter, a CASI promoter, a P promoter, an EF-1 promoter, an alpha9 nicotinic receptor promoter, a prestin promoter, a Gfil promoter, and a Vglut3 promoter. In addition, a promoter that naturally directs expression of one of the above transgenes (e.g., a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, or an Atohl promoter) can be used as a heterologous promoter to direct expression of a transgene. In other embodiments, the promoter is an Espin promoter, a PCDH15 promoter, a PTPRQ promoter, a Myo6 promoter, a KCNQ4 promoter, a Myo7a promoter, a synapsin promoter, a GFAP promoter, a CMV promoter, a CAG promoter, a CBH promoter, a CBA promoter, a U6 promoter, or a TMHS (LHFPL5) promoter.

[0162] Methods of making AAV-PHP.B vectors for packaging a transgene (e.g., TMC1, TMC2, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c), MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCNQ4, TMPRSS3, STRC, EYA4) within an AAV-PHP.B vector are known in the art and use traditional molecular biology techniques and recombinant nucleic acid techniques.

[0163] A transgene can be packaged within an AAV-PHP.B vector using, for example, a packaging host cell. One or more of the components of the viral particle (e.g., a rep sequence, a cap sequence, an inverted terminal repeat (ITR) sequence) can be transiently or stably introduced into the packaging host cell using one or more of the constructs described herein.

[0164] Generally, as used herein, "nucleic acid" can include DNA and RNA, and can also include nucleic acids containing one or more nucleic acid analogs or backbones modified. Nucleic acids can be single-stranded or double-stranded, generally depending on the use. Nucleic acids useful in the methods described herein can be identical to known nucleic acid sequences, or nucleic acids useful in the methods described herein can differ in sequence from known sequences. For example, a nucleic acid (or encoded polypeptide) can be at least 75% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a known sequence.

[0165] In calculating percent sequence identity, the two sequences are aligned and the number of identical matches between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of nucleotides or amino acid residues that are aligned) and multiplied by 100 to yield the percent sequence identity. It will be appreciated that the length of the aligned region can be a portion of one sequence or both sequences to the full length of the shortest sequence. It will also be appreciated that a single sequence can be aligned with more than one other sequence, and thus can have different percent sequence identity values within different aligned regions.

[0166] An alignment of two or more sequences to determine percent sequence identity is performed using the computer program ClustalW with default parameters, which allows for alignment over the entire length of the nucleic acid or polypeptide sequences to be aligned (global alignment). Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW calculates the best fit between the query sequence and one or more test sequences and aligns them to determine identity, similarity, and difference. Gap(s) of one or more residues can be inserted into the query sequence, the test sequence(s), or both, to maximize sequence alignment. For pairwise alignment of nucleotide sequences, the default parameters are used (i.e., word length: 2; window length: 4; scoring method: percent; top diagonal line: 4; gap penalty: 5); for alignment of multiple nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; weight transition: yes. For pairwise alignment of polypeptide sequences, the following parameters are used: word length: 1; window length: 5; scoring method: percent; top diagonal line: 5; gap penalty: 3. For multiple alignment of polypeptide sequences, the following parameters are used: weight matrix: BLOSUM (BLOcks SUbstitution Matrix); gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gap: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; and, residue specific gap penalties: on. ClustalW is available on the internet at the Baylor College of Medicine Search Launcher website or at the European Bioinformatics Institute website, for example.

[0167] Changes can be introduced into the nucleic acid sequence, which, if the nucleic acid is a coding sequence, can result in changes to the amino acid sequence of the encoded polypeptide. Changes can be introduced into the nucleic acid coding sequence, for example, using mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis) or by chemically synthesizing the nucleic acid molecule with such changes. Such nucleic acid changes can result in conservative and / or non-conservative amino acid substitutions at one or more amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain (see, e.g., Dayhoff et al. (1978) Atlas of Protein Sequence and Structure, 5 (Suppl. 3): 345-352), which provides a codetable of amino acid substitutions, while a non-conservative substitution is one in which the amino acid residue is replaced with an amino acid residue that does not have a similar side chain.

[0168] The nucleic acid can be contained in a construct, which can also be referred to as a vector or plasmid. Constructs can be purchased commercially, or can be produced by routine recombinant techniques in the art. Constructs containing nucleic acids can have expression elements that direct and / or modulate expression of the nucleic acid, and can also include sequences such as those for maintenance of the construct (e.g., origin of replication, selectable marker). Expression elements are known in the art and include, for example, promoter sequences, intron sequences, enhancer sequences, response elements, or inducible elements.

[0169] Pharmaceutical compositions

[0170] AAV-PHP.B vectors comprising a promoter (e.g., Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNO4 promoter, Myo7a promoter, synapsin promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and TMHS (LHFPL5) promoter) and a polynucleotide that is one or more of USH1, MY07A, USH1C (harmonin-a, harmonin-b, harmonin-c), CDH23, PCDH15, SANS, and CIB2, typically suspended in a physiologically compatible excipient, can be administered to a subject (e.g., a human or non-human mammal) by injection into the inner ear of the subject via the round window or oval window. Suitable vehicles include saline, which can be formulated using a variety of buffered solutions (e.g., phosphate buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, and water. The AAV-PHP.B vectors are administered in sufficient amounts to transduce or infect the cells and provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue side effects.

[0171] The dosage of the AAV-PHP.B vectors administered to a subject will depend primarily on factors such as the condition to be treated and the age, weight, and health of the subject. For example, a therapeutically effective dose of the AAV-PHP.B vectors to be administered to a human subject is typically in the range of about 0.1 ml to 10 ml of solution, where the solution contains about 1 x 10 12 AAV concentration of genomic copies (GC) (e.g., about 1 x 10 3 to 1 x 10 9 GC).

[0172] Methods of delivering nucleic acids to cells of the inner ear

[0173] Methods of delivering nucleic acids to cells are generally known in the art, and methods of delivering viruses (which are also referred to as viral particles) containing transgenes to cells of the inner ear in vivo are described herein. As described herein, about 10 8 to 10 12 viral particles can be administered to a subject, and the viruses can be suspended in a suitable volume (e.g., 10 μΐ, 50 μΐ, 100 μΐ, 500 μΐ, or 1000 μΐ) of, for example, artificial perilymph fluid.

[0174] Any means can be used to deliver viruses containing promoters (Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNQ4 promoter, Myo7a promoter, Synapsin promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and TMHS (LHFPL5) promoter) and transgenes (e.g., TMC1, TMC2, USH1C (e.g., harmonin-a, harmonin-b, or harmonin-c), MY07A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, KCNQ4, TMPRSS3, STRC, EYA4, OTOF, GPR98, MYO6, MYO15A, LOXHD1, POU3F4, EYA1, WFS1, ACTG1, TMIE, PJVK, SYNE4, and FAM65B) to cells of the inner ear (e.g., cells in the cochlea) as described herein. For example, a therapeutically effective amount of a composition comprising viruses containing one or more different types of transgenes described herein can be injected through the round window or oval window or the utricle typically in a relatively simple (e.g., outpatient) procedure. In some embodiments, a therapeutically effective amount of viral particles containing transgenes or a composition containing one or more sets of different viral particles as described herein can be delivered to the appropriate location in the ear during a surgical procedure (e.g., a cochleostomy or a tympanostomy), where each particle in a set of viral particles can contain the same type of transgene, but each set of particles contains a different type of transgene than the other sets.

[0175] In some embodiments, an AAV-PHP.B vector comprising a promoter (e.g., an Espin promoter, a PCDH15 promoter, a PTPRQ promoter, a Myo6 promoter, a KCNO4 promoter, a Myo7a promoter, a synapsin promoter, a GFAP promoter, a CMV promoter, a CAG promoter, a CBH promoter, a CBA promoter, a U6 promoter, or a TMHS (LHFPL5) promoter) and a polynucleotide that is one or more of USH1, MY07A, USH1C (harmonin-a, harmonin-b, harmonin-c), CDH23, PCDH15, SANS, and CIB2 is injected into a subject in need thereof through the round window or the cochlear round window sac.

[0176] Further, delivery vehicles (e.g., polymers) that facilitate the transfer of an agent across the tympanic membrane and / or through the round window or oval window are available, and any such delivery vehicles can be used to deliver the viruses described herein. See, e.g., Arnold et al., 2005, Audiol. Neurootol., 10:53-63.

[0177] The compositions and methods described herein enable efficient delivery of nucleic acids to cells of the inner ear, such as cells of the cochlea. For example, the compositions and methods described herein are capable of delivering a transgene to and expressing in at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of inner hair cells, or delivering to and expressing in at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of outer hair cells.

[0178] As demonstrated herein, expression of a transgene delivered using an AAV-PHP.B vector can result in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons (e.g., Atoh1, NF2), such that hearing or vestibular function is restored for a long period of time (e.g., months, years, decades, or a lifetime).

[0179] Kit

[0180] The present application also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the present application (e.g., inner ear hair cell targeted AAVs (e.g., KCNQ4 vectors) comprising a promoter (e.g., CMV, Espin, PCDH15, PTPRQ, TMHS (LHFPL5)) and a polynucleotide, wherein the polynucleotide is one or more of USH1, MY07A, USH1C (harmonin-a, harmonin-b, harmonin-c), CDH23, PCDH15, SANS, and CIB2). Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the

[0181] The present application also provides a kit for treating or preventing a disease or pathology (or symptom) associated with a defect in hearing and / or vestibular mechanoreception. In one embodiment, the kit comprises an effective amount of a unit dosage form of an AAV-PHP.B vector comprising a promoter (e.g., Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNO4 promoter, Myo7a promoter, Synapsin promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and TMHS (LHFPL5) promoter) and a polynucleotide (which is one or more of USH1, MY07A, USH1C (harmonin-a, harmonin-b, harmonin-c), CDH23, PCDH15, SANS, and CIB2), and instructions for administering the AAV-PHP.B vector to a subject suffering from or susceptible to a disease or pathology associated with hearing impairment or a symptom thereof. In preferred embodiments, the kit comprises a sterile container that houses the AAV-PHP.B vector; such container can be a box, ampule, bottle, vial, tube, bag, pack, or other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. The instructions typically will include information on the use of the AAV-PHP.B vector for treating a disease or pathology associated with hearing impairment or a symptom thereof. The instructions can be printed directly on the container (when present), or as a label accompanying the container, or as a separate sheet, pamphlet, card, or folder supplied with the container.

[0182] Conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques which are well within the sphere of skill in the art can be used in accordance with the present disclosure. Such techniques are explained fully in the literature. The application will be further described in the following examples which do not limit the scope of the methods and compositions of matter described in the claims.

[0183] [Examples]

[0184] Example 1: AAV-PHP.B vector directs transgene expression in outer and inner hair cells

[0185] In vivo injections

[0186] P0 to P2) with AAV-PHP.B vector CMV GF via the round window membrane using a beveled glass microinjection needle. The needle was pulled from a capillary glass tube (WPI) on a P-2000 needle puller (Sutter Instruments, Novato, CA) and a bevel (~20 pm tip diameter, 28° angle) was cut using a micro-needle beveler (Sutter Instrument, Novato, CA). EMLA cream (2.5% lidocaine and 2.5% prilocaine) for analgesia was applied externally using a sterile swab to cover the surgical site (left-sided mastoid process). Prior to surgery, body temperature was maintained on a 38 °C warming pad. Anesthesia was induced by rapidly lowering the pups in ice / water for 2 to 3 minutes until unconscious and this state was maintained on a cooling platform for 5 to 10 minutes during surgery. The surgical site was sterilized using an iodine (Betadine) brush and wiped with 70% ethanol, repeated three times. A post-auricular incision was made to expose the transparent bulla, the micro-needle was manually advanced through the bulla and overlying fascia, and the RWM was penetrated by the tip of the micro-needle. Approximately 1 pL of virus was unilaterally injected into the left ear of C57BL / 6 animals over 1 minute, manually. Following injection, the skin incision was closed using 6-0 black monofilament suture (Surgical Specialties, Wyomissing, PA). The pups were then returned to a 38 °C warming pad for 5 to 10 minutes before being returned to their mother for rearing.

[0187] AAV-PHP.B vectors transduced nearly 100% of IHC and 100% of OHC Figures 1 to 3 with very few non-hair cells being transduced.

[0188] Next, samples transduced with AAV-PHP.B CMV GFP vector were fixed, stained with either phalloidin or Myo7, and imaged by confocal microscopy. Outer and inner hair cell targeting exemplified efficient transduction.

[0189] Example 2: Hair cell electrophysiology

[0190] Following transduction with AAV-PHP.B vectors containing transgenes encoding genes of interest, the electrophysiology of hair cells was analyzed. Cochleae were excised, mounted on a coverslip, and viewed using an Axio Examiner.A1 upright microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 63x water immersion objective and differential interference contrast optics. Electrophysiological recordings were performed at room temperature (22°C to 24°C) in standard solutions containing (in mM): 137 NaCl, 5.8 KCl, 10 HEPES, 0.7 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, and 5.6 D-glucose, vitamins (1:100), and amino acids (1:50) in MEM (Life Technologies, Carlsbad, CA) (pH 7.4; ~310 mOsm / kg). Recording electrodes (3 to 4 MΩ) were pulled from R-6 glass (King Precision Glass, Claremont, CA) and filled with intracellular solution containing (in mM): 140 CsCl, 5 EGTA-KOH, 5 HEPES, 2.5 Na2ATP, 3.5 MgCl2, and 0.1 CaCl2(pH 7.4; ~280 mOsm / kg). Whole-cell currents were recorded using a high-impedance seal patch-clamp technique using an Axopatch 200B (Molecular Devices, Sunnyvale, CA). Hair cells were held at -84 mV. Currents were filtered at 5 kHz using a low-pass Bessel filter, digitized at >20 kHz using a 12-bit acquisition board (Digidata 1440A, Molecular Devices, Sunnyvale, CA), and recorded using pCLAMP 10 software (Molecular Devices, Sunnyvale, CA). The hair bundle of IHCs and OHCs was deflected using a hard glass probe mounted on a PICMA chip piezoelectric actuator (Physik Instrumente, Karlsruhe, Germany), driven by an LVPZT amplifier (E-500.00, Physik Instrumente, Karlsruhe, Germany), and filtered at 40 kHz using an 8-pole Bessel filter (Model 3384 filter, Krohn-Hite Corporation, Brockton, MA) to minimize residual pipette resonance. The hard glass probe was designed to fit just inside the concave surface of the array of stereocilia of the hair cell for whole bundle recordings (3 to 4 pm in diameter for OHCs; 4 to 5 pm in diameter for IHCs).For whole-cell electrophysiology recordings at >P10, cochlear tissue was excised at P5 to P7 and incubated in MEM (1X) + GlutaMAX™-I medium with 1% FBS at 37°C and 5% C02 for up to 30 days.

[0191] Example 3: Hearing test

[0192] Hearing was also assessed after transduction in mice with a genetic hearing defect. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) data were collected. DPOAE is a test of appropriate cochlear amplification and tuning and is a sensitive measure of outer hair cell viability. Stimulations tested in anesthetized mice varied between 10 and 90 dB sound pressure levels at frequencies of 5.6, 8, 11.3, 16, 22.6, and 32 kHz. The minimum sound threshold required to elicit an ABR was plotted.

[0193] Example 4: Rotarod test

[0194] Mice were tested on a rotarod apparatus for balance behavior. Mice with known vestibular function impairment have great difficulty completing the rotarod test. Previous studies highlighted the ability of this rotarod test to detect balance dysfunction when one ear is affected. Mice injected at P1 were tested at P36, while non-injected control mice were tested at P79. All mice were tested using the following rotarod protocol. On the first day, mice were balance trained for five minutes on a rotarod turning at four RPM. On the second day, mice were tested five times, with five minutes between each test. For each test, the speed of the rod was increased by 1 RPM from an initial rate of 2 RPM. The time (in seconds) until the mouse fell off the apparatus was recorded.

[0195] Since the perilymph of the cochlea is continuous with the perilymph of the vestibular labyrinth, it was evaluated whether AAV-PHP.B vectors expressing a protein of interest via cochlear RWM injection would transduce the vestibular sensory organs. Therefore, to address the safety concern that AAV-PHP.B vector transduction could affect balance, vestibular function rotarod tests were performed on injected mice for which expression had been demonstrated in the vestibular apparatus, relative to non-injected control mice.

[0196] Example 5: Mouse model of Usher syndrome

[0197] Tissue preparation

[0198] Oval window and Corti's organ were collected from Ushlc c.216G>A heterozygous or homozygous mutant mice for electrophysiological studies between postnatal day 0 and 8 (P0 to P8). Postnatal pups were killed by rapid decapitation. The temporal bones were excised and immersed in MEM (Invitrogen, Carlsbad, CA) supplemented with 10 mM HEPES (pH 7.4). The Corti's organ was dissected off without the use of enzymes as previously described (53). The oval window was removed after a 10 min treatment with protease (Protease XXIV, Sigma) at 0.1 mg / ml. Excised organs were mounted on a circular coverslip. A pair of thin glass fibers previously glued to the coverslip were placed on the tissue edges to stabilize it in a flat position. Tissues were used promptly or stored in culture in the presence of 1% fetal calf serum. Cultures were maintained for 7 to 8 days, changing the medium every 2 to 3 days in order to perform experiments involving in vitro viral vector infection.

[0199] Animals

[0200] Ushlc c.216G>A knock-in mice were obtained from the LSU Health Sciences Center. The Cdh23 (Ahl) mutation, which causes age-related hearing loss, was introduced in the C57BL6 background (48, 49) and bred in advance. Mice were genotyped using toe clips (before P8) or ear tags (after P8) and PCR was performed as previously described (32). For all studies, male and female mice were used in approximately equal proportions. No additional randomization was applied.

[0201] Round window membrane (RWM) injection

[0202] AAV-PHP.B vectors expressing the gene of interest under the selected promoter were generated. 0.8 to 1 μΐ of AAV vector was injected in the newborn mice at P0 to P1 and P10 to P12. P0 to P1 mice were first anesthetized using hypothermia exposure while P10 to P12 mice were anesthetized using isoflurane. After anesthesia, a postauricular incision was made to expose the bulla and make the cochlea visible. Injections were performed through the RWM using a glass micropipette controlled by a micromanipulator (Askew et al. 2015). The volume of injected material was controlled at approximately 0.02 μΐ / min for 10 min. Standard postoperative care was applied. Sample size for in vivo studies was determined on a continuing basis to optimize the sample size and reduce variance.

[0203] Electrophysiological recordings

[0204] Recordings were performed in standard artificial perilymph fluid containing (in mM): 144 NaCl, 0.7 NaH2PO4, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 5.6 D-glucose, and 10 HEPES-NaOH, adjusted to pH 7.4 and 320 mOsmol / kg. Vitamins (1:50) and amino acids (1:100) from a concentrated stock (Invitrogen, Carlsbad, CA) were added. Hair cells were viewed from the apical surface using an upright Axioskop FS microscope (Zeiss, Oberkochen, Germany) equipped with a 63X water immersion objective with differential interference phase contrast optics. Recording pipettes (3-5 MΩ) were pulled from borosilicate glass capillaries (Garner Glass, Claremont, CA) and filled with intracellular solution containing (in mM): 135 KCl, 5 EGTA-KOH, 10 HEPES, 2.5 K2ATP, 3.5 MgCl2, 0.1 CaCl2, pH 7.4. Currents were recorded at room temperature under whole-cell voltage clamp at a holding potential of -64 mV. Data were acquired using an Axopatch Multiclamp 700A or Axopatch 200A (Molecular devices, Palo Alto, CA) filtered at 10 kHz using a low-pass Bessel filter, digitized at >20 kHz using a 12-bit acquisition board (Digidata 1322) and pClamp 8.2 and 10.5 (Molecular Devices, Palo Alto, CA). Data were analyzed offline using OriginLab software and are presented as mean ± standard deviation unless explicitly noted.

[0205] Example 6: Auditory startle response

[0206] Acoustic startle response (ASR) was measured using a startle monitor (Kinder Scientific). Mice were placed in a small size, non-restrictive, cubic plexiglass recording chamber (27 cm x 10 cm x 652 12.5 cm) fixed on a piezoelectric / plexiglass sensing assembly and allowed to acclimate for 5 minutes under a 60 dB SPL background white noise. Each session consisted of 35 trials during which single noise pulses in a 10 dB SPL range from 60 to 120 db SPL were delivered, with an average of 30 seconds (range 25 to 35 seconds) between each trial. Pulses were arranged in a pseudo-random order and the background noise was constant at 60 dB SPL to limit external noise interference. The startle monitoring system reduced the response to each pulse to the first N, max N and the maximum time in ms of the response in order to calculate the peak startle response (ASR amplitude) and the time from stimulus to peak startle response (ASR latency). ASR was all blind.

[0207] To assess whether ABR / DPOAE recovery resulted in behaviorally relevant auditory function recovery, acoustic startle responses were measured in mice injected with AAV-PHP.B vectors alone and expressing the protein of interest and in mice injected with both vectors. Response recovery was assessed in 6-week-old mice by startle response analysis to white noise.

[0208] Example 7: Immunofluorescence

[0209] Immunostaining was performed to determine the expression profile of the transgene delivered by the AAV-PHP.B vectors. To do so, immunostaining was performed on freshly excised and fixed for 1 h at room temperature using 4% paraformaldehyde diluted in PBS, Corti's organs. The tissue was then rinsed in PBS, permeabilized in 0.01 to 0.1% Triton X-100 for 30 min and counterstained for 1 h using Alexa Fluor 546-Phalloidin (Molecular Probes, 1 :200 dilution) to label filamentous actin.

[0210] For localization of the exogenously expressed TMC::FLAG fusion protein, the tissue was blocked for 1 hour with 2% BSA and 5% normal goat serum, and incubated overnight at 4°C with FLAG motif antibody (BD Biosciences, 1:200 dilution). For hair cell counting, the tissue was blocked in normal goat serum for 1 hour, stained overnight at 4°C with rabbit anti-Myosin VIIa primary antibody (Proteus Biosciences, 1:1000 dilution), and labeled for 1 hour with goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Life Technologies, 1:200 dilution). Samples were mounted on coverslips using Vectashield mounting medium (Vector Laboratories) and imaged at 10X to 63X magnification using a Zeiss LSM700 confocal microscope.

[0211] Example 8: Utricle injection

[0212] Developing novel injection methods to deliver therapeutic carriers to the inner ear. Existing injection methods deliver carriers through the round window membrane, oval window, or posterior semicircular canal. While these methods have some effectiveness, they all suffer from significant drawbacks, including difficulty in surgically reaching the target, uneven viral distribution, and significant differences in targeting within the perilymphatic or endolymphatic spaces. To overcome these limitations, novel methods are needed that allow for effective delivery into the inner ear space without causing auditory or vestibular dysfunction.

[0213] This method involves targeted injection into the utricle, one of the vestibular organs. The injection is administered into the endolymphatic space. Two different delivery routes are used depending on the age of the mice. As illustrated below, between P0 and P5, the utricle is located near the lateral and posterior semicircular canals. Later than P5, the utricle is injected between the round and oval windows. Figure 4 Because the fluid filling the elliptical window space is continuous with other vestibular organs and the cochlea, a significantly improved distribution of the virus was observed throughout the inner ear.

[0214] To compare this novel method with existing methods involving injection into the round window membrane (RWM), P1 mice were injected with AAV2-Anc80L65-GFP into the utricle or RWM. Temporal bone was harvested at 4 weeks of age for imaging. Hair cells were stained using anti-myosin VIIa antibody. (See reference...) Figure 5A and Figure 5B The expression of GFP in the apex of the cochlea of ​​mice that received utricle injection and RWM injection, respectively, was detected. High-resolution images of the apex, middle, and basal regions of the cochlea showed that GFP expression was significantly increased in mice that received utricle injection compared to mice that received RWM injection. Figure 5C and Figure 5D).

[0215] To determine if PHB.B-Cmv-eGFP is efficiently and specifically transducing mice via oval window or RWM injection, mice were injected with AAV9.PHP.B-Cmv-eGFP via oval window or RWM at P1. Referring to Figure 6A and Figure 6B , mice received oval window injections at P1 of viruses produced at the Polytechnique Federale de Lausanne (EPFL) and at Boston Children’s Hospital (BCH) and at P14, the temporal bones of the mice were harvested and efficient and specific transduction was observed from the temporal bones. RWM injections of EPFL viruses were also efficient and specific Figure 6C ). For both oval window injections and RWM injections, similar percentages of transduced cells were observed in the apical, mid and basal regions of the cochlea Figure 6D and Figure 6E

[0216] The AAV9-PHP.B vector transduces mice at a higher rate than the Anc80 vector.

[0217] Mice were injected with AAV9.PHP.B-Cmv-eGFP or AAV-Anc80-Cmv-eGFP via oval window at P1. At P14, the temporal bones were harvested and an increase in transduction rate of PHP.B over Anc80 was observed within the cochlea Figure 7A and Figure 7B ) and within the apical, mid and basal regions of the cochlea Figure 7C , as determined by fluorescent detection.

[0218] To determine if PHP.B has a higher specificity than Anc80, P1 mice were injected with Anc80-Cmv-eGFP-EPRE or PHP.B-Cmv-eGFP via oval window. At P15, the cochleae of the mice were harvested and cross sections were prepared. Referring to Figure 8A and Figure 8B , an increase in specificity of PHP.B was observed, as indicated by the green fluorescence observed in the lower middle panel.

[0219] To determine which developmental stage of inner and outer hair cells PHP.B-Cmv-eGFP targets, mice were injected with PHP.B vectors via oval window at P7 and P16. Referring to Figure 9A ​, increased eGFP-positive cells were observed in mice injected at P7 relative to mice injected at P16. However, PHP.B-Cmv-eGFP targets both inner ear hair cells at postnatal and mature stages Figure 9B

[0220] In wild-type mice, the effect of AAV9-PHP.B-Cmv-GFP injection on hair cell transduction was evaluated. Mice were injected at P1 with the vector via the oval window. Figure 10A Example of representative current families from GFP-positive cells in response to sensory transduction currents elicited by mechanical displacement of the bundle of stereocilia. Figure 10B It is shown that there is no difference in sensitivity between GFP-positive and GFP-negative outer hair cells at P7. Previously, no difference in current amplitudes between GFP-positive and GFP-negative inner and outer hair cells was observed Figure 10C

[0221] Auditory brainstem recording (ABR) and distortion product otoacoustic emission (DPOAE) thresholds were evaluated. Figure 11A ABR and DPOAE thresholds are shown for four non-injected C57 mice tested at P28 to P31 (black dashed lines), four C57 mice injected at P1 via the oval window with Anc80-eGFP and tested at P30 (light gray), and four C57 mice injected at P1 via the oval window with AAV9.PHP.B-Cmv-eGFP prepared from BCH and tested at P29 (dark gray). Reference is made to Figure 6. Figure 11B ABR thresholds (left) and DPOAE thresholds (right) are observed for four non-injected C57 mice tested at P28 to P31 (black dashed lines), four C57 mice injected at P7 via the oval window with Anc80-eGFP and tested at P30 (light gray), and nine C57 mice injected at P7 via the oval window with PHP.B-eGFP (EPFL) and tested at P27 to P28 (dark gray). Reference is made to Figure 7. Figure 11C ABR thresholds (left) and DPOAE thresholds (right) are also observed for four non-injected C57 mice tested at P28 to P31 (black dashed lines), four C57 mice injected at P16 via the oval window with Anc80-eGFP and tested at P31 (light gray), and four C57 mice injected at P16 via the oval window with PHP.B-eGFP (EPFL) and tested at P28 (dark gray). Overall, these data indicate that ABR thresholds and DPOAE thresholds are not affected by injection of AAV9.PHP.B-Cmv-eGFP at P1, P7 or P16. ​​

[0222] The effect of injection timing on PHP.B and Anc80 transduction was compared. C57 mice were injected with AAV9.PHP.B-Cmv-eGFP or Anc80-Cmv-eGFP at Pl, P7, and P16 via the oval window. Reference Figure 12A and Figure 12B PHP.B-Cmv-eGFP had higher transduction in the oval window and utricle than obtained with the Anc80 construct. eGFP expression in the posterior semicircular canal hair cells was also more robust and specific in quality in mice receiving PHP.B-Cmv-eGFP than in mice receiving Anc80-Cmv-eGFP Figure 13

[0223] Viral vectors prepared by different entities were evaluated. C57 mice were injected with Anc80-Cmv-eGFP, PHP.B-Cmv-eGFP prepared at BCH, or PHP.B-Cmv-eGFP prepared at EPFL via the oval window at Pl. Tissues were harvested at P15. Reference Figure 14 PHP.B-Cmv-eGFP, especially the vector prepared at BCH, had higher transduction in the vestibular hair cells than Anc80 vectors.

[0224] Example 9: Neuronal transduction

[0225] To determine whether cochlear and vestibular neurons could be effectively transduced, AAV9-PHP.B was modified to include the synapsin promoter. C57 mice were dosed with PHP.B-Syn-eGFP via the oval window at Pl. Tissues were harvested at P15, and cross-sections and whole-mount preparations were made. Reference Figure 15 Cochlear and vestibular cross-sections and whole-mount preparations demonstrated that AAV-PHP.B-Syn-eGFP had high specificity and efficiency for transducing spiral ganglion and vestibular neurons.

[0226] Example 10: Restoring auditory function

[0227] The ability of PHP.B vectors to drive expression of therapeutic polypeptides was evaluated in homozygous Tmc1 mutant mice in vivo. Mice were injected at Pl, and auditory function was measured at P30. AAV9-PHP.B-Cmv-Tmc1 vectors restored auditory function in mutant mice as measured by ABR and DPOAE thresholds Figure 16A and Figure 16B This vector outperformed AAV1-Cmv-Tmc1 and Anc80-Cmv-Tmc1, and one mouse dosed with AAV9-PHP.B-Cmv-Tmc1 vector performed similarly to wild type Figure 16A ​​

[0228] Example 11: Promoters for expression of transgenes in inner and outer hair cells

[0229] Promoters that drive expression within specific cells and tissues are of particular value for targeted delivery of therapeutic transgenes and minimization of off-target expression. The ability of several promoters to drive expression specifically in vestibular cells was explored. Referring to Figures 17A to 17C , the Pcdh15, myosin 6, and myosin 7a promoters drive expression within inner hair cells and outer hair cells. The KCNQ4 promoter drives expression specifically in outer hair cells Figure 17D .

[0230] Other embodiments

[0231] It is to be understood that while the present application has been described in terms of a method and composition of matter in a number of different aspects, the foregoing disclosure is intended to illustrate, but not limit, the scope of the method and composition of matter. Other aspects, advantages, and modifications are within the scope of the claims that follow.

[0232] Herein, the description of a series of elements in any definition of a variable includes the definition of the variable as any single element or as a combination (or subcombination) of the listed elements. The description of embodiments herein includes the embodiments as any single embodiment or as a combination with any other embodiment or portion thereof.

[0233] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

[0234] Methods and compositions useful for, useful in the production of, or useful or useful as products of the disclosed methods and compositions are disclosed. These and other materials are disclosed herein, and it is understood that where such combinations, subsets, interactions, groups, etc. are disclosed that all possible combinations and permutations of these materials are similarly disclosed. In other words, if a particular composition of matter or a particular method is disclosed, and a number of combinations or permutations of that composition or method are discussed, each and every combination and permutation is specifically contemplated and described herein. Likewise, any subset of these is also specifically contemplated and disclosed.

Claims

1. A vector expressing the TMC1 polypeptide, comprising an AAV9-php.b promoter selected from the group consisting of the Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNQ4 promoter, Myo7a promoter, synaptic protein promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and LHFPL5 promoter.

2. The vector according to claim 1, wherein the vector transduces inner hair cells and outer hair cells with an efficiency of at least 70% or higher.

3. A cell comprising the carrier according to claim 1, wherein the cell is an external hair cell, an internal hair cell, a vestibular hair cell, a spiral ganglion, or a vestibular ganglion.

4. Use of the vector according to claim 1 in the preparation of a medicament for treating inner ear lesions associated with a TMC1 gene defect in a subject.

5. The use according to claim 4, wherein the agent increases, improves or maintains the hearing and / or vestibular function of the subject.

6. Use of the vector according to claim 1 in preparing a medicament for introducing a wild-type form of the TMC1 gene into the outer hair cells, inner hair cells, vestibular hair cells, spiral ganglia, or vestibular ganglia of a subject with a TMC1 gene defect, wherein the TMC1 gene defect is associated with partial hearing loss, complete deafness, partial vestibular dysfunction, or complete vestibular dysfunction.

7. Use of the vector according to claim 1 in the preparation of a medicament for treating a subject with a TMC1 gene defect, wherein the vector comprises a promoter selected from the group consisting of: Espin promoter, PCDH15 promoter, PTPRQ promoter, Myo6 promoter, KCNQ4 promoter, Myo7a promoter, synaptic protein promoter, GFAP promoter, CMV promoter, CAG promoter, CBH promoter, CBA promoter, U6 promoter, and LHFPL5 promoter, wherein the promoter directs the expression of a polynucleotide encoding TMC1, wherein the TMC1 gene defect is associated with partial hearing loss, complete deafness, partial vestibular dysfunction, or complete vestibular dysfunction.

8. The use according to claim 7, wherein the agent improves or maintains the hearing and / or vestibular function of the subject.

9. The use according to claim 8, wherein the increase in hearing function is related to the maintenance of ciliary tract morphology and / or the restoration of mechanotransduction.

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