Myosin 15 promoter and uses thereof

Abstract

To provide a new therapy targeting hair cells for the treatment of sensorineural hearing loss or vestibular dysfunction.SOLUTION: The present disclose provides polynucleotides containing a region of the myosin 15 (Myo15) promoter, as well as vectors containing the same, which can be used to promote expression of a transgene specifically in hair cells. The polynucleotides described herein can be operably linked to a transgene, such as a transgene encoding a therapeutic protein, to facilitate hair cell-specific expression of the transgene. The polynucleotides described herein can be operably linked to a therapeutic transgene and used in the treatment of a subject having or at risk of developing hearing loss or vestibular dysfunction.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on February 7, 2020, is named 51471-002WO4_Sequence_Listing_2.7.20_ST25, and is 26,901 bytes in size.

[0002] Described herein are polynucleotides containing regions of the myosin 15 (Myo15) promoter, as well as vectors containing them, which can be used to drive transgene expression in hair cells (e.g., cochlear hair cells, such as inner and outer hair cells, and / or vestibular hair cells). Also disclosed are methods of using the polynucleotides and vectors of the invention to achieve transgene expression in hair cells for the treatment of hearing loss and / or vestibular dysfunction. [Background technology]

[0003] Hearing loss is a major public health problem estimated to affect nearly 15% of school-age children and one in three people by age 65. The most common type of hearing loss is sensorineural hearing loss, which is a type of hearing loss caused by defects in cells of the inner ear, such as cochlear hair cells, or in the nerve pathways projecting from the inner ear to the brain. Sensorineural hearing loss is often acquired and has a variety of causes, including acoustic trauma, disease or infection, head trauma, ototoxic drugs, and aging. There are also genetic causes of sensorineural hearing loss, such as mutations in genes involved in the development and function of the inner ear. Mutations in over 90 such genes have been identified, including mutations inherited in autosomal recessive, autosomal dominant, and X-linked patterns.

[0004] Factors that interfere with the development, survival, or integrity of cochlear hair cells, such as genetic mutations, disease or infection, ototoxic drugs, head trauma, and aging, can similarly affect vestibular hair cells and thus contribute to vestibular dysfunction, including vertigo, dizziness, and imbalance. Indeed, patients with mutations that interfere with hair cell development or function may present with both hearing loss and vestibular dysfunction, or only one of the disorders.

[0005] In recent years, efforts to treat hearing loss have increasingly focused on gene therapy as a possible solution, but few approaches remain that specifically target hair cells, which are frequently involved in hearing loss and vestibular dysfunction. There is a need for new therapies that target hair cells for the treatment of sensorineural hearing loss or vestibular dysfunction. Summary of the Invention

[0006] The present invention provides compositions and methods for promoting the expression of a gene of interest in a specific cell type, such as a gene that promotes or improves hair cell function or survival. The compositions and methods described herein relate to polynucleotides that stimulate the transcription of a transgene in hair cells of the inner ear (e.g., cochlear hair cells and vestibular hair cells). The polynucleotides described herein may be operably linked to a therapeutic transgene and administered to a patient to treat or prevent hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance).

[0007] In a first aspect, the present invention provides a nucleic acid sequence comprising a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or a functional part or derivative thereof including the sequence of SEQ ID NO:3 and / or SEQ ID NO:4, and a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or a functional part or derivative thereof including the sequence of SEQ ID NO:8 and / or SEQ ID NO:9, bound thereto (e.g., operably linked thereto). and a second region having a specific amino acid sequence (e.g., a nucleotide sequence similar to that of the first region), and optionally containing a linker comprising 1 to 100 nucleotides (e.g., 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, or 20 to 100 nucleotides) between the first region and the second region.

[0008] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:1.

[0009] In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:2.

[0010] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:13.

[0011] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:15.

[0012] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:16.

[0013] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:30.

[0014] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:31.

[0015] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:36.

[0016] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:37.

[0017] In another aspect, the invention provides a nucleic acid sequence encoding a nucleic acid fragment comprising a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or a functional portion or derivative thereof including the sequence of SEQ ID NO:8 and / or SEQ ID NO:9, and a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or a functional portion or derivative thereof including the sequence of SEQ ID NO:3 and / or SEQ ID NO:4, bound thereto (e.g., operably linked thereto). ), and optionally containing a linker comprising 1 to 100 nucleotides (e.g., 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, or 20 to 100 nucleotides) between the first and second regions.

[0018] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:2.

[0019] In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:1.

[0020] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:14.

[0021] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:35.

[0022] In another aspect, the present invention provides a polynucleotide comprising a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or a functional portion or derivative thereof including the sequence of SEQ ID NO:3 and / or SEQ ID NO:4.

[0023] In some embodiments, the region comprises or consists of the sequence of SEQ ID NO:1.

[0024] In another aspect, the present invention provides a polynucleotide comprising a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or a functional portion or derivative thereof, including the sequence of SEQ ID NO:8 and / or SEQ ID NO:9.

[0025] In some embodiments, the region comprises or consists of the sequence of SEQ ID NO:2.

[0026] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:3.

[0027] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:4.

[0028] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:3 and the sequence of SEQ ID NO:4.

[0029] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:5.

[0030] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:6.

[0031] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:7.

[0032] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 contains the sequence of SEQ ID NO:27.

[0033] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:8.

[0034] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:9.

[0035] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:8 and the sequence of SEQ ID NO:9.

[0036] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:28.

[0037] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:29.

[0038] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:10.

[0039] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:11.

[0040] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:12.

[0041] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 contains the sequence of SEQ ID NO:32.

[0042] In another aspect, the invention provides a nucleic acid sequence encoding a nucleotide sequence comprising a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17, or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 19, and a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 18, or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 20 and / or SEQ ID NO: 21, bound thereto (e.g., operably linked thereto). and a second region having a sequence identity of 1 to 400 nucleotides (e.g., 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 2 25, 1-250, 1-275, 1-300, 1-325, 1-350, 1-375, 1-400, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 30-100, 40-100, 50-100, 50-150, 50-200, 50-250, 50-300, 50-350, and a polynucleotide comprising a linker containing 50 to 400, 100 to 150, 100 to 200, 100 to 250, 100 to 300, 100 to 350, 100 to 400, 150 to 200, 150 to 250, 150 to 300, 150 to 350, 150 to 400, 200 to 250, 200 to 300, 200 to 350, 200 to 400, 250 to 300, 250 to 350, 250 to 400, 300 to 400, or 350 to 400 nucleotides.

[0043] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:17.

[0044] In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:18.

[0045] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:25.

[0046] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:26.

[0047] In another aspect, the invention provides a nucleic acid sequence comprising a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 18, or a functional part or derivative thereof comprising the sequence of SEQ ID NO: 20 and / or SEQ ID NO: 21, and a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17, or a functional part or derivative thereof comprising the sequence of SEQ ID NO: 19, which is bound to (e.g., operably linked to) the first region. and a second region having a sequence identity of 1 to 400 nucleotides (e.g., 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 2 25, 1-250, 1-275, 1-300, 1-325, 1-350, 1-375, 1-400, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 30-100, 40-100, 50-100, 50-150, 50-200, 50-250, 50-300, 50-350, and a polynucleotide comprising a linker containing 50 to 400, 100 to 150, 100 to 200, 100 to 250, 100 to 300, 100 to 350, 100 to 400, 150 to 200, 150 to 250, 150 to 300, 150 to 350, 150 to 400, 200 to 250, 200 to 300, 200 to 350, 200 to 400, 250 to 300, 250 to 350, 250 to 400, 300 to 400, or 350 to 400 nucleotides.

[0048] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:18.

[0049] In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:17.

[0050] In another aspect, the present invention provides a polynucleotide comprising a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:17, or a functional portion or derivative thereof including the sequence of SEQ ID NO:19.

[0051] In some embodiments, the region comprises or consists of the sequence of SEQ ID NO:17.

[0052] In another aspect, the present invention provides a polynucleotide comprising a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:18, or a functional portion or derivative thereof including the sequence of SEQ ID NO:20 and / or SEQ ID NO:21.

[0053] In some embodiments, the region comprises or consists of the sequence of SEQ ID NO:18.

[0054] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:17 contains the sequence of SEQ ID NO:19.

[0055] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:18 contains the sequence of SEQ ID NO:20.

[0056] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:18 contains the sequence of SEQ ID NO:21.

[0057] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:18 contains the sequence of SEQ ID NO:20 and the sequence of SEQ ID NO:21.

[0058] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:18 contains the sequence of SEQ ID NO:22.

[0059] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:18 contains the sequence of SEQ ID NO:23.

[0060] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:18 contains the sequence of SEQ ID NO:24.

[0061] In another aspect, the present invention provides a polynucleotide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence of any one of SEQ ID NOs: 27-35.

[0062] In some embodiments, the polynucleotide has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence of SEQ ID NO:28.

[0063] In some embodiments, the polynucleotide has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence of SEQ ID NO: 32.

[0064] In some embodiments, the polynucleotide has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence of SEQ ID NO: 33.

[0065] In some embodiments, the polynucleotide has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence of SEQ ID NO: 34.

[0066] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:28.

[0067] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:32.

[0068] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:33.

[0069] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:34.

[0070] In some embodiments of any of the aforementioned aspects, the polynucleotide is operably linked to a transgene and, when introduced into a hair cell, induces transgene expression.

[0071] In another aspect, the present invention provides a nucleic acid vector containing a polynucleotide of the present invention.

[0072] In some embodiments, the polynucleotide is operably linked to a transgene.

[0073] In some embodiments, the transgene comprises a nucleic acid sequence encoding a therapeutic protein.

[0074] In some embodiments, the polynucleotide is capable of directing hair cell-specific expression of a therapeutic protein from a nucleic acid sequence in a mammalian hair cell.

[0075] In some embodiments, the hair cells are cochlear hair cells.

[0076] In some embodiments, the cochlear hair cells are inner hair cells.

[0077] In some embodiments, the cochlear hair cells are outer hair cells.

[0078] In some embodiments, the hair cells are vestibular hair cells.

[0079] In some embodiments, the therapeutic protein is selected from the group consisting of ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS 8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

[0080] In some embodiments, the nucleic acid vector is a plasmid, cosmid, artificial chromosome, or viral vector.

[0081] In some embodiments, the nucleic acid vector is a viral vector selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, and a lentivirus.

[0082] In some embodiments, the viral vector is an AAV vector.

[0083] In some embodiments, the serotype of the AAV vector is selected from the group including AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S.

[0084] In some embodiments, the serotype of the AAV vector is AAV1.

[0085] In some embodiments, the serotype of the AAV vector is AAV9.

[0086] In some embodiments, the serotype of the AAV vector is AAV6.

[0087] In some embodiments, the serotype of the AAV vector is Anc80.

[0088] In some embodiments, the serotype of the AAV vector is Anc80L65.

[0089] In some embodiments, the serotype of the AAV vector is DJ / 9.

[0090] In some embodiments, the serotype of the AAV vector is 7m8.

[0091] In some embodiments, the serotype of the AAV vector is AAV2.

[0092] In some embodiments, the serotype of the AAV vector is AAV2quad(YF).

[0093] In some embodiments, the serotype of the AAV vector is PHP.B.

[0094] In some embodiments, the serotype of the AAV vector is AAV8.

[0095] In another aspect, the present invention provides a composition comprising a nucleic acid vector of the present invention.

[0096] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0097] In another aspect, the invention provides methods for increasing expression of a therapeutic protein in a mammalian hair cell by contacting the mammalian hair cell with a nucleic acid vector of the invention or a composition of the invention.

[0098] In some embodiments, expression of the therapeutic protein is increased specifically in hair cells.

[0099] In some embodiments, the mammalian hair cells are human hair cells.

[0100] In some embodiments, the mammalian hair cells are cochlear hair cells.

[0101] In some embodiments, the cochlear hair cells are inner hair cells.

[0102] In some embodiments, the cochlear hair cells are outer hair cells.

[0103] In some embodiments, the mammalian hair cells are vestibular hair cells.

[0104] In some embodiments, expression of the therapeutic protein is not substantially increased in inner ear cells that are not hair cells.

[0105] In another aspect, the present invention provides a method of treating a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss) by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention.

[0106] In some embodiments, the hearing loss is hereditary hearing loss.

[0107] In some embodiments, the genetic hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss.

[0108] In some embodiments, the hearing loss is acquired hearing loss.

[0109] In some embodiments, the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease- or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss.

[0110] In some embodiments, the acquired hearing loss is age-related hearing loss.

[0111] In some embodiments, the hearing loss is noise-induced hearing loss.

[0112] In some embodiments, the hearing loss is ototoxic drug-induced hearing loss.

[0113] In another aspect, the present invention provides a method of treating a subject having or at risk of developing a vestibular dysfunction by administering to the subject an effective amount of a nucleic acid vector of the invention or a composition of the invention.

[0114] In some embodiments, the vestibular dysfunction is vertigo, dizziness, or imbalance (eg, loss of balance).

[0115] In another aspect, the present invention provides a method of promoting hair cell regeneration in a subject in need thereof by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention.

[0116] In some embodiments, the hair cells are cochlear hair cells.

[0117] In some embodiments, the hair cells are vestibular hair cells.

[0118] In another aspect, the present invention provides a method for preventing or reducing ototoxic drug-induced hair cell damage or death by administering to a subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention.

[0119] In some embodiments, the ototoxic drug is selected from the group including aminoglycosides (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), antineoplastic agents (e.g., platinum-containing chemotherapy agents such as cisplatin, carboplatin, and oxaliplatin), ethacrynic acid, furosemide, salicylates (e.g., aspirin, especially at high doses), and quinine.

[0120] In some embodiments, the hair cells are cochlear hair cells.

[0121] In some embodiments, the hair cells are vestibular hair cells.

[0122] In another aspect, the present invention provides a method of treating a subject with tinnitus by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention.

[0123] In some embodiments of any of the aforementioned aspects, the hearing loss, vestibular dysfunction, or tinnitus is associated with loss of hair cells (e.g., cochlear hair cells and / or vestibular hair cells).

[0124] In another aspect, the present invention provides a method of preventing or reducing hair cell damage or death in a subject in need thereof by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention.

[0125] In some embodiments, the hair cells are cochlear hair cells.

[0126] In some embodiments, the hair cells are vestibular hair cells.

[0127] In another aspect, the present invention provides a method of increasing hair cell survival in a subject in need thereof by administering to the subject an effective amount of a nucleic acid vector of the present invention or a composition of the present invention.

[0128] In some embodiments, the hair cells are cochlear hair cells.

[0129] In some embodiments, the hair cells are vestibular hair cells.

[0130] In some embodiments of any of the aforementioned aspects, the hair cells are cochlear hair cells.

[0131] In some embodiments of any of the aforementioned aspects, the cochlear hair cells are inner hair cells.

[0132] In some embodiments of any of the aforementioned aspects, the cochlear hair cells are outer hair cells.

[0133] In some embodiments of any of the aforementioned aspects, the mammalian hair cells are vestibular hair cells.

[0134] In some embodiments of any of the aforementioned aspects, the method further includes assessing the subject's hearing (e.g., assessing hearing using a standard test such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), or otoacoustic emissions) prior to administering the nucleic acid vector or composition.

[0135] In some embodiments of any of the aforementioned aspects, the method further includes assessing the subject's hearing after administering the nucleic acid vector or composition (e.g., assessing hearing using a standard test such as audiometry, ABR, ECOG, or otoacoustic emissions).

[0136] In some embodiments of any of the aforementioned aspects, the method further comprises assessing the subject's vestibular function (e.g., assessing vestibular function using a standard test such as an electronystagmogram (ENG) or videonystagmogram (VNG), posturography, rotary chair test, ECOG, vestibular evoked myogenic potentials (VEMPs), or specialized clinical balance testing) prior to administering the nucleic acid vector or composition.

[0137] In some embodiments of any of the aforementioned aspects, the method further comprises assessing the subject's vestibular function (e.g., assessing vestibular function using a standard test such as ENG or VNG, posturography, rotary chair test, ECOG, VEMP, or specialized clinical balance testing) prior to administering the nucleic acid vector or composition.

[0138] In some embodiments of any of the foregoing aspects, the nucleic acid vector or composition is administered locally (e.g., administered to the inner ear, e.g., into the perilymph or endolymph, such as through the window oval, the round window cochlea, or the horizontal semicircular canal).

[0139] In some embodiments of any of the foregoing aspects, the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, prevent or reduce vestibular dysfunction, prevent or reduce tinnitus, delay the onset of hearing loss, delay the onset of vestibular dysfunction, slow the progression of hearing loss, slow the progression of vestibular dysfunction, improve hearing, improve vestibular function, improve hair cell function, prevent or reduce hair cell damage, prevent or reduce hair cell death, or increase hair cell number.

[0140] In some embodiments of any of the aforementioned aspects, the subject is a human.

[0141] In another aspect, the present invention provides a kit containing the nucleic acid vector of the present invention or the composition of the present invention.

[0142] definition As used herein, the term "about" refers to a value that is 10% above or below the stated value.

[0143] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., a nucleic acid vector containing a myosin 15 (Myo15) promoter operably linked to a transgene) to a subject by any effective route. Exemplary administration routes are described herein below.

[0144] As used herein, the term "cell type" refers to a group of cells that share a phenotype that is statistically separable based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of a common cell type may include those isolated from a common tissue (e.g., epithelial, nervous, connective, or muscle tissue) and / or those isolated from a common organ, tissue system, blood vessel, or other structure and / or region in an organism.

[0145] As used herein, the term "cochlear hair cells" refers to a group of specialized cells in the inner ear that are involved in detecting sound. There are two types of cochlear hair cells: inner hair cells and outer hair cells. Damage to cochlear hair cells and genetic mutations that interfere with the function of cochlear hair cells are involved in hearing loss and hearing loss.

[0146] As used herein, the terms "conservative mutation," "conservative substitution," and "conservative amino acid substitution" refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk. These properties are summarized for each of the 20 naturally occurring amino acids in Table 1 below. [Table 1]

[0147] From this table, it can be seen that conservative amino acid families include: (i) G, A, V, L, and I, (ii) D and E, (iii) C, S, and T, (iv) H, K, and R, (v) N and Q, and (vi) F, Y, and W. Thus, a conservative variation or substitution is one that replaces one amino acid with a member of the same amino acid family (e.g., replacing Ser with Thr or Lys with Arg).

[0148] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein refer to an amount sufficient to produce beneficial or desired results, including clinical results, when administered to a subject in need thereof, including a mammal, e.g., a human. Accordingly, "effective amount" or its synonyms depend on the context in which it is applied. For example, in the context of treating sensorineural hearing loss or vestibular dysfunction, an "effective amount" is the amount of a composition, vector construct, or viral vector sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, and the identity (e.g., age, sex, weight) of the subject or host being treated, but can still be routinely determined by one of skill in the art. Also, as used herein, a "therapeutically effective amount" of a composition, vector construct, or viral vector of the present disclosure is an amount that produces beneficial or desired results in a subject compared to a control. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each component that is effective when administered individually.As defined herein, the therapeutically effective amount of the composition, vector construct, or viral vector of the present disclosure can be easily determined by those skilled in the art by conventional methods known in the art.Dosage regimens can be adjusted to provide optimal therapeutic responses.

[0149] As used herein, the term "endogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell such as a human cell, e.g., a human hair cell).

[0150] As used herein, the term "expression" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), (3) translation of the RNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein.

[0151] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell such as a human cell, e.g., a human hair cell). Exogenous materials include those provided to an organism from a foreign source or culture materials extracted therefrom.

[0152] As used herein, the term "hair cell-specific expression" refers to the production of an RNA transcript or polypeptide primarily within hair cells (e.g., cochlear hair cells and / or vestibular hair cells) relative to other cell types of the inner ear (e.g., spiral ganglion neurons, glia, or other inner ear cell types). Hair cell-specific expression of a transgene can be confirmed by comparing transgene expression (e.g., RNA or protein expression) between various cell types of the inner ear (e.g., hair cells and non-hair cells) using any standard technique (e.g., quantitative real-time PCR, immunohistochemistry, Western blot analysis, or measuring the fluorescence of a reporter (e.g., GFP) operably linked to a promoter). A hair cell-specific promoter induces expression of a transgene (e.g., RNA or protein expression) to which it is operably linked that is at least 50% greater (e.g., 50%, 75%, 100%, 125%, 150%, 175%, 200% greater or more) in hair cells compared to at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) of the following inner ear cell types: border cells, inner phalangeal cells, inner pillar cells, outer pillar cells, first row Deiters cells, second row Deiters cells, third row Deiters cells, Hensen cells, Clausius cells, medial sulcus cells, lateral sulcus cells, spiral projecting cells, root cells, interdental cells, basal cells of the stria vascularis, intermediate cells of the stria vascularis, marginal cells of the stria vascularis, spiral ganglion neurons, Schwann cells.

[0153] As used herein, the terms "increase" and "decrease" refer to modulations that result in an increase or decrease, respectively, in the amount of a metric's function, expression, or activity relative to a baseline. For example, after administering a composition according to the methods described herein, the amount of a marker for a metric described herein (e.g., transgene expression) may increase or decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more in a subject compared to the amount of the marker before administration. Generally, the metric is measured after administration at a time when administration has had the recited effect, e.g., at least 1 week, 1 month, 3 months, or 6 months after the treatment regimen has begun.

[0154] As used herein, the term "intron" refers to a region within the coding region of a gene whose nucleotide sequence is not translated into the amino acid sequence of the corresponding protein. The term intron also refers to the corresponding region of the RNA transcribed from the gene. Introns are transcribed into pre-mRNA, but are removed during processing and are not included in the mature mRNA.

[0155] As used herein, the term "linker" refers to a series of nucleotides that connects two different regions of a polynucleotide. The linker does not interfere with the function of the two regions of the polynucleotide that it connects.

[0156] As used herein, "topical" or "local administration" refers to administration at a particular site in the body where a local effect, rather than a systemic effect, is intended. Examples of local administration are epithelial, inhaled, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional, lymph node, intratumoral, administration to the inner ear, and administration to a mucous membrane of a subject, where the administration is intended to have a local effect, rather than a systemic effect.

[0157] As used herein, the term "operably linked" refers to a first molecule that can be joined to a second molecule, where the first molecule is positioned so that it affects the function of the second molecule. The term "operably linked" includes the juxtaposition of two or more components (e.g., a promoter and another sequence element) that allows for both components to function normally and for at least one of the components to mediate a function exerted on at least one of the other components. The two molecules may or may not be part of a single, continuous molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter controls the transcription of the transcribable polynucleotide molecule of interest in a cell.

[0158] In further embodiments, two portions of a transcriptional regulatory element are operably linked to each other when they are joined such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to each other without any intervening nucleotides.

[0159] As used herein, the term "plasmid" refers to an extrachromosomal, circular, double-stranded DNA molecule into which additional DNA segments can be ligated. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Certain plasmids are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain plasmids are capable of directing the expression of genes to which they are operably linked.

[0160] As used herein, the terms "nucleic acid" and "polynucleotide," used interchangeably herein, refer to a polymeric form of nucleosides of any length. Typically, polynucleotides are composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. The term encompasses molecules containing nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids; such molecules may be preferred for certain applications. When the application refers to polynucleotides, it is understood that both DNA and RNA, and in each case, both single- and double-stranded forms (as well as the complementary strand of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information (i.e., the series of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in a 5' to 3' direction unless otherwise indicated.

[0161] As used herein, the term "complementarity" or "complementary" of a nucleic acid means that a nucleotide sequence in one strand of a nucleic acid will form hydrogen bonds with another sequence on an opposing nucleic acid strand due to the orientation of its nucleobase groups. Complementary bases in DNA are typically A and T and C and G. In RNA, they are typically C and G and U and A. Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids means that the two nucleic acids can form a duplex in which all bases in the duplex are bound to complementary bases by Watson-Crick pairing. "Substantial" or "sufficient" complementarity means that the sequence in one strand is not completely and / or perfectly complementary to the sequence in the opposing strand, but sufficient binding occurs between the bases on the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using sequences and standard mathematical calculations to predict the Tm (melting temperature) of hybridized strands, or by empirical determination of Tm using routine methods. Tm comprises the temperature at which the population of hybridization complexes formed between two nucleic acid strands is 50% denatured (i.e., the population of double-stranded nucleic acid molecules becomes half-dissociated into single strands). At temperatures below Tm, the formation of hybridization complexes is favored, while at temperatures above Tm, melting or separation of the strands in the hybridization complexes is favored. Tm can be estimated for nucleic acids with a known G+C content in 1 M NaCl aqueous solution, for example, by using Tm = 81.5 + 0.41 (% G+C), although other known Tm calculations take into account nucleic acid structural characteristics.

[0162] As used herein, the term "promoter" refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of a transgene.

[0163] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. Illustratively, the percent sequence identity of a given nucleic acid or amino acid sequence A to a given nucleic acid or amino acid sequence B (alternatively, it can be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0164] As used herein, the term "derivative" refers to a nucleic acid, peptide, or protein, or variant or analog thereof, that contains one or more mutations and / or chemical modifications compared to the corresponding full-length wild-type nucleic acid, peptide, or protein. Non-limiting examples of chemical modifications involving nucleic acids include, for example, modifications to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or combinations thereof.

[0165] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, administered to a subject, such as a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition that is or may be affecting the subject.

[0166] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit / risk ratio. Preferably, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals, and more specifically, in humans.

[0167] As used herein, the term "sample" refers to a specimen isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or skin), pancreatic juice, chorionic villus samples, and cells).

[0168] As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that at least partially controls the transcription of a gene of interest. Transcriptional regulatory elements can include promoters, enhancers, and other nucleic acids that control or help control gene transcription (e.g., polyadenylation signals). Examples of transcriptional regulatory elements can be found, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0169] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, impalefection, etc.

[0170] As used herein, the terms "subject" and "patient" refer to animals (e.g., mammals such as humans), veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). A subject treated according to the methods described herein may be a subject diagnosed with hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction (e.g., dizziness, vertigo, or imbalance), or a subject at risk for developing these conditions. Diagnosis may be made by any method or technique known in the art. One skilled in the art will understand that a subject treated according to the present disclosure may have undergone standard testing or may have been identified as a risk factor due to the presence of one or more risk factors associated with a disease or condition without testing.

[0171] As used herein, the terms "transduction" and "transduce" refer to a method of introducing a vector construct, or a portion thereof, into a cell. When the vector construct is contained in a viral vector, such as, for example, an AAV vector, transduction refers to viral infection of the cell and subsequent transfer and integration of the vector construct, or a portion thereof, into the cellular genome.

[0172] As used herein, "treatment" and "treating" a condition, disorder, or condition can include: (1) preventing, delaying, or reducing the incidence and / or likelihood of at least one clinical or subclinical symptom of the condition, disorder, or condition developing in a subject who may be affected by or predisposed to the condition, disorder, or condition but who has not yet experienced or displayed clinical or subclinical symptoms of the condition, disorder, or condition; or (2) inhibiting the condition, disorder, or condition, i.e., preventing, reducing, or delaying the onset of the disease or its recurrence, or at least one clinical or subclinical symptom thereof; or (3) ameliorating the disease, i.e., causing regression of the condition, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to a subject being treated is statistically significant or at least perceptible to the patient or physician.

[0173] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors such as plasmids, cosmids, or artificial chromosomes, RNA vectors, viruses, or any other suitable replicon (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in Gellissen, "Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems" (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the compositions and methods described herein contain polynucleotide sequences and additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the transgenes described herein include vectors containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for transgene expression contain polynucleotide sequences that increase the translation rate of the transgene or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0174] As used herein, the term "vestibular hair cells" refers to a group of specialized cells in the inner ear that are involved in sensing motion and contribute to balance and spatial orientation. Vestibular hair cells are located in the semicircular canals and otoliths of the inner ear. Damage to vestibular hair cells and genetic mutations that interfere with vestibular hair cell function are involved in vestibular dysfunction, such as vertigo and balance disorders.

[0175] As used herein, the term "wild type" refers to the genotype that has the highest frequency of a particular gene in a given organism. [Brief explanation of the drawings]

[0176] [Figure 1] A series of fluorescence images of mouse cochleae transduced with either an adeno-associated virus (AAV) vector expressing GFP under the control of the cytomegalovirus (CMV) promoter (A) or an AAV vector expressing GFP under the control of the mouse Myo15 promoter (SEQ ID NO: 13, B). The AAV-Myo15-GFP virus was injected into 6-8 week-old C57Bl / 6J male mice via the posterior semicircular canal. Mice recovered from surgery were euthanized and perfused with 10% normal buffered formalin 10 days later. The cochleae were harvested and decalcified in 8% EDTA for 3 days. The cochleae were dissected from the decalcified temporal bones and mounted on slides for confocal imaging. Using a ubiquitous promoter, AAV-CMV-GFP induced GFP expression in many cell types within the cochlea, including inner hair cells, outer hair cells, spiral ganglion neurons, mesenchymal cells, and glia (A). Using a hair cell-specific promoter, AAV-Myo15-GFP induced expression only in inner and outer hair cells (B). [Figure 2]Fluorescence images of regions of the mouse vestibular system (utricle, saccule, posterior crest (PC), anterior crest (AC), and horizontal crest (HC)) transduced with an AAV vector expressing GFP under the control of the mouse Myo15 promoter (SEQ ID NO: 13, Figure 2). The AAV-Myo15-GFP virus was injected into 6-8 week-old C57Bl / 6J male mice via the posterior semicircular canal. Mice recovered from surgery were euthanized and perfused with 10% normal buffered formalin 10 days later. The cochlear temporal bones were harvested and decalcified in 8% EDTA for 3 days. The vestibular organs were dissected from the decalcified temporal bones and mounted on slides for imaging. Using a hair cell-specific promoter, AAV-Myo15-GFP induced expression only in vestibular hair cells (Figure 2). [Figure 3] A series of fluorescence images of mouse cochleae transduced with an AAV2 / 9 viral vector expressing GFP under the control of the mouse Myo15 1 kb promoter (SEQ ID NO: 15, A) or the Myo15 mini-intron promoter (SEQ ID NO: 16, B). As shown in A-B, both the mouse Myo15 1 kb (SEQ ID NO: 15) and the mouse Myo15 mini-intron (SEQ ID NO: 16) promoters induced GFP expression specifically in hair cells. [Figure 4A]A series of images of mouse cochleae demonstrating the specificity of the mouse and human Myo15 promoters. Neonatal mouse cochlear explants were treated in culture with AAV1 vectors encoding GFP under the control of either the 0.6 kb human Myo15 promoter (SEQ ID NO: 26, Figure 4A), the 1.2 kb human Myo15 promoter (SEQ ID NO: 25, Figure 4B), or the 1 kb mouse Myo15 promoter (SEQ ID NO: 15, Figure 4C) (Scale bar = 100 μm). GFP signal as detected with an anti-GFP antibody is shown. In separate experiments, AAV1-1.2 kb human Myo15-GFP or AAV1-1 kb mouse Myo15-GFP was delivered into adult mouse cochleae by round window injection. Whole-mount images (Figures 4D-4E) or sections (Figures 4F-4G) show hair cell-specific signals in the apex, middle, and base of the cochlea, as detected with an anti-GFP antibody. The arrow in Figure 4F points to a GFP-labeled cochlear hair cell. As shown in Figures 4F and 4G, expression of both the 1.2 kb human Myo15 promoter and the 1 kb mouse Myo15 promoter was primarily restricted to inner hair cells. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 5A] Figure 5A is a series of fluorescence images of non-human primate cochleae showing that the Myo15 promoter restricts GFP expression to hair cells within the cochlea of ​​non-human primates. Figure 5A is a confocal image of a cochlea from a non-human primate that received a local injection of AAV1-CMV-GFP through the round window membrane. The tissue was harvested 28 days after injection. Native GFP fluorescence is shown. GFP expression was detected in a wide range of cell types throughout the organ. Figure 5B is a confocal image of a cochlea from a non-human primate that was injected with AAV1-Myo15 (SEQ ID NO: 13)-GFP and treated in the same manner as the cochlea in Figure 5A. GFP expression was restricted to hair cells. Figure 5C is a magnified view of a hair cell in the boxed region shown in Figure 5B. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6] This graph shows that the 1.6 kb Myo15 promoter (SEQ ID NO: 13) enhanced the biological efficacy of the AAV mouse TMC1 vector in Tmc1 knockout (KO) mice compared with the ubiquitous CMV promoter. Tmc1 KO mice were injected on postnatal day 2 (P2), and auditory brainstem responses (ABRs) were assessed at the indicated ages. ABR thresholds were plotted as a function of stimulation frequency for naive homozygous (light gray line with open circles) and heterozygous (dark gray line through black circles) Tmc1 KO mice, as well as homozygous Tmc1 KO mice injected with AAV-CMV mouse TMC1 (CMV_mTmc1; dark gray line with open circles) or AAV-Myo15 (SEQ ID NO: 13) mouse TMC1 (PdBx_mTmc1; P23, light gray line through light gray circles; P28, dark gray line with filled circles). Compared with AAV-CMV-mouseTMC1, homozygous Tmc1 KO mice injected with AAV-Myo15-mouseTMC1 showed significantly improved ABR threshold restoration. [Figure 7A]A series of fluorescent images of the inner ear of a cynomolgus monkey treated with an AAV1 vector expressing GFP under the control of the 1.2 kb human myosin 15 promoter (SEQ ID NO: 25). Using bilateral (n = 1 animal) injections, 30 μL of AAV1-1.2 kb myosin 15-GFP (viral titer at 1.06 × 10 13 genome copies / mL) was administered into the round window of the inner ear at a flow rate of 15 μL / min. Four weeks after injection, the inner ear was removed and a basilar membrane surface preparation was performed. GFP expression was used to monitor myosin 15 promoter activity (Figure 7A, one representative ear is shown). AAV1-1.2 kb myosin 15-GFP transduction of the inner ear of a cynomolgus monkey resulted in GFP expression throughout the base-apical axis of the cochlea, with high frequencies at the base and low frequencies at the apex (Figure 7A, frequency in kHz). High-magnification images at 2.8 kHz showed that GFP expression was observed within inner hair cells (IHCs) and outer hair cells (OHCs) (Figure 7B, upper panel). Immunohistochemistry for Myo7A was used to visualize hair cells, and nuclei were stained with DAPI (Figure 7B, middle and lower panels). [Figure 7B] Same as above. [Figure 8A] A series of fluorescent images of the inner ear of a cynomolgus monkey treated with an AAV1 vector expressing GFP under the control of the 0.6 kb human myosin 15 promoter (SEQ ID NO: 26). Using bilateral (n = 1 animal) injections, 30 μL of AAV1-0.6 kb myosin 15-GFP (viral titer at 1.22 × 10 13 genome copies / mL) was administered into the round window of the inner ear at a flow rate of 15 μL / min. Four weeks after injection, the inner ear was removed and a basilar membrane surface preparation was performed. GFP expression was used to monitor myosin 15 promoter activity (Figure 8A, one representative ear is shown). AAV1-0.6 kb myosin 15-GFP transduction of the inner ear of a cynomolgus monkey resulted in GFP expression throughout the base-apical axis of the cochlea, with high frequencies at the base and low frequencies at the apex (Figure 8A, frequency in kHz). High-magnification images at 2.8 kHz showed that GFP expression was observed within inner hair cells (IHCs) and outer hair cells (OHCs) (Figure 8B, upper panel). Immunohistochemistry for Myo7A was used to visualize hair cells, and nuclei were stained with DAPI (Figure 8B, middle and lower panels). [Figure 8B] Same as above. [Figure 9]

[0039] Figure 1 shows a bar graph depicting quantification of GFP expression from three different mouse Myo15 (mMyo15) promoters with hair cell-specific expression using quantitative PCR (qPCR). Transcription levels were normalized to the hair cell-specific gene Myo7a. Expression levels of the GFP reporter from the mMyo15 1.6 kb (SEQ ID NO: 13), 1 kb (SEQ ID NO: 15), and 0.5 kb (SEQ ID NO: 16) promoters were comparable and robust. [Figure 10] Graph showing the results of quantitative image analysis of GFP fluorescence in myosin 7a (Myo7a)-positive hair cells driven by various different promoters, including a truncated chimeric CMV-chicken β-actin (smCBA) promoter, a 1.6 kb mMyo15 promoter (SEQ ID NO: 13), a 1 kb mMyo15 promoter (SEQ ID NO: 15), a 0.5 kb mMyo15 promoter (SEQ ID NO: 16), a 1.2 kb hMyo15 promoter (SEQ ID NO: 25), and a 0.6 kb hMyo15 promoter (SEQ ID NO: 26). The expression level of GFP protein per cell shows the difference in expression between the Myo15 promoters, with the mMyo15 1 kb promoter producing the highest level of GFP protein per cell. [Figure 11A]A series of whole-mount fluorescent images of neonatal cochlear explant cultures infected with viral vectors containing the Myo15 promoter variants SEQ ID NOs: 16 and 27-35. The promoter sequence SEQ ID NO: 27 (Myo15 A) was neither sufficient nor necessary for general promoter activity or hair cell specificity, as shown in Figures 11B (SEQ ID NO: 27), 11E (SEQ ID NO: 30), 11F (SEQ ID NO: 31), and 11G (SEQ ID NO: 32). The promoter sequence SEQ ID NO: 27 provided a splice donor site that increased expression levels when paired with a splice acceptor site, thus introducing an intron into the promoter sequence, as shown in Figures 11A (SEQ ID NO: 16) and 11H (SEQ ID NO: 33). The promoter sequence of SEQ ID NO:28 (Myo15 B) alone was found to be a core promoter that conferred general promoter activity, as shown in Figure 11C (SEQ ID NO:28), but did not drive hair cell-specific GFP expression in the absence of the promoter sequence of SEQ ID NO:29 (Myo15 C), as shown in Figures 11C (SEQ ID NO:29) and 11G (SEQ ID NO:32). The promoter sequence of SEQ ID NO:29 (Myo15 C) was found to be an enhancer that restricted the activity of the core promoter (SEQ ID NO:28) to inner and outer hair cells, as shown in Figure 11G (SEQ ID NO:32), but was not sufficient for general promoter activity in the absence of the core promoter, as shown in Figure 11D (SEQ ID NO:29). Rearrangement of the promoter sequences of SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 in various combinations still resulted in hair cell-specific promoter activity as shown in Figures 11H (SEQ ID NO:33) and 11I (SEQ ID NO:34), although not all rearrangements were tolerated as shown in Figure 11J (SEQ ID NO:35). [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 11G] Same as above. [Figure 11H] Same as above. [Figure 11I] Same as above. [Figure 11J] Same as above. [Figure 12] Bar graph showing quantification of GFP expression from promoter variants with hair cell-specific expression, as measured by qPCR. Transcription levels were normalized to the hair cell-specific gene Myo7a. Expression levels of the GFP reporter from the Myo15 0.5kb (SEQ ID NO: 16), Myo15 BC (SEQ ID NO: 32), and Myo15 BAC (SEQ ID NO: 33) promoters were comparable and robust. The Myo15 CAB promoter (SEQ ID NO: 34) was generated by rearranging the positions of the splice donor and acceptor sites, resulting in cryptic splicing of the GFP cDNA toward the center, as evidenced by higher expression levels detected with GFP probes located at the 3' end of the transcript versus the 5' end or center of the transcript. [Figure 13A] A series of graphs showing quantitative image analysis of GFP expression levels in Myo7a-positive hair cells. The expression level of GFP protein per cell showed differences among the Myo15 promoters (Figures 13A-13B), including the 0.5 kb mMyol5 promoter (also referred to as the Myo15 ABC promoter; SEQ ID NO: 16), the Myo15 AB promoter (SEQ ID NO: 30), the Myo15 AC promoter (SEQ ID NO: 31), the Myo15 BC promoter (SEQ ID NO: 32), the Myo15 A promoter (SEQ ID NO: 27), the Myo15 B promoter (SEQ ID NO: 28), the Myo15 C promoter (SEQ ID NO: 29), the Myo15 BAC promoter (SEQ ID NO: 33), the Myo15 CAB promoter (SEQ ID NO: 34), and the Myo15 BCA promoter (SEQ ID NO: 35). The Myo15 ABC promoter (SEQ ID NO: 16) produced the highest GFP expression per cell (Figure 13A). Background levels of fluorescence were determined for each data set based on tissue autofluorescence (less than 2000 units for Figure 13A and less than 6000 units for Figure 13B). [Figure 13B]Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0177] Described herein are compositions and methods for specifically inducing expression of a transgene in hair cells (e.g., cochlear hair cells and / or vestibular hair cells). The invention features polynucleotides containing regions of the myosin 15 (Myo15) promoter that can express a transgene specifically in cochlear hair cells. The invention also features nucleic acid vectors containing these promoters operably linked to polynucleotides encoding polypeptides. The compositions and methods described herein can be used to express polynucleotides that encode hair cell proteins specifically in cochlear hair cells and vestibular hair cells, and thus the compositions described herein can be administered to a subject (such as a mammalian subject, e.g., a human) to treat disorders caused by hair cell dysfunction, such as hearing loss or vestibular dysfunction.

[0178] hair cells Hair cells are sensory cells of the auditory and vestibular systems present in the inner ear. Cochlear hair cells are sensory cells of the auditory system and are composed of two main cell types: inner hair cells, which are responsible for detecting sound, and outer hair cells, which are thought to amplify low-level sounds. Vestibular hair cells are located in the semicircular canals and otolithic organs of the inner ear and are involved in the sense of movement, which contributes to balance and spatial orientation. Hair cells are named for the stereocilia that protrude from the apical surface of the cell and form the hair cell bundle. Deflection of the stereocilia (e.g., by sound waves in cochlear hair cells or by rotational or linear acceleration in vestibular hair cells) results in the opening of mechanically gated ion channels, which allow the hair cells to release neurotransmitters to activate neurons, thereby converting mechanical sound or movement signals into electrical signals that can be transmitted to the brain. Cochlear hair cells are essential for normal hearing, and damage to cochlear hair cells and genetic mutations that disrupt their function contribute to hearing loss and hearing loss. Damage to vestibular hair cells and genetic mutations that disrupt their function contribute to vestibular dysfunction, such as loss of balance and vertigo (e.g., dizziness). Gene therapy has recently emerged as an attractive therapeutic approach for treating hearing loss and vestibular dysfunction, but the field lacks methods for specifically targeting nucleic acid vectors used in gene therapy to hair cells.

[0179] Myosin 15 Myo15 is a non-traditional actin-based molecular motor that regulates stereocilia development. Mice with mutations in Myo15 have been found to have short stereocilia, severe hearing loss, and vestibular dysfunction, while mutations in the human ortholog, Myo15A, cause a nonsyndromic autosomal recessive hearing loss, DFNB3. Myo15 has been observed to localize to stereocilia and is essential for their development and maintenance. The localization pattern suggests that Myo15 may be specifically expressed in hair cells. However, the Myo15 promoter has not previously been isolated or characterized. We identified evolutionarily conserved blocks in orthologous genomic sequences that may constitute the promoter's regulatory elements and found them to be located more than 7,200 base pairs (bp) upstream of the translation start site. This genomic region is too large to be used in conjunction with adeno-associated virus (AAV) vectors, which have a maximum packaging capacity of 4.7 kb and cannot deliver transgenes of interest for gene therapy.

[0180] The present invention is based, in part, on the discovery of a region upstream of the Myol5 translation start site, which can be used to drive transgene expression specifically in hair cells (e.g., cochlear hair cells and / or vestibular hair cells). Accordingly, the compositions and methods described herein can be used to express a gene of interest in hair cells (e.g., a gene involved in hair cell development, function, cell fate specification, regeneration, survival, or maintenance, or a gene known to be disrupted, e.g., mutated, in subjects with hearing loss or vestibular dysfunction) to treat subjects with or at risk of developing hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction (e.g., vertigo, dizziness, or loss of balance).

[0181] Mouse myosin 15 promoter The polynucleotides of the compositions and methods described herein include nucleic acid sequences from regions of the mouse Myol5 locus capable of transgene expression specifically in hair cells, or variants thereof, such as nucleic acid sequences having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to regions of the mouse Myol5 locus capable of transgene expression specifically in hair cells. These regions include the nucleic acid sequence immediately preceding the mouse Myol5 translation start site and upstream regulatory elements located 5 kb from the mouse Myol5 translation start site. The polynucleotides of the compositions and methods described herein can optionally include a linker operably linking regions of the mouse Myol5 locus capable of transgene expression specifically in hair cells, or the regions of the mouse Myol5 locus can be directly linked without an intervening linker.

[0182] In some embodiments, the polynucleotides described herein contain a first region (upstream regulatory element) having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to a region containing the first non-coding exon of the Myo15 gene (nucleic acids -6755 to -7209 relative to the mouse Myo15 translation start site, this sequence being set forth in SEQ ID NO:1), or a functional portion or derivative thereof, joined (e.g., operably linked) to a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence immediately preceding the translation start site of mouse Myo15 (nucleic acids -1 to -1157 relative to the mouse Myo15 translation start site, this sequence being set forth in SEQ ID NO:2), or a functional portion or derivative thereof. A functional portion of SEQ ID NO:1 can have the nucleic acid sequence of -7166 to -7091 relative to the mouse Myo15 translation start site (as set forth in SEQ ID NO:3) and / or the nucleic acid sequence of -7077 to -6983 relative to the mouse Myo15 translation start site (as set forth in SEQ ID NO:4). The first region can contain the nucleic acid sequence of SEQ ID NO:3 fused to the nucleic acid sequence of SEQ ID NO:4 without an intervening nucleic acid, as set forth in SEQ ID NO:5, or the first region can contain the nucleic acid sequence of SEQ ID NO:4 fused to the nucleic acid sequence of SEQ ID NO:3 without an intervening nucleic acid, as set forth in SEQ ID NO:6. Alternatively, the first region can contain the sequences of SEQ ID NO:3 and SEQ ID NO:4 joined by an endogenous intervening nucleic acid sequence (e.g., the first region can have or include the nucleic acid sequence of -7166 to -6983 relative to the mouse Myo15 translation start site, as set forth in SEQ ID NO:7 and SEQ ID NO:27) or a nucleic acid linker.

[0183] In a polynucleotide in which the first region contains both SEQ ID NO:3 and SEQ ID NO:4, the two sequences can be included in any order (e.g., SEQ ID NO:3 can be joined to (e.g., precede) SEQ ID NO:4, or SEQ ID NO:4 can be joined to (e.g., precede) SEQ ID NO:3). A functional portion of SEQ ID NO:2 can have the nucleic acid sequence from -590 to -509 relative to the mouse Myo15 translation start site (as set forth in SEQ ID NO:8) and / or the nucleic acid sequence from -266 to -161 relative to the mouse Myo15 translation start site (as set forth in SEQ ID NO:9). In some embodiments, the sequence containing SEQ ID NO:8 has the sequence of SEQ ID NO:28. In some embodiments, the sequence containing SEQ ID NO:9 has the sequence of SEQ ID NO:29. The second region can contain the nucleic acid sequence of SEQ ID NO:8 fused to the nucleic acid sequence of SEQ ID NO:9 without any intervening nucleic acids, as set forth in SEQ ID NO:10, or the second region can contain the nucleic acid sequence of SEQ ID NO:9 fused to the nucleic acid sequence of SEQ ID NO:8 without any intervening nucleic acids, as set forth in SEQ ID NO:11. The second region can contain the nucleic acid sequence of SEQ ID NO: 28 fused to the nucleic acid sequence of SEQ ID NO: 29 without an intervening nucleic acid, as set forth in SEQ ID NO: 32, or the second region can contain the nucleic acid sequence of SEQ ID NO: 29 fused to the nucleic acid sequence of SEQ ID NO: 28 without an intervening nucleic acid. Alternatively, the second region can contain the sequences of SEQ ID NO: 8 and SEQ ID NO: 9 joined by an endogenous intervening nucleic acid sequence (e.g., the second region can have the nucleic acid sequence from -590 to -161 relative to the mouse Myo15 translation start site, as set forth in SEQ ID NO: 12) or a nucleic acid linker.

[0184] In a polynucleotide in which the second region contains both SEQ ID NO:8 and SEQ ID NO:9, the two sequences may be included in any order (e.g., SEQ ID NO:8 may be joined to (e.g., precede) SEQ ID NO:9, or SEQ ID NO:9 may be joined to (e.g., precede) SEQ ID NO:8).

[0185] The first and second regions of the polynucleotide can be joined directly or via a nucleic acid linker. For example, the polynucleotide can contain the sequence of SEQ ID NO: 1, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, e.g., SEQ ID NOs: 3 and 4) fused to the sequence of SEQ ID NO: 2, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29, and 32, e.g., SEQ ID NOs: 8 and 9) without any intervening nucleic acid. For example, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO: 1 to SEQ ID NO: 2 is set forth in SEQ ID NO: 13. Alternatively, a linker can be used to join the sequence of SEQ ID NO: 1, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, e.g., SEQ ID NOs: 3 and 4) to the sequence of SEQ ID NO: 2, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29, and 32, e.g., SEQ ID NOs: 8 and 9). Exemplary polynucleotides containing functional portions of both SEQ ID NO:1 and SEQ ID NO:2 are provided in SEQ ID NOs:15, 16, 30, and 31.

[0186] The length of a nucleic acid linker for use in the polynucleotides described herein can be about 5 kb or less (e.g., about 5 kb, 4.5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, 5 bp, 4 bp, 3 bp, 2 bp, or less). Nucleic acid linkers that can be used in the polynucleotides described herein do not interfere with the ability of the polynucleotides of the invention to direct transgene expression in hair cells.

[0187] In some embodiments, the sequence of SEQ ID NO: 1, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, e.g., SEQ ID NOs: 3 and 4) is joined (e.g., operably linked) to the sequence of SEQ ID NO: 2, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29, and 32, e.g., SEQ ID NOs: 8 and 9), and in some embodiments, the order of the regions is reversed (e.g., the sequence of SEQ ID NO: 2, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29, and 32, e.g., SEQ ID NOs: 8 and 9) is joined (e.g., operably linked) to the sequence of SEQ ID NO: 1 or is joined (e.g., operably linked) to a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOS: 3-7 and 27, e.g., SEQ ID NOS: 3 and 4). For example, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO: 2 to SEQ ID NO: 1 is set forth in SEQ ID NO: 14. An example of a polynucleotide in which a functional portion or derivative of SEQ ID NO: 2 precedes a functional portion or derivative of SEQ ID NO: 1 is provided in SEQ ID NO: 35. Regardless of order, the sequence of SEQ ID NO: 1, or a functional portion or derivative thereof, and the sequence of SEQ ID NO: 2, or a functional portion or derivative thereof, can be joined by direct fusion or by a nucleic acid linker, as described above.

[0188] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to a region containing the first non-coding exon of the Myo15 gene (nucleic acid sequence -6755 to -7209 relative to the mouse Myo15 translation start site, this sequence being set forth in SEQ ID NO:1), or a functional portion or derivative thereof. A functional portion of SEQ ID NO:1 can have the nucleic acid sequence -7166 to -7091 relative to the mouse Myo15 translation start site (set forth in SEQ ID NO:3) and / or the nucleic acid sequence -7077 to -6983 relative to the Myo15 translation start site (set forth in SEQ ID NO:4). The polynucleotide can contain the nucleic acid sequence of SEQ ID NO:3 fused to the nucleic acid sequence of SEQ ID NO:4 without intervening nucleic acids, as set forth in SEQ ID NO:5, or the polynucleotide can contain the nucleic acid sequence of SEQ ID NO:4 fused to the nucleic acid sequence of SEQ ID NO:3 without intervening nucleic acids, as set forth in SEQ ID NO:6. Alternatively, the polynucleotide may contain the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 joined by an endogenous intervening nucleic acid sequence (e.g., the first region may have or include the nucleic acid sequence from -7166 to -6983 relative to the mouse Myo15 translation start site, as shown in SEQ ID NO: 7 and SEQ ID NO: 27) or a nucleic acid linker.

[0189] In a polynucleotide containing both SEQ ID NO:3 and SEQ ID NO:4, the two sequences may be included in any order (e.g., SEQ ID NO:3 may be joined to (e.g., precede) SEQ ID NO:4, or SEQ ID NO:4 may be joined to (e.g., precede) SEQ ID NO:3).

[0190] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the nucleic acid sequence immediately upstream of the mouse Myo15 translation start site (nucleic acid -1 to -1157 relative to the mouse Myo15 translation start site, this sequence being set forth in SEQ ID NO:2), or a functional portion or derivative thereof. A functional portion of SEQ ID NO:2 can have the nucleic acid sequence -590 to -509 relative to the mouse Myo15 translation start site (set forth in SEQ ID NO:8) and / or the nucleic acid sequence -266 to -161 relative to the mouse Myo15 translation start site (set forth in SEQ ID NO:9).

[0191] In some embodiments, the sequence containing SEQ ID NO:8 has the sequence of SEQ ID NO:28.

[0192] In some embodiments, the sequence containing SEQ ID NO:9 has the sequence of SEQ ID NO:29. The polynucleotide can contain the nucleic acid sequence of SEQ ID NO:8 fused to the nucleic acid sequence of SEQ ID NO:9 without an intervening nucleic acid, as shown in SEQ ID NO:10, or the polynucleotide can contain the nucleic acid sequence of SEQ ID NO:9 fused to the nucleic acid sequence of SEQ ID NO:8 without an intervening nucleic acid, as shown in SEQ ID NO:11. The polynucleotide can contain the nucleic acid sequence of SEQ ID NO:28 fused to the nucleic acid sequence of SEQ ID NO:29 without an intervening nucleic acid, as shown in SEQ ID NO:32, or the second region can contain the nucleic acid sequence of SEQ ID NO:29 fused to the nucleic acid sequence of SEQ ID NO:28 without an intervening nucleic acid. Alternatively, the polynucleotide can contain the sequences of SEQ ID NO:8 and SEQ ID NO:9 joined by an endogenous intervening nucleic acid sequence (e.g., the second region can have the nucleic acid sequence from -590 to -161 relative to the mouse Myo15 translation start site, as shown in SEQ ID NO:12) or a nucleic acid linker.

[0193] In a polynucleotide containing both SEQ ID NO:8 and SEQ ID NO:9, the two sequences may be included in any order (e.g., SEQ ID NO:8 may be joined to (e.g., precede) SEQ ID NO:9, or SEQ ID NO:9 may be joined to (e.g., precede) SEQ ID NO:8).

[0194] In some embodiments, the polynucleotides described herein contain a portion or derivative of a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a region containing the first non-coding exon of the Myo15 gene (nucleic acids -6755 to -7209 relative to the mouse Myo15 translation start site, this sequence being set forth in SEQ ID NO:1), flanked on either side by a portion or derivative of a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the nucleic acid sequence immediately upstream of the translation start site of mouse Myo15 (nucleic acids -1 to -1157 relative to the mouse Myo15 translation start site, this sequence being set forth in SEQ ID NO:2). For example, a functional portion or derivative of SEQ ID NO: 2, e.g., SEQ ID NO: 8 or 28, may be directly fused or joined by a nucleic acid linker to a portion of SEQ ID NO: 1, e.g., any one of SEQ ID NOs: 3-7 and 27, which in turn is directly fused or joined by a nucleic acid linker to a different functional portion of SEQ ID NO: 2, e.g., SEQ ID NO: 9 or 29.

[0195] In other embodiments, a functional portion or derivative of SEQ ID NO:2, e.g., SEQ ID NO:9 or 29, can be fused directly or joined by a nucleic acid linker to a portion of SEQ ID NO:1, e.g., any one of SEQ ID NOs:3-7 and 27, which is fused directly or joined by a nucleic acid linker to a different functional portion of SEQ ID NO:2, e.g., SEQ ID NO:8 or 28. For example, polynucleotides having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the nucleic acid sequences of SEQ ID NOs:28, 27, and 29 can be fused to produce a polynucleotide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the nucleic acid sequence of SEQ ID NO:33.

[0196] In some embodiments, polynucleotides having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the nucleic acid sequences of SEQ ID NOs: 29, 27, and 28 can be fused to produce a polynucleotide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the nucleic acid sequence of SEQ ID NO: 34.

[0197] Human myosin 15 promoter The polynucleotides of the compositions and methods described herein can also include a nucleic acid sequence from a region of the mouse Myol5 locus capable of transgene expression specifically in hair cells, or a variant thereof, such as a nucleic acid sequence having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a region of the human Myol5 locus capable of transgene expression specifically in hair cells. The polynucleotides of the compositions and methods described herein can optionally include a linker operably linking the region of the human Myol5 locus capable of transgene expression specifically in hair cells, or the region of the human Myol5 locus can be directly joined without an intervening linker.

[0198] In some embodiments, the polynucleotides described herein contain a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the sequence set forth in SEQ ID NO: 17, or a functional portion or derivative thereof, and a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the sequence set forth in SEQ ID NO: 18, or a functional portion or derivative thereof, to which it is linked (e.g., operably linked). A functional portion of SEQ ID NO: 17 can have the sequence set forth in SEQ ID NO: 19. A functional portion of SEQ ID NO: 18 can have the sequence set forth in SEQ ID NO: 20 and / or the sequence set forth in SEQ ID NO: 21. The second region may contain the nucleic acid sequence of SEQ ID NO:20 fused to the nucleic acid sequence of SEQ ID NO:21 with no intervening nucleic acid, as shown in SEQ ID NO:22, or the second region may contain the nucleic acid sequence of SEQ ID NO:21 fused to the nucleic acid sequence of SEQ ID NO:20 with no intervening nucleic acid, as shown in SEQ ID NO:23. Alternatively, the second region may contain the sequences of SEQ ID NO:20 and SEQ ID NO:21 joined by an endogenous intervening nucleic acid sequence (as shown in SEQ ID NO:24) or a nucleic acid linker.

[0199] In polynucleotides in which the second region contains both SEQ ID NO:20 and SEQ ID NO:21, the two sequences may be included in any order (e.g., SEQ ID NO:20 may be joined to (e.g., precede) SEQ ID NO:21, or SEQ ID NO:21 may be joined to (e.g., precede) SEQ ID NO:20).

[0200] The first and second regions of the polynucleotide can be joined directly or by a nucleic acid linker. For example, a polynucleotide can contain the sequence of SEQ ID NO: 17, or a functional portion or derivative thereof (e.g., SEQ ID NO: 19), fused to the sequence of SEQ ID NO: 18, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NO: 20 and / or 21) without an intervening nucleic acid. Alternatively, a linker can be used to join the sequence of SEQ ID NO: 17, or a functional portion or derivative thereof (e.g., SEQ ID NO: 19), to the sequence of SEQ ID NO: 18, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NO: 20 and / or 21). Exemplary polynucleotides containing functional portions of both SEQ ID NO: 17 and SEQ ID NO: 18 are provided in SEQ ID NOs: 25 and 26.

[0201] In some embodiments, the sequence of SEQ ID NO: 17, or a functional portion or derivative thereof (e.g., SEQ ID NO: 19), is joined (e.g., operably linked) to the sequence of SEQ ID NO: 18, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NOs: 20 and 21); in some embodiments, the order of the regions is reversed (e.g., the sequence of SEQ ID NO: 18, or a functional portion or derivative thereof (e.g., any one of SEQ ID NOs: 20-24, e.g., SEQ ID NO: 20 and / or 21) is joined (e.g., operably linked) to the sequence of SEQ ID NO: 17, or a functional portion or derivative thereof (e.g., SEQ ID NO: 19). Regardless of the order, the sequence of SEQ ID NO: 17, or a functional portion or derivative thereof, and the sequence of SEQ ID NO: 18, or a functional portion or derivative thereof, can be joined by direct fusion or by a nucleic acid linker, as described above.

[0202] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to a region containing the sequence set forth in SEQ ID NO: 17, or a functional portion or derivative thereof. A functional portion of SEQ ID NO: 17 can have the nucleic acid sequence set forth in SEQ ID NO: 19.

[0203] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the sequence set forth in SEQ ID NO: 18, or a functional portion or derivative thereof. A functional portion of SEQ ID NO: 18 may have the sequence set forth in SEQ ID NO: 20 and / or the sequence set forth in SEQ ID NO: 21. A polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 20 fused to the nucleic acid sequence of SEQ ID NO: 21 without an intervening nucleic acid, as set forth in SEQ ID NO: 22, or a polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 21 fused to the nucleic acid sequence of SEQ ID NO: 20 without an intervening nucleic acid, as set forth in SEQ ID NO: 23. Alternatively, a polynucleotide may contain the sequences of SEQ ID NO: 20 and SEQ ID NO: 21 joined by an internal intervening nucleic acid sequence (as set forth in SEQ ID NO: 24) or a nucleic acid linker.

[0204] In a polynucleotide containing both SEQ ID NO:20 and SEQ ID NO:21, the two sequences may be included in any order (e.g., SEQ ID NO:20 may be joined to (e.g., precede) SEQ ID NO:21, or SEQ ID NO:21 may be joined to (e.g., precede) SEQ ID NO:20).

[0205] The length of a nucleic acid linker for use in the polynucleotides described herein can be about 5 kb or less (e.g., about 5 kb, 4.5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, 5 bp, 4 bp, 3 bp, 2 bp, or less). Nucleic acid linkers that can be used in the polynucleotides described herein do not interfere with the ability of the polynucleotides of the invention to direct transgene expression in hair cells.

[0206] The aforementioned nucleic acid sequences are summarized in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] [Table 2-18]

[0207] Additional polynucleotides useful in conjunction with the compositions and methods described herein include nucleic acid sequences set forth in Table 2, as well as nucleic acid molecules having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to functional portions or derivatives of the nucleic acid sequences set forth in Table 2.

[0208] The aforementioned polynucleotides can be included in a nucleic acid vector and operably linked to a transgene to express the transgene specifically in hair cells (e.g., cochlear hair cells and / or vestibular hair cells). In some embodiments, the transgene encodes a protein involved in hair cell function, hair cell development, hair cell fate specification, hair cell regeneration, hair cell survival, or hair cell maintenance, or the transgene is a wild-type version of a gene found to be mutated in subjects with hearing loss, hearing loss, auditory neuropathy, tinnitus, or vestibular dysfunction (e.g., vertigo, dizziness, or loss of balance). According to the methods described herein, a subject can be administered a composition containing one or more of the aforementioned polynucleotides (e.g., one or more of the polynucleotides listed in Table 2) operably linked to a transgene encoding a therapeutic protein for the treatment of hearing loss and / or vestibular dysfunction.

[0209] In some embodiments, the transgene is selected from the group consisting of actin gamma 1 (ACTG1), fascin actin-bundling protein 2, retinal (FSCN2), radixin (RDX), POU class 4 homeobox 3 (POU4F3), TRIO and F-actin binding protein (TRIOBP), tapellin (TPRN), Xin actin-binding repeat containing 2 (XIRP2), Atonal BHLH transcription factor 1 (ATOH1), growth factor-independent 1 transcriptional repressor (GFI1), cholinergic receptor nicotinic alpha 9 subunit (CHRNA9), calcium and integrin binding family member 3 (CIB3), cadherin 23 (CDH23), protocadherin 15 (PCDH15), kinocilin (KNCN), peyvakin (Pejvakin) (DFNB59), otoferlin (OTOF), MKRN2 inverse chain (MKRN2OS), LIM homeobox protein 3 (LHX3), transmembrane channel-like 1 (TMC1), myosin 15 (MYO15), myosin 7A (MYO7A), myosin 6 (MYO6), myosin IIIA (MYO3A), myosin IIIB (MYO3B), glutaredoxin domain-containing cysteine-rich protein 1 (GRXCR1), protein tyrosine phosphatase, receptor type Q (PTPRQ), late corneal envelope 6A (LCE6A), lipoxygenase LOXHD1 homology domain-containing protein 1 (LOXHD1), ADP-ribosyltransferase 1 (ART1), ATPase plasma membrane Ca2+ transporter 2 (ATP2B2), calcium and integrin binding family member 2 (CIB2), calcium voltage-dependent channel auxiliary subunit α2δ4 (CACNA2D4), calcium binding protein 2 (CABP2), epidermal growth factor receptor pathway substrate 8 (EPS8), EPS8-like 2 (EPS8L2), espin (ESPN), espin-like (ESPNL), peripherin 2 (PRPH2), stereocillin (STRC), solute carrier family 8 member A2 (SLC8A2), zinc finger CCHC-type-containing protein 12 (ZCCHC12), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT2, LRTOMT1), USH1 protein network component harmonin (USH1C),Extracellular leucine-rich repeat and fibronectin type III domain-containing 1 (ELFN1), tetratricopeptide repeat protein 24 (TTC24), dystrotelin (DYTN), chierin / chordin-like protein (KCP), coiled-coil glutamic acid-rich protein 2 (CCER2), leucine-rich repeat and transmembrane domain-containing protein 2 (LRTM2), potassium voltage-gated channel subfamily A member 10 (KCNA10), neurotrophin 3 (NTF3), cularin 1 (CLRN1), cularin 2 (CLRN2), SKI family transcriptional corepressor 1 (SKOR1), Tctex1 domain-containing protein 1 (TCTEX1D1), Fc The gene encoding the protein is selected from the group consisting of: FcCRLB, solute carrier family 17 member 8 (SLC17A8), glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2), brain-derived neurotrophic factor (BDNF), serpin family E member 3 (SERPINE3), Nescient helix-loop-helix 1 (NHLH1), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), activating transcription factor 6 (ATF6), eukaryotic translation initiation factor 2 alpha kinase 3 (PERK), serine / threonine-protein kinase / endoribonuclease IRE1 (IRE1), and binding immunoglobulin protein (BIP).

[0210] Expression of exogenous nucleic acids in mammalian cells Mutations in various genes, such as MYO7A, POU4F3, SLC17A8, and TMC1, have been associated with sensorineural hearing loss, and some of these mutations, such as mutations in MYO7A, have also been associated with vestibular dysfunction. The compositions and methods described herein can be used to construct nucleic acid vectors containing the Myol5 promoter operably linked to a nucleic acid sequence encoding a protein of interest (e.g., a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:1, or a functional portion or derivative thereof, and / or a nucleic acid sequence having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:2, or a functional portion or derivative thereof). %, 97%, 98%, 99%, or more), and optionally a linker joining the first and second regions, can be administered to induce or increase expression of a protein encoded by a gene of interest (e.g., a wild-type form of a gene involved in hearing loss and / or vestibular dysfunction, or a gene involved in hair cell development, function, cell fate specification, regeneration, survival, or maintenance) specifically in hair cells (e.g., cochlear hair cells and / or vestibular hair cells). A wide range of methods has been established for the delivery of proteins to mammalian cells and the stable expression of genes encoding proteins in mammalian cells.

[0211] Proteins that may be expressed in connection with the compositions described herein are proteins expressed in healthy hair cells (e.g., cochlear hair cells and / or vestibular hair cells, e.g., proteins that play a role in hair cell development, function, regeneration, cell fate specification, survival, or maintenance, or proteins that are deficient in subjects with sensorineural hearing loss or vestibular dysfunction) or other therapeutic proteins of interest (e.g., when a transgene encoding the protein is operably linked to a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1 or a functional portion or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion or derivative thereof). Proteins that can be expressed in hair cells using the compositions and methods described herein include ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CA CNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

[0212] A polynucleotide encoding a protein of interest One platform that can be used to achieve therapeutically effective intracellular concentrations of a protein of interest in mammalian cells is by stable expression of a gene encoding the protein of interest (e.g., by integration into the nuclear or mitochondrial genome of the mammalian cell, or by episomal concatemer formation in the nucleus of the mammalian cell). A gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein.

[0213] To introduce an exogenous gene into mammalian cells, the gene can be incorporated into a vector. Vectors can be introduced into cells by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and encapsulation of the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described, for example, in Green, et al., Molecular Cloning: A Laboratory Manual. Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014), and Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0214] Proteins of interest can also be introduced into mammalian cells by targeting the vector containing the gene encoding the protein of interest to cell membrane phospholipids.For example, the vector can target the phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to the VSV-G protein, a viral protein that has affinity for all cell membrane phospholipids.Such constructs can be produced using methods well known to those skilled in the art.

[0215] Recognition and binding of a polynucleotide encoding a protein of interest by mammalian RNA polymerase is important for gene expression. Therefore, a polynucleotide may contain sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and promote assembly of a transcription complex at the transcription initiation site. Such sequence elements include, for example, mammalian promoters, whose sequences can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters are described in Smith, et al., Mol. Sys. Biol., 3:73, published online, the disclosure of which is incorporated herein by reference. A promoter used in the methods and compositions described herein is a polynucleotide that contains a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1 or a functional portion or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion or derivative thereof, and optionally containing a linker between the first and second regions.

[0216] Once a polynucleotide encoding a protein of interest has been integrated into the nuclear DNA of a mammalian cell, transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing mammalian cells to an external chemical reagent, such as an agent that modulates the binding of transcription factors and / or RNA polymerase to a mammalian promoter, thereby regulating gene expression. The chemical reagent can serve to promote the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example, by removing promoter-bound repressor proteins. Alternatively, the chemical reagent can serve to increase the affinity of the mammalian promoter for RNA polymerase and / or transcription factors, such that the transcription rate of genes located downstream of the promoter increases in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells to promote gene expression according to established protocols.

[0217] Other DNA sequence elements that can be included in polynucleotides for use in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in polynucleotides, including genes of interest, such that the DNA adopts a three-dimensional orientation favorable for the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein, including those encoding proteins of interest, can also include mammalian enhancer sequences. Many enhancer sequences are now known from mammalian genes, including enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce the activation of eukaryotic gene transcription include the CMV enhancer and the RSV enhancer. The enhancer can be spliced ​​into a vector containing a polynucleotide encoding a protein of interest, for example, at a 5' or 3' position relative to the gene. In a preferred orientation, the enhancer is located 5' from the promoter, and the promoter is then located 5' from the polynucleotide encoding the protein of interest.

[0218] Nucleic acid vectors containing the Myo15 promoter described herein may contain a woodchuck posttranscriptional regulatory element (WPRE). The WPRE acts at the transcriptional level by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of nascent transcripts, thus increasing the total amount of mRNA in the cell. Addition of a WPRE to a vector can result in substantial improvements in the level of transgene expression from several different promoters, both in vitro and in vivo.

[0219] In some embodiments, nucleic acid vectors containing the Myo15 promoter described herein include a reporter sequence that can be useful, for example, for verifying expression of a gene operably linked to the Myo15 promoter in cells and tissues (e.g., hair cells, such as cochlear hair cells and / or vestibular hair cells). Reporter sequences that can be provided in a transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When associated with a regulatory element that drives expression, such as the Myo15 promoter, the reporter sequence provides a signal that is detectable by conventional means, including enzymatic, radioactive, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, if the marker sequence is the LacZ gene, the presence of the signal-bearing vector can be detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-bearing vector can be visually measured by color or light production in a luminometer.

[0220] Methods for delivery of exogenous nucleic acids into target cells Techniques that can be used to introduce a transgene, for example, a transgene operably linked to the Myol5 promoter described herein, into target cells (e.g., mammalian cells) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the target cells. Mammalian cells, such as human cells, that are subjected to an external electric field in this manner are then predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection™ and protocols useful for performing this technique are described, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and US2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0221] Additional techniques useful for transfection of target cells include squeezing-perforation.This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress.This technique is advantageous in that no vector is required for the delivery of nucleic acid into cells, such as human target cells.Squeezing-perforation is described in detail in, for example, Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0222] Lipofection is another technique useful for transfecting target cells. This method involves loading nucleic acid into liposomes, which often present cationic functional groups, such as quaternary amines or protonated amines, on the outside of the liposome. This promotes electrostatic interactions between liposomes and cells due to the anionic nature of the cell membrane, ultimately leading to the uptake of exogenous nucleic acid, for example, by direct fusion of liposomes with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acid includes contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that favors interaction with cell membranes include activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described, e.g., in Gulick et al., Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference. Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Because this methodology utilizes an applied magnetic field to direct the uptake of nucleic acids, magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner. This technology is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0223] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laser transfection, also known as phototransfection, which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize cells and allow polynucleotides to penetrate the cell membrane.The biological activity of this technique is similar to that of electroporation, and in some cases, is found to be superior to that of electroporation.

[0224] Imparefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing genes intended for intracellular delivery is attached to the nanostructure surface. A chip with an array of these needles is then pressed against cells or tissues. Cells impaled by the nanostructures can express the delivered gene(s). An example of this technique is described in Shalek et al. PNAS 107:1870 (2010), the disclosure of which is incorporated herein by reference.

[0225] Magnetofection can also be used to deliver nucleic acids to target cells. The principle of magnetofection is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made entirely of biodegradable iron oxide and are coated with specific cationic specific molecules that vary depending on the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated in the target cells by the influence of an external magnetic field generated by a magnet. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0226] Another useful tool for inducing the uptake of exogenous nucleic acid by target cells is sonoporation, which is a technique that involves the use of sound (typically ultrasonic frequency) to modify the permeability of cell plasma membrane to make the cell permeable, allowing polynucleotide to penetrate the cell membrane.This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0227] Microvesicles represent another potential vehicle that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of the glycoprotein VSV-G with a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins to cells that subsequently catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the cell's genome for covalent integration of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also called gesicles, for the genetic modification of eukaryotic cells is described, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract], Methylation changes in early embryonic genes in cancer [abstract], and in Proceedings of the 18th This is described in detail in the Annual Meeting of the American Society of Gene and Cell Therapy; May 13, 2015, Abstract No. 122.

[0228] Vectors for delivery of exogenous nucleic acids to target cells In addition to achieving high transcription and translation rates, stable expression of exogenous genes in mammalian cells can be achieved by integrating a polynucleotide containing the gene into the nuclear genome of the mammalian cell. A variety of vectors have been developed for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of expression vectors can be found, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors for use in the compositions and methods described herein contain a Myo15 promoter (e.g., a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:1, or a functional portion or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:2, or a functional portion or derivative thereof, optionally containing a linker between the first and second regions) operably linked to a polynucleotide sequence encoding a protein of interest, and additional sequence elements used, for example, for expression of these agents and / or integration of these polynucleotide sequences into the genome of a mammalian cell. Vectors that can contain the Myo15 promoter operably linked to a transgene encoding a protein of interest include plasmids (e.g., circular DNA molecules capable of autonomous replication within cells), cosmids (e.g., pWE or sCos vectors), artificial chromosomes (e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), and viral vectors. Certain vectors that can be used to express proteins of interest include plasmids containing regulatory sequences, such as enhancer regions, that direct gene transcription. Other useful vectors for expressing proteins of interest contain polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRESs), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector.Expression vectors suitable for use with the compositions and methods described herein may contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0229] Viral vectors for nucleic acid delivery Viral genomes provide a rich source of vectors that can be used to efficiently deliver genes of interest into the genome of target cells (e.g., mammalian cells, such as human cells).Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within these genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction.These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Examples of viral vectors include negative-strand RNA viruses such as retroviruses (e.g., retrovirus family viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma virus, avian C virus, mammalian C virus, B virus, D virus, oncoretrovirus, HTLV-BLV complex, lentivirus, alpharetrovirus, gammaretrovirus, and spumavirus (Coffin, J.M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon monkey leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which is incorporated herein by reference as it relates to viral vectors for use in gene therapy.

[0230] AAV vectors for nucleic acid delivery In some embodiments, the polynucleotides of the compositions and methods described herein are incorporated into rAAV vectors and / or virions to facilitate their introduction into cells. The rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that include (1) a Myo15 promoter described herein (e.g., a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1 or a functional portion or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion or derivative thereof, optionally containing a linker between the first and second regions), (2) a heterologous sequence to be expressed, and (3) viral sequences that promote the stability and expression of the heterologous gene. Viral sequences may include sequences of AAV required in cis for replication and packaging of DNA into virions (eg, functional ITRs).

[0231] In typical applications, the transgene encodes a therapeutic protein that can promote hair cell development, hair cell function, hair cell regeneration, hair cell fate specification, hair cell survival, or hair cell maintenance, or a wild-type form of a hair cell protein that is mutated in subjects with a form of inherited hearing loss or vestibular dysfunction, which may be useful for improving hearing or vestibular function in subjects with mutations associated with hearing loss, hearing loss, or vestibular dysfunction (e.g., dizziness, vertigo, or imbalance). Such rAAV vectors may contain a marker or reporter gene. Useful rAAV vectors lack one or more AAV WT genes in whole or in part, but retain functional adjacent ITR sequences. AAV ITRs can be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs can be AAV2 ITRs. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0232] The polynucleotides and vectors described herein (e.g., a Myo15 promoter operably linked to a transgene encoding a protein of interest) can be incorporated into rAAV virions to facilitate the introduction of the polynucleotide or vector into cells. The capsid protein of AAV constitutes the outer, non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for virion assembly. Construction of rAAV virions is described, for example, in US Pat. No. 5,173,414, US Pat. No. 5,139,941, US Pat. No. 5,863,541, US Pat. No. 5,869,305, US Pat. No. 6,057,152, and US Pat. No. 6,376,237, as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0233] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from various AAV serotypes, including AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S. For targeting hair cells, AAV1, AAV2, AAV2quad(YF), AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, and PHP.B may be particularly useful. Serotypes evolved for transduction of the retina may also be used in the methods and compositions described herein. The construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619 (2000), Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000), Xiao et al., J. Virol. 72:2224 (1998), Halbert et al., J. Virol. 74:1524 (2000), Halbert et al., J. Virol. 75:6615 (2001), and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0234] Pseudotyped rAAV vectors are also useful in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0235] AAV virions with mutations in the virion capsid can be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations to facilitate targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in the methods described herein include those capsid hybrids generated by viral molecular hybridization and exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0236] Pharmaceutical Compositions A polynucleotide described herein (e.g., a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1 or a functional portion or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion or derivative thereof, optionally containing a linker between the first and second regions) may be operably linked to a transgene (e.g., a transgene encoding a protein of interest) or incorporated into a vehicle for administration to a patient, such as a human patient suffering from sensorineural hearing loss and / or vestibular dysfunction. Pharmaceutical compositions containing a vector, such as a viral vector, containing a polynucleotide described herein operably linked to a therapeutic transgene can be prepared using methods known in the art. For example, such compositions can be prepared in a desired form, such as a lyophilized formulation or aqueous solution, using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013), incorporated herein by reference).

[0237] Mixtures of nucleic acid vectors (e.g., viral vectors) containing a polynucleotide described herein (e.g., a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1 or a functional portion or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion or derivative thereof, optionally containing a linker between the first and second regions) operably linked to a therapeutic transgene may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (as described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference).

[0238] In all cases, the formulation may be sterile and fluid to the extent that easy syringability exists. The formulation may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0239] In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0240] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0241] In this regard, sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure. For example, one dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous dissolution fluid or injected at the proposed injection site. Some dosage variation will inevitably occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition can be formulated to contain synthetic perilymph. An exemplary synthetic perilymph solution contains 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl, 1-10 mM glucose, and 2-50 mM HEPE, with a pH of approximately 6-9 and an osmolality of approximately 300 mOsm / kg. The administering individual will, in any event, determine the appropriate dose for the individual subject. Furthermore, for human administration, preparations can meet sterility, pyrogenicity, general safety, and purity standards required by FDA Office of Biologics standards.

[0242] Treatment method The compositions described herein can be administered to a subject with sensorineural hearing loss and / or vestibular dysfunction by a variety of routes, including local administration to the inner ear (e.g., administration to the perilymph or endolymph via the vestibular window, the cochlear window, or a semicircular canal (e.g., the horizontal canal), e.g., administration to cochlear or vestibular hair cells), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most suitable route for administration in any given case depends on the particular composition being administered, the patient, the pharmaceutical formulation, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. The compositions may be administered once or more than once (e.g., once per year, twice per year, three times per year, once every two months, or once per month).

[0243] Subjects who can be treated as described herein are those who have or are at risk of developing sensorineural hearing loss and / or vestibular dysfunction (e.g., subjects who have or are at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods described herein can be used to treat subjects who have or are at risk of developing damage to cochlear hair cells (e.g., damage associated with acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging), who have or are at risk of developing damage to vestibular hair cells (e.g., damage associated with disease or infection, head trauma, ototoxic drugs, or aging), who have or are at risk of developing sensorineural hearing loss, hearing loss, or auditory neuropathy, who have or are at risk of developing vestibular dysfunction (e.g., dizziness, vertigo, or imbalance), who have tinnitus (e.g., tinnitus alone or tinnitus associated with sensorineural hearing loss or vestibular dysfunction), who have a genetic mutation associated with hearing loss and / or vestibular dysfunction, or who have a family history of hereditary hearing loss, hearing loss, auditory neuropathy, tinnitus, or vestibular dysfunction.

[0244] In some embodiments, the subject has hearing loss and / or vestibular dysfunction associated with or resulting from loss of hair cells (e.g., cochlear hair cells or vestibular hair cells). The methods described herein may include screening the subject for mutations in genes known to be associated with hearing loss or vestibular dysfunction prior to treatment or administration with a composition described herein. Subjects can be screened for genetic mutations using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein may also include assessing hearing and / or vestibular function in the subject prior to treatment or administration with a composition described herein. Hearing can be assessed using standard tests such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions. Vestibular function can be assessed using eye movement tests (e.g., electronystagmography (ENG) or videonystagmography (VNG)), posturography, rotary chair testing, ECOG, vestibular-evoked myogenic potentials (VEMPs), and other methods described, for example, by Mancini and Hearing loss and / or vestibular dysfunction may be assessed using standard tests, such as a specialized clinical balance test, such as that described in Horak, Eur J Phys Rehabil Med, 46:239 (2010). The compositions and methods described herein may also be administered as a preventative treatment to patients at risk of developing hearing loss and / or vestibular dysfunction, for example, patients with a family history of hearing loss or vestibular dysfunction (e.g., genetic hearing loss or vestibular dysfunction), patients who have a genetic mutation associated with hearing loss or vestibular dysfunction but have not yet exhibited hearing loss or vestibular dysfunction, or patients exposed to risk factors for acquired hearing loss (e.g., disease or infection, head trauma, ototoxic drugs, or aging) or vestibular dysfunction (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging).

[0245] The compositions and methods described herein can be used to promote or induce hair cell regeneration (e.g., cochlear hair cell and / or vestibular hair cell regeneration) in a subject. Subjects that can benefit from compositions that promote or induce hair cell regeneration include those suffering from hearing loss or vestibular dysfunction as a result of hair cell loss (e.g., hair cell loss associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), and those with reduced hair cell numbers due to hair cell abnormalities (e.g., hair cells that do not function properly compared to normal hair cells), hair cell damage (e.g., hair cell damage associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase hair cell survival (e.g., to increase survival of damaged hair cells, promote repair of damaged hair cells, or preserve hair cells in subjects at risk of hair cell loss (e.g., hair cell loss due to age, exposure to loud noise, disease or infection, head trauma, or ototoxic drugs)).

[0246] The compositions and methods described herein can also be used to prevent or reduce ototoxic drug-induced hair cell damage or death (e.g., cochlear hair cell and / or vestibular hair cell damage or death) in subjects who have been treated with, are currently being treated with, or will soon begin treatment with, an ototoxic drug. Ototoxic drugs are toxic to cells of the inner ear and can cause sensorineural hearing loss, vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), tinnitus, or a combination of these symptoms. Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, antineoplastic agents (e.g., platinum-containing chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, especially at high doses), and quinine. In some embodiments, the methods described herein prevent or reduce hair cell damage or death associated with acoustic trauma, disease or infection, head trauma, or aging.

[0247] For the treatment of a subject described herein, a transgene operably linked to the Myol5 promoter (e.g., a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, or a functional portion or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or a functional portion or derivative thereof) can be a transgene encoding a protein expressed in healthy hair cells (e.g., cochlear hair cells and / or vestibular hair cells, e.g., a protein that plays a role in hair cell development, function, cell fate specification, regeneration, survival, or maintenance) or another therapeutic protein of interest. The transgene can be selected based on the cause of the subject's hearing loss or vestibular dysfunction (e.g., if the subject's hearing loss or vestibular dysfunction is associated with a particular genetic mutation, the transgene can be the wild type of the gene that is mutated in the subject, or if the subject has hearing loss that is associated with hair cell loss, the transgene can encode a protein that promotes hair cell regeneration), the severity of the subject's hearing loss or vestibular dysfunction, the health of the subject's hair cells, the subject's age, the subject's family history of hearing loss or vestibular dysfunction, or other factors.Proteins that may be expressed by a transgene operably linked to the Myo15 promoter for the treatment of a subject described herein include ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2 , CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

[0248] Treatment can involve the administration of a composition containing a nucleic acid vector (e.g., an AAV viral vector) containing the Myol5 promoter described herein in various unit doses. Each unit dose typically contains a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route of administration and formulation, is within the skill of those in the art. The unit dose need not be administered as a single injection, but can include continuous infusion over a predetermined period of time. Administration can be carried out using a syringe pump to control the rate of infusion, minimizing damage to the inner ear (e.g., the cochlea). If the nucleic acid vector is an AAV vector (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, or PHP.S vector), ), the viral vector can be present in a volume of 1 μL to 200 μL (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μL), e.g., about 1 × 10 10 Vector genome (VG) ~ 1 x 10 15 VG (e.g., 1 × 10 10 VG, 2×10 10 VG, 3×10 10 VG, 4×10 10 VG, 5×10 10 VG, 6×10 10 VG, 7×10 10 VG, 8×10 10 VG, 9×10 10 VG, 1×10 11 VG, 2×10 11 VG, 3×10 11 VG, 4×10 11 VG, 5×10 11 VG, 6×10 11 VG, 7×10 11 VG, 8×10 11 VG, 9×10 11 VG, 1×10 12VG、2×10 12 VG、3×10 12 VG、4×10 12 VG、5×10 12 VG、6×10 12 VG、7×10 12 VG、8×10 12 VG、9×10 12 VG、1×10 13 VG、2×10 13 VG、3×10 13 VG、4×10 13 VG、5×10 13 VG、6×10 13 VG、7×10 13 VG、8×10 13 VG、9×10 13 VG、1×10 14 VG、2×10 14 VG、3×10 14 VG、4×10 14 VG、5×10 14 VG、6×10 14 VG、7×10 14 VG、8×10 14 VG、9×10 14 VG、1×10 15 VG).

[0249] The compositions described herein are administered in an amount sufficient to improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), reduce tinnitus, increase expression of a therapeutic protein encoded by a transgene, increase function of a therapeutic protein encoded by a transgene, prevent or reduce hair cell damage, prevent or reduce hair cell death (e.g., ototoxic drug-induced hair cell death, age-related hair cell death, or noise (e.g., acoustic trauma)-associated hair cell death), promote or increase hair cell development, increase hair cell number (e.g., promote or induce hair cell regeneration), increase or promote hair cell survival, or improve hair cell function. Hearing may be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions) and may improve by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to hearing measurements obtained before treatment. Vestibular function can be assessed using standard tests for balance and vertigo (e.g., oculomotor testing (e.g., ENG or VNG), posturography, rotary chair testing, ECOG, VEMP, and specialized clinical balance testing) and can improve by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to measurements obtained before treatment.

[0250] In some embodiments, the composition is administered in an amount sufficient to improve the subject's ability to understand speech. The compositions described herein may also be administered in an amount sufficient to slow or prevent the onset or progression of sensorineural hearing loss and / or vestibular dysfunction (e.g., in subjects with a genetic mutation associated with hearing loss or vestibular dysfunction, subjects with a family history of hearing loss or vestibular dysfunction (e.g., genetic hearing loss or vestibular dysfunction), or subjects who have been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, acoustic trauma, or infection) but who do not exhibit hearing loss or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), or in subjects who exhibit mild to moderate hearing loss or vestibular dysfunction). Expression of a therapeutic protein encoded by a transgene operably linked to the Myol5 promoter in a nucleic acid vector administered to a subject may be assessed using immunohistochemistry, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detecting protein or mRNA, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to expression before administration of a composition described herein. The hair cell number, hair cell function, or function of a therapeutic protein encoded by a nucleic acid vector administered to a subject can be indirectly assessed based on hearing tests or vestibular function tests, and can be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to the hair cell number, hair cell function, or function of the therapeutic protein before administration of a composition described herein. Hair cell damage or death can be reduced by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to the hair cell damage and death typically observed in untreated subjects.These effects may occur, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more weeks after administration of the compositions described herein. Patients may be evaluated 1, 2, 3, 4, 5, 6, or more months after administration of the compositions, depending on the dose and route of administration used for treatment. Depending on the results of the evaluation, patients may receive additional treatment.

[0251] kit The compositions described herein can be provided in a kit for use in treating sensorineural hearing loss or vestibular dysfunction. The compositions can include a polynucleotide described herein (e.g., a polynucleotide containing a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO: 1 or a functional portion or derivative thereof, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO: 2 or a functional portion or derivative thereof, optionally containing a linker between the first and second regions), a nucleic acid vector containing such a polynucleotide, and a nucleic acid vector containing a polynucleotide described herein operably linked to a transgene encoding a protein of interest (e.g., a protein that can be expressed in hair cells to treat hearing loss and / or vestibular dysfunction). The nucleic acid vector may be packaged in an AAV viral capsid (e.g., AAV1, AAV2, AAV2quad(YF), AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B). The kit may further include a package insert instructing a user of the kit, such as a physician, to practice the methods described herein. The kit may optionally include a syringe or other device for administering the composition. [Example]

[0252] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0253] Example 1: Generation of the mouse Myo15 promoter Evolutionarily conserved regions in the vertebrate Myo15 promoter were first identified using the UCSC genome browser (genome.ucsc.edu). The region immediately upstream of the Myo15 translation start site (-1 to -1157, SEQ ID NO: 1) and the upstream region containing non-coding exon 1 of the Myo15 gene (-6755 to -7209, SEQ ID NO: 2) were synthesized and spliced ​​together as a single DNA fragment by de novo gene synthesis (SEQ ID NO: 13). The total size of the truncated Myo15 promoter is 1611 bp versus over 7000 bp for the entire genomic region.

[0254] Experiments evaluating the tropism (cell type targeting) and extent and duration of transgene expression by the Myo15 promoter compared to the cytomegalovirus (CMV) promoter in the mouse cochlea revealed that the Myo15 promoter, but not the CMV promoter, resulted in selective expression in cochlear hair cells. AAV constructs were transfected into Aequorea We generated a Myo15-driven AAV construct using AcGFP, a CMV-based green fluorescent protein, and analyzed the progression of gene expression relative to a matched standard AAV construct. Transgene expression was assessed in experiments in which the virus was delivered to the mouse cochlea in neonatal mice, adult mice, and ex vivo in cochlear explants.

[0255] To assess transgene expression, AAV-Myo15-GFP virus was injected into 6-8 week-old C57Bl / 6J male mice via the posterior semicircular canal. After recovery from surgery, mice were euthanized and perfused with 10% normal buffered formalin 10 days later. The cochlea and vestibular system were harvested and decalcified in 8% EDTA for 3 days. The cochlea or vestibular system was dissected from the decalcified temporal bone and mounted on slides for imaging. Using a ubiquitous promoter, AAV-CMV-GFP induced GFP expression in many cell types within the cochlea, including inner hair cells, outer hair cells, spiral ganglion neurons, mesenchymal cells, and glia (Figure 1A). Using a hair cell-specific promoter, AAV-Myo15-GFP induced expression only in inner and outer hair cells (Figure 1B).

[0256] In the vestibular system, AAV-Myo15-GFP induced expression only in vestibular hair cells (Fig. 2 ).

[0257] Example 2: Generation of a minimal Myo15 promoter A series of promoters are generated and placed upstream of a fluorescent reporter (e.g., GFP, AcGFP, or luciferase). The promoters generated include: 1) SEQ ID NO:3 fused to SEQ ID NO:2; 2) SEQ ID NO: 4 fused to SEQ ID NO: 2; 3) a fusion of SEQ ID NO:3 and SEQ ID NO:4 (e.g., SEQ ID NO:5, 6, or 7) fused to SEQ ID NO:2; 4) SEQ ID NO: 8 fused to SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12); 5) SEQ ID NO: 1 fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12); 6) SEQ ID NO: 3 fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12); 7) SEQ ID NO: 4 fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12); 8) a fusion of SEQ ID NO:3 and SEQ ID NO:4 (e.g., SEQ ID NO:5, 6, or 7) fused to a fusion of SEQ ID NO:8 and SEQ ID NO:9 (e.g., SEQ ID NO:10, 11, or 12); 9) a fusion of SEQ ID NO: 3 and SEQ ID NO: 4 (e.g., SEQ ID NO: 5, 6, or 7); 11) SEQ ID NO: 1; 12) SEQ ID NO: 2; 13) SEQ ID NO: 17, 14) SEQ ID NO: 18, 15) SEQ ID NO: 17 fused to SEQ ID NO: 18; 16) SEQ ID NO: 18 fused to SEQ ID NO: 17; 17) SEQ ID NO: 19 fused to SEQ ID NO: 18; 18) SEQ ID NO: 20 (e.g., SEQ ID NO: 22, 23, or 24) fused to SEQ ID NO: 21; 19) SEQ ID NO: 17 fused to a fusion of SEQ ID NO: 20 and SEQ ID NO: 21 (e.g., SEQ ID NO: 22, 23, or 24), and 20) SEQ ID NO: 19 fused to a fusion of SEQ ID NO: 20 and SEQ ID NO: 21 (e.g., SEQ ID NO: 22, 23, or 24). The promoter construct is packaged into an AAV serotype capable of transducing hair cells (e.g., AAV1, AAV2, AAV6, AAV9, Anc80, or Anc80L65).

[0258] The viral promoter constructs are used to infect organotypic cochlear explants. After 48 hours of incubation with the virus, the explants are imaged and analyzed using reporter fluorescence intensity to measure hair cell-specific expression.

[0259] Example 3: Administration of a composition containing a nucleic acid vector containing the Myo15 promoter to a subject with sensorineural hearing loss According to the methods disclosed herein, one skilled in the art can treat a patient, such as a human patient, with sensorineural hearing loss to improve or restore hearing. To this end, one skilled in the art can identify a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7, e.g., SEQ ID NOs: 3 and 4), and / or a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, e.g., SEQ ID NOs: 8 and 9). A composition containing an AAV vector (e.g., AAV1, AAV2, AAV2quad(YF), AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B) containing a polynucleotide operably linked to a transgene encoding a therapeutic protein, the polynucleotide containing a second region having a sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) and optionally containing a linker between the first and second regions, can be administered to a human patient. For example, the polynucleotide operably linked to the transgene encoding the therapeutic protein can be SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 36, or SEQ ID NO: 37. The composition containing the AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph) to treat sensorineural hearing loss.

[0260] After administering the composition to a patient, those skilled in the art can monitor the expression of the therapeutic protein encoded by the transgene and the patient's improvement in response to treatment by various methods. For example, a physician can monitor the patient's hearing after administering the composition by performing standard tests such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions. A finding that the patient shows improved hearing in one or more tests after administration of the composition compared to the hearing test results before administration of the composition indicates that the patient is responding favorably to treatment. Subsequent doses can be determined and administered as needed.

[0261] Example 4: Mouse Myo15 promoter sequence containing SEQ ID NO: 1 and part of SEQ ID NO: 2 directs transgene expression in hair cells. Neonatal cochlear explants were established from P0 wild-type CD1 mice. Briefly, neonatal mice were euthanized according to an IACUC-approved protocol. The inner ear was removed from the head, and the cochlea was carefully extracted using microdissection. The modiolus was removed from the center of the coil, and the apex of the cochlear duct was removed, exposing the sensory epithelium. Cochlear explants were placed on a Mat-tek dish coated with 15% Matrigel, with the luminal side of the cochlear duct facing up. Cochlear explant culture medium, consisting of DMEM containing 10% FBS and 10 μg / mL ciproflaxin, was added directly to the cultures. Viruses containing the Myo15 1kb (SEQ ID NO: 15) promoter or the Myo15 mini-intron (SEQ ID NO: 16) promoter (AAV2 / 9-Myo15 1kb-GFP or AAV2 / 9-Myo15 mini-intron-GFP) were added to the culture medium at a concentration of 1E+10 genome copies / mL. Cultures were incubated in the virus-containing medium at 37°C under standard culture conditions of 5% CO2 for 48 hours. Cultures were then fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before antibody staining. Antibodies used: rabbit anti-Myo6 (Proteus), donkey anti-rabbit Alexa 568 (Thermo As shown in Figures 3A-3B, both the Myo15 1 kb (SEQ ID NO: 15) and Myo15 mini-intron (SEQ ID NO: 16) promoters induced GFP expression specifically in hair cells.

[0262] Example 5: The Myol5 promoter sequence drives transgene expression in hair cells in the mouse cochlea. Neonatal cochlear explants were established from P0 wild-type B6.CAST-Cdh23A1+ / Kjn mice. Briefly, neonatal mice were euthanized according to an IACUC-approved protocol. The inner ear was removed from the head, and the cochlea was carefully extracted using microdissection. The modiolus was removed from the center of the coil, and the apex of the cochlear duct was removed, exposing the sensory epithelium. Cochlear explants were placed on the prepared 3D rat tail collagen matrix, with the luminal side of the cochlear duct facing up. Cochlear explant culture medium, consisting of DMEM containing 7% FBS and 1 U / μL penicillin G, was added directly to the cultures. Viruses containing the mouse Myo15 1kb (SEQ ID NO: 15) promoter, human Myo15 0.6kb (SEQ ID NO: 26), or human Myo15 1.2kb (SEQ ID NO: 25) promoter (AAV1-Myo15 1kb-GFP, AAV1-Myo15 0.6kb-GFP, or AAV1-Myo15 1.2kb-GFP) were added to the culture medium at a concentration of 1E+11 vg / culture. Cultures were incubated in virus-containing medium under standard culture conditions at 37°C with 5% CO for 72 hours. Cultures were then fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before anti-GFP antibody staining. GFP signals as detected with anti-GFP antibodies are shown (Figures 4A-4C). In a separate experiment, AAV1-1.2 kb human Myo15-GFP or AAV1-1 kb mouse Myo15-GFP was delivered into the cochlea of ​​adult mice via round window injection at a dose of 2.65E+12 vg / mL. Whole-mount images (Figures 4D-4E) or sections (Figures 4F-4G) show hair cell-specific signals in the apex, middle, and base of the cochlea, as detected with an anti-GFP antibody. The arrow in Figure 4F points to a GFP-labeled cochlear hair cell. As shown in Figures 4F and 4G, expression of both the 1.2 kb human Myo15 promoter and the 1 kb mouse Myo15 promoter was primarily restricted to inner hair cells.

[0263] Example 6: The mouse Myol5 promoter sequence drives transgene expression in the cochlea of ​​non-human primates in vivo. The specificity of the mouse Myo15 promoter was tested in non-human primates. Thirty microliters of AAV1-CMV-GFP or AAV1-Myo15 (SEQ ID NO: 13)-GFP was injected into the cochlea through the round window membrane at 15 μL / min. Animals were sacrificed 4 weeks after AAV injection, and cochleae were harvested and processed as surface preparations to examine transgene expression without antibody enhancement. AAV1 had high infectivity. Under the ubiquitous CMV promoter, GFP was expressed in hair cells, supporting cells, and fiber cells in the lateral wall of the rhesus monkey cochlea (Figure 5A). In contrast, the 1.6 kb mouse Myo15 promoter (SEQ ID NO: 13) restricted GFP transgene expression to hair cells in the cynomolgus monkey cochlea (Figures 5B-5C).

[0264] Example 7: The mouse Myo15 promoter improves the biological efficacy of AAV mouse Tmc1 in Tmc1 knockout mice compared to the ubiquitous promoter. Tmc1 knockout (KO) mice were anesthetized with isoflurane, their hair clipped, and povidone-iodine applied to the skin. An incision was made below the left ear, over the cheek muscle and behind the ear. The skin was separated, and the muscles were loosened and cleaned to expose the posterior sulcus. A small hole was drilled in the tubing using a drill bit, and the bone was dried with a fine cotton tip. A polyamide / polyethylene tubing was inserted into the hole and sealed with bone glue. Using a micropump equipped with a Hamilton syringe, 1 μL of vector (AAV-CMV1 mouse TMC1 or AAV-Myo15 (SEQ ID NO: 13) mouse TMC1) and 0.05 μL of trypan blue were delivered into the IL space at a rate of 100 nL / min. Five minutes were allowed to elapse after delivery of 1 μL to allow the fluid to reach the apex of the cochlea and prevent backflow and leakage. The tubing was bent and cut close to the bone. The muscle was retracted and the skin was glued back into place. Mice were given 0.01 cc of meloxicam and then allowed to recover under a heat lamp. Animals were checked for signs of pain or infection for 5 days postoperatively. Animals 21–28 days postoperatively were anesthetized with ketamine and xylazine and auditory brainstem responses (ABRs) were measured. As shown in Figure 6, ABR threshold restoration was significantly improved in homozygous Tmc1 KO mice injected with AAV-Myo15-mouseTMC1 compared to AAV-CMV-mouseTMC1.

[0265] Example 8: Human Myo15 promoter sequences drive transgene expression in the cochlea of ​​non-human primates in vivo. A composition containing an AAV1 vector carrying an EGFP transgene under the control of a 1.2 kb human myosin 15 promoter (SEQ ID NO: 25) or a 0.6 kb human myosin 15 promoter (SEQ ID NO: 26) was delivered bilaterally to the inner ear of cynomolgus monkeys by injection through the round window membrane. AAV1-1.2 kb myosin 15-GFP (1.06 × 10 13 viral titer in genome copies / mL) or AAV1-0.6kb myosin 15-GFP (1.22 × 10 13Injections (30 μL) of 1.2 kb myosin 15-GFP (viral titer in genome copies / mL) were performed at an injection rate of 15 μL / min. Four weeks after injection, the inner ear was removed and a basilar membrane surface preparation was performed. GFP expression was used to monitor myosin 15 promoter activity (Figures 7A and 8A, one representative ear is shown). Transduction of the inner ear of cynomolgus monkeys with AAV1-1.2 kb myosin 15-GFP resulted in GFP expression throughout the base-apical axis of the cochlea, with high frequencies at the base and low frequencies at the apex (Figure 7A, frequency in kHz). High-magnification images at 2.8 kHz showed that GFP expression was observed within inner hair cells (IHCs) and outer hair cells (OHCs) (Figure 7B, upper panel). Similarly, AAV1-0.6 kb myosin15-GFP transduction of the inner ear of cynomolgus monkeys resulted in GFP expression throughout the base-apical axis of the cochlea, with high frequencies at the base and low frequencies at the apex (Figure 8A, frequency in kHz). High-magnification images at 2.8 kHz showed that GFP expression was observed within IHCs and OHCs (Figure 8B, upper panel). Immunohistochemistry for myosin 7A (Myo7A) was used to visualize hair cells, and nuclei were stained with DAPI (Figures 7B and 8B, middle and lower panels).

[0266] Example 9: Myol5 promoter variants drive transgene expression in mouse cochlear explants. Neonatal cochlear explant cultures and viral transduction Neonatal cochlear explants were established from P0 wild-type B6.CAST-Cdh23Ah1+ / Kjn mice. Briefly, neonatal mice were euthanized according to a protocol approved by the Animal Care and Use Committee. The inner ear was removed from the head, and the cochlea was carefully extracted using microdissection. The modiolus was removed from the center of the coil, and the apex of the cochlear duct was removed, exposing the sensory epithelium. Cochlear explants were placed on the prepared 3D rat tail collagen matrix, with the luminal side of the cochlear duct facing up. Cochlear explant culture medium, consisting of DMEM containing 7% FBS and 1 U / μL penicillin G, was added directly to the cultures. Mouse Myo15 1 kb (SEQ ID NO: 15) promoter, human Myo15 0.6 kb (SEQ ID NO: 26), human Myo15 1.2 kb (SEQ ID NO: 25) promoter, mouse Myo15 0.5 kb promoter (SEQ ID NO: 16), mouse Myo15 A promoter (SEQ ID NO: 27), mouse Myo15 B promoter (SEQ ID NO: 28), mouse Myo15 C promoter (SEQ ID NO: 29), mouse Myo15 AB promoter (SEQ ID NO: 30), mouse Myo15 AC promoter (SEQ ID NO: 31), mouse Myo15 BC promoter (SEQ ID NO: 32), mouse Myo15 BAC promoter (SEQ ID NO: 33), mouse Myo15 CAB promoter (SEQ ID NO: 34), or mouse Myo15 BCA promoter (SEQ ID NO: 35, AAV1-Myo15 1 kb-GFP, AAV1-Myo15 0.6 kb-GFP, AAV1-Myo15 Viruses containing AAV1-Myo15 1.2kb-GFP, AAV1-Myo15 0.5kb-GFP, AAV1-Myo15 A-GFP, and AAV1-Myo15 B-GFP were added to the vector at 1 × 10 11 The virus was added to the culture medium at a concentration of vg / culture. Cultures were incubated in the virus-containing medium for 72 hours at 37°C under standard culture conditions in 5% CO2.

[0267] GFP quantification After 72 hours of incubation in virus-containing medium, cultures were fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before anti-Myo7a and / or anti-GFP antibody. Samples were imaged using a confocal microscope (Zeiss LSM 810) with uniform settings (laser intensity and gain) across samples. GFP intensity values ​​per cell were quantified using ImageJ.

[0268] mRNA quantification After 72 hours of incubation in virus-containing medium, cultures were flash-frozen on dry ice, and RNA was extracted using a PicoPure RNA isolation kit (Arcturus catalog no. 0204). GFP and Myo7A mRNA were quantified by qPCR using the Quantinova Probe RT-PCR kit (Qiagen catalog no. 208354) for all cDNA synthesis and RT-PCR reactions. GFP expression levels were normalized to Myo7a expression.

[0269] result Quantitation of GFP expression from the mouse Myo15 promoter, which has hair cell-specific expression, using quantitative PCR (qPCR) showed that the expression levels of the GFP transgene from the mMyo15 1.6 kb, 1 kb, and 0.5 kb promoters were robust and comparable in extent to each other in GFP expression (Figure 9). Comparison of GFP expression driven by various mouse or human Myo15 promoters showed differences in GFP expression in Myo7a-positive hair cells, with the mMyo15 1 kb promoter resulting in the highest level of GFP expression per cell (Figure 10).

[0270] Analysis of whole-mount images of neonatal cochlear explant cultures infected with a subset of the Myo15 promoter variants listed in Table 2 indicated that the promoter sequence of SEQ ID NO:27 (Myo15 A) is neither sufficient nor necessary for general promoter activity or hair cell specificity, as shown in Figures 11B (SEQ ID NO:27), 11E (SEQ ID NO:30), 11F (SEQ ID NO:31), and 11G (SEQ ID NO:32). The promoter sequence of SEQ ID NO:27 provides a splice donor site that increases expression levels when paired with a splice acceptor site, thus introducing an intron into the promoter sequence, as shown in Figures 11A (SEQ ID NO:16) and 11H (SEQ ID NO:33). The promoter sequence of SEQ ID NO:28 (Myo15 B) alone was found to be a core promoter that conferred general promoter activity, as shown in Figure 11C (SEQ ID NO:28), but did not drive hair cell-specific GFP expression in the absence of the promoter sequence of SEQ ID NO:29 (Myo15 C), as shown in Figures 11C (SEQ ID NO:29) and 11G (SEQ ID NO:32). The promoter sequence of SEQ ID NO:29 (Myo15 C) was found to be an enhancer that restricted the activity of the core promoter (SEQ ID NO:28) to inner and outer hair cells, as shown in Figure 11G (SEQ ID NO:32), but was not sufficient for general promoter activity in the absence of the core promoter, as shown in Figure 11D (SEQ ID NO:29). The promoter sequences of SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 were rearranged in various combinations and still resulted in hair cell-specific promoter activity as shown in Figures 11H (SEQ ID NO:33) and 11I (SEQ ID NO:34), although not all rearrangements were tolerated as shown in Figure 11J (SEQ ID NO:35). These data are summarized in Table 3 below. [Table 3]

[0271] Myo15 promoter activity was further evaluated to determine the expression intensity of Myo15 promoter variants that exhibit hair cell specificity, including Myo15 0.5kb (SEQ ID NO: 16), Myo15 BC (SEQ ID NO: 32), and Myo15 BAC (SEQ ID NO: 33). The expression levels of the GFP reporter from the Myo15 0.5kb, Myo15 BC, and Myo15 BAC promoters were robust and comparable to each other. The Myo15 CAB promoter was generated by rearranging the positions of the splice donor and acceptor sites, resulting in cryptic splicing of the GFP cDNA toward the middle, as evidenced by higher expression levels detected with a GFP probe located at the 3' end of the transcript versus the 5' end of the transcript (Figure 12).

[0272] Quantitative image analysis of GFP expression levels in Myo7a-positive hair cells was performed to evaluate the expression level of GFP per hair cell for various Myo15 promoters, including the 0.5 kb mMyol5 promoter (also referred to as the Myo15 ABC promoter; SEQ ID NO: 16), Myo15 AB promoter (SEQ ID NO: 30), Myo15 AC promoter (SEQ ID NO: 31), Myo15 BC promoter (SEQ ID NO: 32), Myo15 A promoter (SEQ ID NO: 27), Myo15 B promoter (SEQ ID NO: 28), Myo15 C promoter (SEQ ID NO: 29), Myo15 BAC promoter (SEQ ID NO: 33), Myo15 CAB promoter (SEQ ID NO: 34), and Myo15 BCA promoter (SEQ ID NO: 35). The Myo15 ABC promoter (SEQ ID NO: 16) conferred the highest expression of GFP per cell (Figure 13A). Background levels of fluorescence were determined for each data set based on tissue autofluorescence (less than 2000 units for Figure 13A and less than 6000 units for Figure 13B).

[0273] Other embodiments Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. Other embodiments are within the scope of the following claims. (Addendum) The technical ideas that can be understood from the above-described embodiment and modified examples will be described. [Item 1] A polynucleotide comprising: a first region having at least 85% sequence identity to SEQ ID NO: 1 or a functional part or derivative thereof, the first region comprising the sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4; and a second region having at least 85% sequence identity to SEQ ID NO: 2 or a functional part or derivative thereof, the second region comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 9, operably linked thereto; and optionally a linker comprising 1 to 100 nucleotides between the first region and the second region. [Item 2] 2. The polynucleotide of item 1, wherein the first region comprises or consists of the sequence of SEQ ID NO: 1. [Item 3] Item 2. The polynucleotide according to item 1, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3. [Item 4] Item 2. The polynucleotide according to item 1, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4. [Item 5] 2. The polynucleotide according to item 1, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4. [Item 6] 6. The polynucleotide according to item 5, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5. [Item 7] 6. The polynucleotide according to item 5, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6. [Item 8] 6. The polynucleotide according to item 5, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 7. [Item 9] 6. The polynucleotide according to item 5, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 27. [Item 10] 10. The polynucleotide according to any one of items 1 to 9, wherein the second region comprises or consists of the sequence of SEQ ID NO:2. [Item 11] 10. The polynucleotide according to any one of items 1 to 9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8. [Item 12] 10. The polynucleotide according to any one of items 1 to 9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9. [Item 13] 10. The polynucleotide according to any one of items 1 to 9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9. [Item 14] 14. The polynucleotide of item 13, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10. [Item 15] Item 14. The polynucleotide of item 13, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11. [Item 16] Item 14. The polynucleotide according to Item 13, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12. [Item 17] 10. The polynucleotide according to any one of items 1 to 9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28. [Item 18] 10. The polynucleotide according to any one of items 1 to 9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 29. [Item 19] 10. The polynucleotide according to any one of items 1 to 9, wherein the functional part of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28 and the sequence of SEQ ID NO: 29. [Item 20] 20. The polynucleotide of item 19, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 32. [Item 21] 2. The polynucleotide according to item 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 13. [Item 22] 2. The polynucleotide according to item 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 15. [Item 23] 2. The polynucleotide according to item 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 16. [Item 24] 2. The polynucleotide of item 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 36. [Item 25] 2. The polynucleotide of item 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 37. [Item 26] A polynucleotide comprising: a first region having at least 85% sequence identity to SEQ ID NO:2 or a functional part or derivative thereof, the first region comprising the sequence of SEQ ID NO:8 and / or SEQ ID NO:9; and a second region having at least 85% sequence identity to SEQ ID NO:1 or a functional part or derivative thereof, the second region comprising the sequence of SEQ ID NO:3 and / or SEQ ID NO:4, operably linked thereto; and optionally a linker comprising 1 to 100 nucleotides between the first region and the second region. [Item 27] 27. The polynucleotide of item 26, wherein the first region comprises or consists of the sequence of SEQ ID NO: 2. [Item 28] 27. The polynucleotide according to item 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8. [Item 29] 27. The polynucleotide according to item 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9. [Item 30] 27. The polynucleotide according to item 26, wherein the functional part of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9. [Item 31] 31. The polynucleotide according to item 30, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10. [Item 32] 31. The polynucleotide according to item 30, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11. [Item 33] 31. The polynucleotide according to item 30, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12. [Item 34] 27. The polynucleotide according to item 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28. [Item 35] 27. The polynucleotide according to item 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 29. [Item 36] 27. The polynucleotide according to item 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28 and the sequence of SEQ ID NO: 29. [Item 37] 37. The polynucleotide of item 36, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 32. [Item 38] 38. The polynucleotide according to any one of Items 26 to 37, wherein the second region comprises or consists of the sequence of SEQ ID NO:1. [Item 39] 38. The polynucleotide according to any one of items 26 to 37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3. [Item 40] 38. The polynucleotide according to any one of Items 26 to 37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4. [Item 41] 38. The polynucleotide according to any one of Items 26 to 37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4. [Item 42] 42. The polynucleotide according to item 41, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5. [Item 43] 42. The polynucleotide according to item 41, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6. [Item 44] 42. The polynucleotide according to item 41, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 7. [Item 45] 42. The polynucleotide according to item 41, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 27. [Item 46] 27. The polynucleotide according to item 26, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 14. [Item 47] A polynucleotide comprising a region having at least 85% sequence identity to SEQ ID NO: 1 or a functional part or derivative thereof, including the sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4. [Item 48] 48. The polynucleotide according to item 47, wherein the first region comprises or consists of the sequence of SEQ ID NO: 1. [Item 49] 48. The polynucleotide according to item 47, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3. [Item 50] 48. The polynucleotide according to item 47, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4. [Item 51] 48. The polynucleotide according to item 47, wherein the functional part of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4. [Item 52] 52. The polynucleotide according to item 51, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5. [Item 53] 52. The polynucleotide of item 51, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6. [Item 54] 52. The polynucleotide according to item 51, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 7. [Item 55] 52. The polynucleotide according to item 51, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 27. [Item 56] A polynucleotide comprising a region having at least 85% sequence identity to SEQ ID NO: 2 or a functional part or derivative thereof, including the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 9. [Item 57] 57. The polynucleotide of item 56, wherein the first region comprises or consists of the sequence of SEQ ID NO:2. [Item 58] 57. The polynucleotide of item 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8. [Item 59] 57. The polynucleotide of item 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9. [Item 60] 57. The polynucleotide according to item 56, wherein the functional part of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9. [Item 61] 61. The polynucleotide according to item 60, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10. [Item 62] 61. The polynucleotide according to item 60, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11. [Item 63] 61. The polynucleotide according to item 60, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12. [Item 64] 57. The polynucleotide of item 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28. [Item 65] 57. The polynucleotide of item 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 29. [Item 66] 57. The polynucleotide according to item 56, wherein the functional part of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28 and the sequence of SEQ ID NO: 29. [Item 67] 67. The polynucleotide according to item 66, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 32. [Item 68] A polynucleotide comprising: a first region having at least 85% sequence identity to SEQ ID NO: 17 or a functional part or derivative thereof, the first region comprising the sequence of SEQ ID NO: 19; and a second region having at least 85% sequence identity to SEQ ID NO: 18 or a functional part or derivative thereof, the second region comprising the sequence of SEQ ID NO: 20 and / or SEQ ID NO: 21, operably linked thereto; and optionally a linker comprising 1 to 400 nucleotides between the first region and the second region. [Item 69] 69. The polynucleotide of item 68, wherein the first region comprises or consists of the sequence of SEQ ID NO: 17. [Item 70] 69. The polynucleotide according to item 68, wherein the functional portion of SEQ ID NO: 17 comprises the sequence of SEQ ID NO: 19. [Item 71] 71. The polynucleotide according to any one of Items 68 to 70, wherein the second region comprises or consists of the sequence of SEQ ID NO: 18. [Item 72] 71. The polynucleotide according to any one of items 68 to 70, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20. [Item 73] 71. The polynucleotide according to any one of items 68 to 70, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 21. [Item 74] 71. The polynucleotide according to any one of Items 68 to 70, wherein the functional part of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20 and the sequence of SEQ ID NO: 21. [Item 75] 75. The polynucleotide according to item 74, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 22. [Item 76] 75. The polynucleotide of item 74, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 23. [Item 77] 75. The polynucleotide according to item 74, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 24. [Item 78] 69. The polynucleotide according to item 68, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 25. [Item 79] 69. The polynucleotide according to item 68, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 26. [Item 80] A polynucleotide comprising: a first region having at least 85% sequence identity to SEQ ID NO: 18, or a functional part or derivative thereof, comprising the sequence of SEQ ID NO: 20 and / or SEQ ID NO: 21; and a second region having at least 85% sequence identity to SEQ ID NO: 17, or a functional part or derivative thereof, comprising the sequence of SEQ ID NO: 19 operably linked thereto; and optionally a linker comprising 1 to 400 nucleotides between the first region and the second region. [Item 81] 81. The polynucleotide of item 80, wherein the first region comprises or consists of the sequence of SEQ ID NO: 18. [Item 82] 81. The polynucleotide according to item 80, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20. [Item 83] 81. The polynucleotide according to item 80, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 21. [Item 84] 81. The polynucleotide according to item 80, wherein the functional part of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20 and the sequence of SEQ ID NO: 21. [Item 85] 85. The polynucleotide of item 84, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 22. [Item 86] 85. The polynucleotide of item 84, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 23. [Item 87] 85. The polynucleotide of item 84, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 24. [Item 88] 88. The polynucleotide according to any one of items 80 to 87, wherein the second region comprises or consists of the sequence of SEQ ID NO: 17. [Item 89] 88. The polynucleotide according to any one of items 80 to 87, wherein the functional portion of SEQ ID NO: 17 comprises the sequence of SEQ ID NO: 19. [Item 90] A polynucleotide comprising the sequence of SEQ ID NO:19, comprising a region having at least 85% sequence identity to SEQ ID NO:17 or a functional part or derivative thereof. [Item 91] 91. The polynucleotide according to item 90, wherein the region comprises or consists of the sequence of SEQ ID NO: 17. [Item 92] 91. The polynucleotide according to item 90, wherein the functional portion of SEQ ID NO: 17 comprises the sequence of SEQ ID NO: 19. [Item 93] A polynucleotide comprising a region having at least 85% sequence identity to SEQ ID NO: 18 or a functional part or derivative thereof, including SEQ ID NO: 20 and / or SEQ ID NO: 21. [Item 94] 94. The polynucleotide of item 93, wherein the region comprises or consists of the sequence of SEQ ID NO: 18. [Item 95] 94. The polynucleotide of item 93, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20. [Item 96] 94. The polynucleotide of item 93, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 21. [Item 97] 94. The polynucleotide according to item 93, wherein the functional part of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20 and the sequence of SEQ ID NO: 21. [Item 98] 98. The polynucleotide of item 97, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 22. [Item 99] 98. The polynucleotide of item 97, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 23. [Item 100] 98. The polynucleotide of item 97, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 24. [Item 101] A polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:28. [Item 102] A polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:32. [Item 103] A polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:33. [Item 104] A polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:34. [Item 105] 105. The polynucleotide of any one of items 1 to 104, wherein the polynucleotide, when operably linked to a transgene and introduced into a hair cell, induces transgene expression. [Item 106] 106. A nucleic acid vector comprising the polynucleotide according to any one of items 1 to 105. [Item 107] 107. The nucleic acid vector of claim 106, wherein the polynucleotide is operably linked to a transgene. [Item 108] 108. The nucleic acid vector of claim 107, wherein the transgene comprises a nucleic acid sequence encoding a therapeutic protein. [Item 109] 109. The nucleic acid vector of claim 108, wherein the polynucleotide is capable of directing hair cell-specific expression of the therapeutic protein from a nucleic acid sequence in a mammalian hair cell. [Item 110] Item 109, the nucleic acid vector of item 109, wherein the hair cells are cochlear hair cells. [Item 111] 111. The nucleic acid vector of item 110, wherein the cochlear hair cells are inner hair cells and / or outer hair cells. [Item 112] Item 109, the nucleic acid vector of item 109, wherein the hair cells are vestibular hair cells. [Item 113] The therapeutic protein is selected from the group consisting of ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, and ESPNL , PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP. [Item 114] 114. The nucleic acid vector according to any one of items 106 to 113, wherein the nucleic acid vector is a plasmid, a cosmid, an artificial chromosome, or a viral vector. [Item 115] Item 115. The nucleic acid vector of Item 114, wherein the nucleic acid vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), adenovirus, and lentivirus. [Item 116] Item 116. The nucleic acid vector of Item 115, wherein the viral vector is an AAV vector. [Item 117] 117. The nucleic acid vector of item 116, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S. [Item 118] A composition comprising the nucleic acid vector according to any one of items 106 to 117. [Item 119] 119. The composition of claim 118, further comprising a pharmaceutically acceptable excipient. [Item 120] 117. A method for increasing expression of a therapeutic protein in mammalian hair cells, the method comprising contacting said mammalian hair cells with the nucleic acid vector of any one of items 106 to 117 or the composition of item 118 or 119. [Item 121] 121. The method of claim 120, wherein expression of the therapeutic protein is increased specifically in hair cells. [Item 122] 122. The method of claim 120 or 121, wherein the mammalian hair cells are human hair cells. [Item 123] 123. The method according to any one of items 120 to 122, wherein the mammalian hair cells are cochlear hair cells. [Item 124] Item 124. The method of item 123, wherein the cochlear hair cells are inner hair cells. [Item 125] Item 124. The method of item 123, wherein the cochlear hair cells are outer hair cells. [Item 126] 123. The method according to any one of items 120 to 122, wherein the mammalian hair cells are vestibular hair cells. [Item 127] 127. The method of any one of items 120 to 126, wherein expression of the therapeutic protein is not substantially increased in inner ear cells that are not hair cells. [Item 128] 117. A method for treating a subject having or at risk of developing hearing loss, the method comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 106 to 117 or the composition of item 118 or 119. [Item 129] Item 129. The method of item 128, wherein the hearing loss is hereditary hearing loss. [Item 130] 130. The method of claim 129, wherein the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss. [Item 131] Item 129. The method of item 128, wherein the hearing loss is acquired hearing loss. [Item 132] 132. The method of claim 131, wherein the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease- or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss. [Item 133] 116. A method for treating a subject having or at risk of developing vestibular dysfunction, the method comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 106 to 117 or the composition of item 118 or 119. [Item 134] Item 134. The method of item 133, wherein the vestibular dysfunction is vertigo, dizziness, or imbalance. [Item 135] 116. A method for promoting hair cell regeneration in a subject in need thereof, the method comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 106 to 117 or the composition of item 118 or 119. [Item 136] Item 136. The method of item 135, wherein the hair cells are cochlear hair cells. [Item 137] Item 136. The method of item 135, wherein the hair cells are vestibular hair cells. [Item 138] 117. A method for preventing or reducing ototoxic drug-induced hair cell damage or death, comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 106 to 117 or the composition of item 118 or 119. [Item 139] 139. The method of claim 132 or 138, wherein the ototoxic drug is selected from the group consisting of aminoglycosides, antineoplastic agents, ethacrynic acid, furosemide, salicylates, and quinine. [Item 140] 117. A method for treating a subject having tinnitus, the method comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 106 to 117 or the composition of item 118 or 119. [Item 141] 116. A method for preventing or reducing hair cell damage or death in a subject in need thereof, the method comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 106 to 117 or the composition of item 118 or 119. [Item 142] 116. A method for increasing hair cell survival in a subject in need thereof, the method comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 106 to 117 or the composition of item 118 or 119. [Item 143] 143. The method of any one of items 128-132, 135, 136, and 138-142, wherein the method further comprises assessing the hearing of the subject prior to administering the nucleic acid vector or composition. [Item 144] 144. The method of any one of items 128 to 132, 135, 136, and 138 to 143, wherein the method further comprises assessing the hearing of the subject after administering the nucleic acid vector or composition. [Item 145] 143. The method of any one of items 133-135, 137-139, 141, and 142, wherein the method further comprises assessing vestibular function of the subject prior to administering the nucleic acid vector or composition. [Item 146] 146. The method of any one of items 133-135, 137-139, 141, 142, and 145, wherein the method further comprises assessing vestibular function of the subject prior to administering the nucleic acid vector or composition. [Item 147] 147. The method of any one of items 128 to 146, wherein the nucleic acid vector or composition is administered topically. [Item 148] 148. The method of any one of items 128-147, wherein the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, prevent or reduce vestibular dysfunction, prevent or reduce tinnitus, delay the onset of hearing loss, delay the onset of vestibular dysfunction, slow the progression of hearing loss, slow the progression of vestibular dysfunction, improve hearing, improve vestibular function, improve hair cell function, prevent or reduce hair cell damage, prevent or reduce hair cell death, or increase hair cell number. [Item 149] 149. The method according to any one of items 128 to 148, wherein the subject is a human. [Item 150] A kit comprising the nucleic acid vector according to any one of items 106 to 117 or the composition according to item 118 or 119.

Claims

[Claim 1] The invention described herein.

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