Myosin 15 promoter and uses thereof

Abstract

To provide novel therapeutic methods targeting hair cells for treating sensorineural hearing impairment or vestibular dysfunction.SOLUTION: The disclosure provides a polynucleotide comprising a myosin 15 (Myo15) promoter region, and a vector comprising the same, that can be used to promote expression of a transgene specifically in hair cells. The polynucleotide described herein may be operably linked to a transgene, such as a transgene encoding a therapeutic protein, so as to promote hair cell-specific expression of the transgene. The polynucleotide described herein may be operably linked to a therapeutic transgene and used for treatment of a subject having or at risk of developing hearing loss or vestibular dysfunction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Described herein are polynucleotides comprising a myosin 15 (Myo15) promoter region that can be used to drive transgene expression in hair cells (e.g., cochlear hair cells, e.g., inner and outer hair cells, and / or vestibular hair cells), as well as vectors comprising the same. Methods of using the polynucleotides and vectors of the invention to achieve transgene expression in hair cells to treat hearing loss and / or vestibular dysfunction are also disclosed. [Background technology]

[0002] Hearing loss is a significant public health issue, affecting nearly 15% of school-age children and an estimated 1 in 3 people by age 65. The most common type of hearing loss is sensorineural hearing loss, which is 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. Genetic causes of sensorineural hearing loss also exist, including mutations in genes involved in the development and function of the inner ear. Mutations in over 90 such genes have been identified, including autosomal recessive, autosomal dominant, and X-linked inheritance patterns.

[0003] Factors that disrupt 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 are therefore also associated with vestibular dysfunction, such as vertigo, dizziness, and imbalance. Indeed, patients with mutations that disrupt hair cell development or function may exhibit both hearing loss and vestibular dysfunction, or only one of these disorders. In recent years, efforts to treat hearing loss have increasingly focused on gene therapy as a possible solution; however, few approaches remain that specifically target hair cells, which are frequently involved in hearing loss and vestibular dysfunction. Novel therapies that target hair cells to treat sensorineural hearing loss or vestibular dysfunction are needed. Summary of the Invention

[0004] 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 enhances 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).

[0005] In a first aspect, the present 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:1 or a functional part or derivative thereof, comprising 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, comprising 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 sequence identity with the first region and a sequence identity with the second region, and optionally a linker having 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.

[0006] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:1. In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:2.

[0007] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:13. In another aspect, the present invention provides 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, comprising 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, comprising the sequence of SEQ ID NO:3 and / or SEQ ID NO:4, bound thereto (e.g., operably linked thereto). and a second region having a sequence identity (identity), and optionally a linker having 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.

[0008] In some embodiments, the first region comprises or consists of the sequence of SEQ ID NO:2. In some embodiments, the second region comprises or consists of the sequence of SEQ ID NO:1.

[0009] In some embodiments, the polynucleotide comprises or consists of the sequence of SEQ ID NO:14. In another aspect, the present invention provides polynucleotides 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 sequences of SEQ ID NO:3 and / or SEQ ID NO:4.

[0010] In some embodiments, this region comprises or consists of the sequence of SEQ ID NO:1. 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.

[0011] In some embodiments, this region comprises or consists of the sequence of SEQ ID NO:2. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:3. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:4. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:3 and the sequence of SEQ ID NO:4. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:5. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:6. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:7.

[0012] In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:8. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:9. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:8 and the sequence of SEQ ID NO:9. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:10. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:11. In some embodiments of any of the aforementioned aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:12.

[0013] In some embodiments of any of the aforementioned aspects, the polynucleotide is operably linked to a transgene, and when introduced into a hair cell, expression of the transgene is induced. In another aspect, the present invention provides a nucleic acid vector comprising a polynucleotide of the present invention. In some embodiments, the polynucleotide is operably linked to a transgene. In some embodiments, the transgene comprises a nucleic acid sequence encoding a therapeutic protein. In some embodiments, the polynucleotide is capable of directing hair cell-specific expression of a therapeutic protein derived from the nucleic acid sequence in a mammalian hair cell. In some embodiments, the hair cell is a cochlear hair cell. In some embodiments, the cochlear hair cell is an inner hair cell. In some embodiments, the cochlear hair cell is an outer hair cell. In some embodiments, the hair cell is a vestibular hair cell.

[0014] 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, NT3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

[0015] In some embodiments, the nucleic acid vector is a plasmid, cosmid, artificial chromosome, or viral vector. In some embodiments, the nucleic acid vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), adenovirus, and lentivirus. In some embodiments, the viral vector is an AAV vector. In some embodiments, the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, 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. In some embodiments, the serotype of the AAV vector is AAV1. In some embodiments, the serotype of the AAV vector is AAV9. In some embodiments, the serotype of the AAV vector is AAV6. In some embodiments, the serotype of the AAV vector is Anc80. In some embodiments, the serotype of the AAV vector is Anc80L65. In some embodiments, the serotype of the AAV vector is DJ / 9. In some embodiments, the serotype of the AAV vector is 7m8. In some embodiments, the serotype of the AAV vector is AAV2. In some embodiments, the serotype of the AAV vector is PHP.B. In some embodiments, the serotype of the AAV vector is AAV8.

[0016] In another aspect, the present invention provides compositions comprising the nucleic acid vectors of the present invention. In some embodiments, the compositions further comprise a pharmaceutically acceptable excipient. 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. In some embodiments, expression of the therapeutic protein is increased specifically in the hair cell.

[0017] In some embodiments, the mammalian hair cells are human hair cells. In some embodiments, the mammalian hair cells are cochlear hair cells. In some embodiments, the cochlear hair cells are inner hair cells. In some embodiments, the cochlear hair cells are outer hair cells.

[0018] In some embodiments, the mammalian hair cells are vestibular hair cells. In some embodiments, expression of the therapeutic protein is not substantially increased in inner ear cells that are not hair cells.

[0019] 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.

[0020] In some embodiments, the hearing loss is hereditary hearing loss. In some embodiments, the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss. In some embodiments, the hearing loss is acquired hearing loss. 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. In some embodiments, the acquired hearing loss is age-related hearing loss. In some embodiments, the hearing loss is noise-induced hearing loss. In some embodiments, the hearing loss is ototoxic drug-induced hearing loss.

[0021] In another aspect, the present invention provides methods of treating a subject having or at risk of developing vestibular dysfunction by administering to the subject an effective amount of a nucleic acid vector of the invention or a composition of the invention. In some embodiments, the vestibular dysfunction is vertigo, dizziness, or imbalance.

[0022] In another aspect, the present invention provides a method for 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. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0023] In another aspect, the present invention provides a method for preventing or reducing ototoxic agent-induced hair cell damage or death by administering an effective amount of a nucleic acid vector of the present invention or a composition of the present invention to a subject. In some embodiments, the ototoxic agent is selected from the group consisting of aminoglycosides (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), antineoplastic agents (e.g., platinum-containing chemotherapeutic agents, e.g., cisplatin, carboplatin, and oxaliplatin), ethacrynic acid, furosemide, salicylates (e.g., aspirin, especially at high doses), and quinines. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0024] 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. 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).

[0025] In another aspect, the present invention provides a method for 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. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0026] In another aspect, the present invention provides a method for 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. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0027] In some embodiments of any of the aforementioned aspects, the hair cells are cochlear hair cells. In some embodiments of any of the aforementioned aspects, the cochlear hair cells are inner hair cells. In some embodiments of any of the aforementioned aspects, the cochlear hair cells are outer hair cells. In some embodiments of any of the aforementioned aspects, the mammalian hair cells are vestibular hair cells.

[0028] In some embodiments of any of the aforementioned aspects, the method further comprises 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.

[0029] 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).

[0030] 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 tests) prior to administering the nucleic acid vector or composition.

[0031] 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 tests) prior to administering the nucleic acid vector or composition.

[0032] In some embodiments of any of the foregoing aspects, the nucleic acid vector or composition is administered locally (e.g., to the inner ear, e.g., into the perilymph or endolymph, such as via the oval window, round window, or horizontal semicircular canal).

[0033] In some embodiments of any of the aforementioned 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.

[0034] In some embodiments of any of the aforementioned aspects, the subject is a human. In another aspect, the present invention provides a kit comprising a nucleic acid vector of the present invention or a composition of the present invention.

[0035] definition As used herein, the term "about" refers to a value that is within 10% above or below the stated value.

[0036] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., a nucleic acid vector comprising a myosin 15 (Myo15) promoter operably linked to a transgene) to a subject by any effective route. Exemplary administration routes are described herein below.

[0037] As used herein, the term "cell type" refers to a group of cells that share a statistically separable phenotype 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 properties. Cells of a common cell type may include cells isolated from a common tissue (e.g., epithelial tissue, nervous tissue, connective tissue, or muscle tissue) and / or a common organ, tissue system, blood vessel, or other structure and / or region within an organism.

[0038] As used herein, the term "cochlear hair cells" refers to a specialized group of cells in the inner ear that are involved in sound detection. There are two types of cochlear hair cells: inner hair cells and outer hair cells. Damage to cochlear hair cells and genetic mutations that disrupt cochlear hair cell function are associated with hearing loss and hearing loss.

[0039] As used herein, the terms "conservative mutation," "conservative substitution," and "conservative amino acid substitution" refer to the substitution 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 in Table 1 below for each of the 20 naturally occurring amino acids.

[0040] [Table 1]

[0041] According to this table, the family of conservative amino acids includes: (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 substitutes an amino acid with a member of the same amino acid family (e.g., Ser for Thr, or Lys for Arg).

[0042] 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, the "effective amount" or its synonyms will depend on the context in which it is applied. For example, with respect to the treatment of sensorineural hearing loss or vestibular dysfunction, it is the amount of the 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, characteristics of the subject (e.g., age, sex, weight), or host being treated, but can nevertheless 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 administering a combination of active ingredients, the effective amount of the combination may or may not include the amount of each ingredient that would be 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 routine methods known in the art. The administration regimen may be adjusted to provide an optimal therapeutic response.

[0043] As used herein, the term "endogenous" refers to 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, e.g., a human cell, e.g., a human hair cell).

[0044] As used herein, the term "express" 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., splicing, editing, 5' cap formation, 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.

[0045] As used herein, the term "exogenous" refers to 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, e.g., a human cell, e.g., a human hair cell). Exogenous material includes materials provided from an external source to an organism or a culture extracted therefrom.

[0046] 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) among various cell types of the inner ear (e.g., in hair cells versus non-hair cells) using any standard technique (e.g., quantitative real-time PCR, immunohistochemistry, Western blot analysis, or fluorescence measurement of a reporter (e.g., GFP) operably linked to a promoter). The hair cell-specific promoter induces expression of a transgene (e.g., RNA or protein expression) to which it is operably linked, and the induced expression is at least 50% (e.g., 50%, 75%, 100%, 125%, 150%, 175%, 200% or more) greater in hair cells than in 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, Claudius cells, inner sulcus cells, outer sulcus cells, spiral ridge 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, and Schwann cells.

[0047] As used herein, the terms "increase" and "decrease" refer to modulation that results in a greater or lesser amount of a metric of function, expression, or activity, respectively, compared to a reference. For example, following administration of a composition according to the methods described herein, the amount of a metric marker described herein (e.g., transgene expression) may be increased or decreased in a subject 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 relative to the amount of the marker before administration. Generally, the metric is measured at a time when administration results in the described effect, e.g., at least 1 week, 1 month, 3 months, or 6 months after initiating a treatment regimen.

[0048] As used herein, the term "intron" refers to a region in 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 mature mRNA.

[0049] 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.

[0050] As used herein, "topical" or "local administration" refers to administration at a particular site in the body for a local rather than a systemic effect. Examples of local administration include administration onto the skin of a subject, inhalation, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional, lymph node, intratumoral, to the inner ear, and to a mucosa, where administration is intended to produce a local rather than a systemic effect.

[0051] As used herein, the term "operably linked" refers to a first molecule that can bind to a second molecule, positioning the molecules so that the first molecule affects the function of the second molecule. The term "operably linked" includes juxtaposing two or more components (e.g., a promoter and another sequence element) so that both components function normally and at least one component mediates the function of at least one other component. The two molecules may or may not be part of a single, uninterrupted molecule, and may be adjacent or non-adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule of interest if it regulates the transcription of the transcribable polynucleotide molecule in a cell. In a further embodiment, two portions of a transcriptional regulatory element are operably linked to each other if they are linked 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 without any intervening nucleotides.

[0052] 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.

[0053] As used herein, the terms "nucleic acid" and "polynucleotide," used interchangeably herein, refer to a polymeric form of nucleosides of any length. Generally, a polynucleotide is composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), linked by phosphodiester bonds. The term encompasses molecules containing nucleosides or nucleoside analogs having chemically or biologically modified bases, modified backbones, and the like, whether or not found in natural nucleic acids; such molecules may be preferred for particular uses. When the application refers to a polynucleotide, it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and the complement 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 shown in the 5' to 3' direction unless otherwise specified.

[0054] 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 opposite nucleic acid strand due to the orientation of its nucleobases. 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 complete or substantial / sufficient. Complete complementarity between two nucleic acids means that the two nucleic acids can form a duplex, in which all bases in the duplex bind to complementary bases by Watson-Crick pairing. "Substantially" or "fully" complementary means that the sequence of one strand is not completely and / or perfectly complementary to the sequence of the opposite strand, but that sufficient binding occurs between the bases of the two strands under a set of hybridization conditions (e.g., salt concentration and temperature) to form a stable hybrid complex. Such conditions can be predicted by predicting the Tm (melting temperature) of the hybridized strands using the sequences and standard mathematical calculations, or by empirically determining the Tm using routine methods. The Tm is the temperature at which 50% of the population of hybridization complexes formed between two nucleic acid strands denatures (i.e., half of the population of double-stranded nucleic acid molecules dissociates into single strands). Temperatures below the Tm favor the formation of hybridization complexes, while temperatures above the Tm favor melting or separation of the strands in the hybridization complexes. The Tm of a nucleic acid may be estimated using a known G+C content in 1 M aqueous NaCl, e.g., Tm = 81.5 + 0.41 (% G+C), although other known Tm calculations take into account the structural properties of the nucleic acid.

[0055] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase promotes transcription of the transgene. "Percentage (%) amino acid sequence identity" to a reference polynucleotide sequence or reference 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 sequence or reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining percentage nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the skill of those 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 sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. For example, percentage sequence identity values may be generated using the sequence comparison computer program BLAST. As an example, the percentage sequence identity of a given nucleic acid or amino acid sequence A to, with, or relative 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 particular percentage of sequence identity to, with, or relative 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 a programmatic alignment of A and B, and Y is the total number of nucleic acids in B. 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 be considered equal to the percent sequence identity of B to A.

[0056] As used herein, the term "derivative" refers to a nucleic acid, peptide, or protein, or a 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.

[0057] 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, for administration to a subject, such as a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition that affects or may affect the subject.

[0058] As used herein, the term "pharmaceutically acceptable" refers to compounds, substances, 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 excessive toxicity, irritation, allergic response, and other problematic hazards, and that have 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 pharmacopeia for use in mammals, more specifically humans.

[0059] 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).

[0060] 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 (e.g., polyadenylation signals) that control or help control gene transcription. Examples of transcriptional regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0061] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used for the introduction of foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, squeezeporation, sonoporation, phototransfection, magnetofection, imparefection, etc.

[0062] As used herein, the terms "subject" and "patient" refer to animals (e.g., mammals such as humans), veterinary subjects (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). A subject treated in accordance with 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 at risk for developing these conditions. Diagnosis may be performed by any method or technique known in the art. One of skill in the art will understand that a subject treated in accordance with the present disclosure may have undergone standard testing or may have not been tested but has been identified as at risk due to the presence of one or more risk factors associated with the disease or condition.

[0063] As used herein, the terms "transduction" and "transducing" 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 a cell and subsequent transfer and integration of the vector construct, or a portion thereof, into the cellular genome.

[0064] 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 asymptomatic symptom of the condition, disorder, or condition developing in a subject who may be suffering from or predisposed to the condition, disorder, or condition, but who has not yet experienced or displayed clinical or asymptomatic symptoms; or (2) inhibiting the condition, disorder, or condition, i.e., preventing, alleviating, or delaying the onset of the disease or its recurrence or the onset of at least one clinical or asymptomatic symptom; or (3) alleviating the disease, i.e., regressing the condition, disorder, or condition, or at least one of its clinical or asymptomatic symptoms. The benefit to a subject being treated will be statistically significant or at least perceptible to the patient or physician.

[0065] 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 foreign 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 in 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. Particular vectors that can be used for expressing the transgenes described herein include vectors containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for expressing transgenes contain polynucleotide sequences that enhance the translation rate of the transgene or improve the stability or nuclear export of 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 genes incorporated into the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0066] As used herein, the term "vestibular hair cells" refers to a specialized group of 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 disrupt vestibular hair cell function are associated with vestibular dysfunction, such as vertigo and balance disorders.

[0067] As used herein, the term "wild type" refers to the most frequently occurring genotype for a particular gene in a given organism. [Brief explanation of the drawings]

[0068] [Figure 1A] This is a series of fluorescent images of mouse cochleae transduced with an adeno-associated virus (AAV) vector expressing GFP under the control of the cytomegalovirus (CMV) promoter. Six- to eight-week-old C57Bl / 6J male mice were injected with AAV-CMV-GFP virus via the posterior semicircular canal. Mice were allowed to recover from surgery, euthanized, and perfused with 10% neutral-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 analysis. 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 1B] This is a series of fluorescent images of mouse cochleae transduced with an AAV vector (SEQ ID NO: 13) expressing GFP under the control of the Myo15 promoter. Six- to eight-week-old C57Bl / 6J male mice were injected with the AAV-Myo15-GFP virus via the posterior semicircular canal. Mice were allowed to recover from surgery, euthanized, and perfused with 10% neutral-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 analysis. Using a hair cell-specific promoter, AAV-Myo15-GFP directed expression only in inner and outer hair cells. [Figure 2]These are fluorescence images of regions of the mouse vestibular system (utricle, saccule, posterior medial fold (PC), anterior medial fold (AC), and horizontal medial fold (HC)) transduced with an AAV vector expressing GFP under the control of the Myo15 promoter (SEQ ID NO: 13, Figure 2). Six- to eight-week-old C57Bl / 6J male mice were injected with AAV-Myo15-GFP virus via the posterior semicircular canal. Mice were allowed to recover from surgery, euthanized, and perfused with 10% neutral-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 image analysis. Using a hair cell-specific promoter, AAV-Myo15-GFP induced expression exclusively in vestibular hair cells (Figure 2). [Figure 3A] Figure 3A shows a series of fluorescence images of the cochlea of a non-human primate, demonstrating that the Myo15 promoter restricts GFP expression to the hair cells of the cochlea of non-human primates. Figure 3B shows 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 variety of cell types throughout the organ. [Figure 3B] Figure 3B is a series of fluorescence images of non-human primate cochleae showing that the Myo15 promoter restricts GFP expression to the hair cells of non-human primate cochleae. Figure 3B is a confocal image of a cochlea from a non-human primate injected with AAV1-Myo15-GFP and treated in the same manner as the cochlea in Figure 3A. GFP expression was restricted to hair cells. [Figure 3C] Figure 3C is a series of fluorescent images of the non-human primate cochlea showing that the Myo15 promoter restricts GFP expression to hair cells in the non-human primate cochlea. Figure 3C is a magnified view of a hair cell within the boxed region shown in Figure 3B. [Figure 4]Figure 1 shows a graph demonstrating 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 to 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 are plotted as a function of stimulation frequency for naive homozygous (open circles and light gray line) and heterozygous (filled circles and dark gray line) Tmc1 KO mice, as well as homozygous Tmc1 KO mice injected with AAV-CMV mouse TMC1 (CMV_mTmc1; open circles and dark gray line) or AAV-Myo15-mouse TMC1 (PdBx_mTmc1; P23, light gray line through light gray circles; P28, filled circles and dark gray line). ABR threshold recovery was significantly improved in homozygous Tmc1 KO mice injected with AAV-Myo15-mouseTMC1 compared with AAV-CMV-mouseTMC1. DETAILED DESCRIPTION OF THE INVENTION

[0069] Described herein are compositions and methods for inducing transgene expression, particularly in hair cells (e.g., cochlear hair cells and / or vestibular hair cells). The invention features polynucleotides including regions of the myosin 15 (Myo15) promoter that enable transgene expression specifically in cochlear hair cells. The invention also features nucleic acid vectors having these promoters operably linked to polynucleotides encoding polypeptides. The compositions and methods described herein can be used to express polynucleotides encoding hair cell proteins, particularly in cochlear hair cells and vestibular hair cells, and thus the compositions described herein can be administered to a subject (e.g., a mammalian subject, e.g., a human) to treat disorders caused by hair cell dysfunction, such as hearing loss or vestibular dysfunction.

[0070] 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 involved in sound detection, and outer hair cells, which are thought to amplify low-pitched 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 a bundle of hair cells. Deflection of the stereocilia (e.g., by sound waves in cochlear hair cells or by rotation or linear acceleration of vestibular hair cells) opens mechano-gated ion channels, causing the hair cells to release neurotransmitters and activate nerves, thereby transducing mechanical sound or motion 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 cochlear hair cell function are associated with hearing loss and hearing loss. Damage to vestibular hair cells and genetic mutations that disrupt vestibular hair cell function are associated with vestibular dysfunction, such as imbalance and dizziness (e.g., vertigo). In recent years, gene therapy has emerged as an attractive therapeutic approach for treating hearing loss and vestibular dysfunction; however, the field lacks methods for specifically targeting nucleic acid vectors used in gene therapy to hair cells.

[0071] Myosin 15 Myo15 is a nonconventional actin-based molecular motor that regulates stereocilia development. Mice with mutations in Myo15 have been observed to have short stereocilia and severe hearing loss and vestibular dysfunction, and mutations in the human ortholog, Myo15A, cause DFNB3, a nonsyndromic autosomal recessive form of hearing loss. 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 yet been isolated and characterized. We identified evolutionarily conserved blocks within orthologous genomic sequences that may constitute the promoter's regulatory elements and found them located more than 7,200 base pairs (bp) upstream of the translation start site. This genomic region is too large to be used in combination with adeno-associated virus (AAV) vectors, which have a maximum packaging capacity of 4.7 kb, to deliver transgenes of interest for gene therapy.

[0072] The present invention is based, in part, on the discovery of a region upstream of the Myol5 translation start site that 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 a subject with, or at risk of developing, hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance).

[0073] The polynucleotides of the compositions and methods described herein comprise nucleic acid sequences derived from regions of the Myol5 locus capable of transgene expression specifically in hair cells, or variants thereof, e.g., 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 Myol5 locus capable of transgene expression specifically in hair cells. These regions include the nucleic acid sequence immediately preceding the Myol5 translation start site and regulatory elements located 5 kb or more upstream from the Myol5 translation start site. The polynucleotides of the compositions and methods described herein can optionally include a linker operably connecting regions of the Myol5 locus capable of transgene expression specifically in hair cells, or the regions of the Myol5 locus can be directly joined without an intervening linker.

[0074] In some embodiments, the polynucleotides described herein comprise 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 comprising the first non-coding exon of the Myo15 gene (nucleic acids -6755 to -7209 relative to the Myo15 translation start site, the sequence of which is set forth in SEQ ID NO:1), or a functional portion or derivative thereof, and a second region associated therewith (e.g., operably linked thereto) having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a nucleic acid sequence immediately preceding the Myo15 translation start site (nucleic acids -1 to -1157 relative to the Myo15 translation start site, the sequence of which is 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 from -7166 to -7091 relative to the Myo15 translation start site (as set forth in SEQ ID NO:3) and / or the nucleic acid sequence from -7077 to -6983 relative to the Myo15 translation start site (as set forth in SEQ ID NO:4). The first region can include 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 include 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 include an endogenous intervening nucleic acid sequence (e.g., the first region can have the nucleic acid sequence from -7166 to -6983 relative to the Myo15 translation start site as set forth in SEQ ID NO:7) or the sequences of SEQ ID NO:3 and SEQ ID NO:4 joined by a nucleic acid linker. In polynucleotides in which the first region includes 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 Myo15 translation start site (set forth in SEQ ID NO:8) and / or the nucleic acid sequence from -266 to -161 relative to the Myo15 translation start site (set forth in SEQ ID NO:9).The second region can comprise 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 set forth in SEQ ID NO:10, or the second region can comprise 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 set forth in SEQ ID NO:11. Alternatively, the second region can comprise an endogenous intervening nucleic acid sequence (e.g., the second region can have a nucleic acid sequence from -590 to -161 relative to the Myo15 translation start site, as set forth in SEQ ID NO:12) or the sequences of SEQ ID NO:8 and SEQ ID NO:9 joined by a nucleic acid linker. In polynucleotides in which the second region comprises both SEQ ID NO:8 and SEQ ID NO:9, the two sequences can be included in any order (e.g., SEQ ID NO:8 can be linked to (e.g., preceding) SEQ ID NO:9, or SEQ ID NO:9 can be linked to (e.g., preceding) SEQ ID NO:8).

[0075] The first and second regions of the polynucleotide can be linked directly or via a nucleic acid linker. For example, the polynucleotide can include the sequence of SEQ ID NO: 1, or a functional portion or derivative thereof (e.g., one or more of SEQ ID NOs: 3-7, 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., one or more of SEQ ID NOs: 8-12, 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 and SEQ ID NO: 2 is set forth in SEQ ID NO: 13. Alternatively, a linker can be used to link the sequence of SEQ ID NO: 1, or a functional portion or derivative thereof (e.g., one or more of SEQ ID NOs: 3-7, 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., one or more of SEQ ID NOs: 8-12, e.g., SEQ ID NOs: 8 and 9).

[0076] 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 disrupt the ability of the polynucleotides of the invention to direct expression of a transgene in hair cells.

[0077] 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, 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, 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, e.g., SEQ ID NOs: 8 and 9) is joined (e.g., operably linked) to 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, 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 and SEQ ID NO: 1 is set forth in SEQ ID NO: 14. 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.

[0078] In some embodiments, the polynucleotides described herein comprise a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to a region comprising the first non-coding exon of the Myo15 gene (nucleic acids -6755 to -7209 relative to the Myo15 translation start site, the sequence of which is set forth in SEQ ID NO:1), or a functional portion or derivative thereof. The functional portion of SEQ ID NO:1 can have the nucleic acid sequence -7166 to -7091 relative to the 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 comprise 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 set forth in SEQ ID NO:5, or the polynucleotide can comprise the nucleic acid sequence of SEQ ID NO:4 fused to the nucleic acid of SEQ ID NO:3 without an intervening nucleic acid set forth in SEQ ID NO:6. Alternatively, the polynucleotide can include the sequences of SEQ ID NO:3 and SEQ ID NO:4 connected by an endogenous intervening nucleic acid sequence (e.g., the first region can have the nucleic acid sequence from -7166 to -6983 relative to the Myo15 translation start site, as set forth in SEQ ID NO:7) or a nucleic acid linker. In a polynucleotide that includes 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 connected to (e.g., preceded by) SEQ ID NO:4, or SEQ ID NO:4 can be connected to (e.g., preceded by) SEQ ID NO:3).

[0079] In some embodiments, the polynucleotides described herein comprise a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to the nucleic acid sequence immediately upstream of the Myo15 translation start site (nucleic acids -1 to -1157 relative to the Myo15 translation start site, the sequence of which is 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 Myo15 translation start site (set forth in SEQ ID NO:8) and / or the nucleic acid sequence -266 to -161 relative to the Myo15 translation start site (set forth in SEQ ID NO:9). A polynucleotide can comprise 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 set forth in SEQ ID NO:10, or a polynucleotide can comprise 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 set forth in SEQ ID NO:11. Alternatively, the polynucleotide can include 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 Myo15 translation start site, as set forth in SEQ ID NO:12) or a nucleic acid linker. In a polynucleotide that includes both SEQ ID NO:8 and SEQ ID NO:9, the two sequences can be included in any order (e.g., SEQ ID NO:8 can be joined to (e.g., preceded by) SEQ ID NO:9, or SEQ ID NO:9 can be joined to (e.g., preceded by) SEQ ID NO:8).

[0080] The aforementioned nucleic acid sequences are summarized in Table 2 below.

[0081] [Table 2-1]

[0082] [Table 2-2]

[0083] [Table 2-3]

[0084] [Table 2-4]

[0085] [Table 2-5]

[0086] Additional polynucleotides useful in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the nucleic acid sequences set forth in Table 2 and functional portions or derivatives of the nucleic acid sequences set forth in Table 2.

[0087] The aforementioned polynucleotides can be included in a nucleic acid vector and operably linked to a transgene to specifically express the transgene 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 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 imbalance). According to the methods described herein, a composition comprising 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 can be administered to a subject. 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), pedivakin (DFNB59), otoferlin (OTOF), MKRN2 opposite chain (MKRN2OS), LIM ho Meobox 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 cornified envelope 6A (LCE6A),Lipoxygenase 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 (PRP) H2), 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), key Lin / 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 (NT3), cularin 1 (CLRN1), cularin 2 (CLRN2), SKI family transcriptional corepressor 1 (SKOR1), Tctex1 domain-containing protein 1 (TCTEX1D1), Fc receptor-like B (FCRLB), solute carrier family 1 7 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).

[0088] Expression of foreign nucleic acids in mammalian cells Mutations in various genes, such as MYO7A, POU4F3, SLC17A8, and TMC1, are associated with sensorineural hearing loss, and some of these mutations, e.g., mutations in MYO7A, are also associated with vestibular dysfunction. The compositions and methods described herein can be used to express a nucleic acid vector comprising a 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 encoding SEQ ID NO: 2, or a functional portion or derivative thereof), particularly in hair cells (e.g., cochlear hair cells and / or vestibular hair cells). By administering a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the first region, optionally including a linker joining the first and second regions, expression of the protein encoded by the gene of interest (e.g., a wild-type 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) can be induced or increased. A wide range of methods have been established for delivering proteins to mammalian cells and for stably expressing protein-encoding genes in mammalian cells.

[0089] Proteins that can be expressed in connection with the compositions described herein (e.g., when a transgene encoding the protein is operably linked to a polynucleotide 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 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) 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. 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, NT3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

[0090] 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 stably expressing 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. To introduce an exogenous gene into mammalian cells, the gene can be incorporated into a vector. Vectors can be introduced into cells by various methods, including transformation, transfection, transduction, direct uptake, particle 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 in further detail, for example, in Green, et al., Molecular Cloning: A Laboratory 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.

[0091] The vector containing the gene encoding the protein of interest can also be targeted to the phospholipids of the cell membrane, thereby introducing the protein of interest into mammalian cells.For example, the vector molecule can be bound to the VSV-G protein, a viral protein that has affinity to all cell membrane phospholipids, thereby targeting the vector to the phospholipids on the extracellular surface of the cell membrane.Such a construct can be produced using methods well known to those skilled in the art.

[0092] It is important for gene expression that a polynucleotide encoding a protein of interest is recognized and bound by mammalian RNA polymerase. Therefore, a sequence element that shows high affinity for transcription factors that recruit RNA polymerase and promote the assembly of a transcription complex at the transcription start site may be included in the polynucleotide. Such a sequence element includes, for example, a mammalian promoter, whose sequence can be recognized and bound by specific transcription initiation factors and ultimately by 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 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 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 comprising a linker between the first and second regions.

[0093] 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 a drug that modulates the binding of transcription factors and / or RNA polymerase to a mammalian promoter, thereby regulating gene expression. The chemical reagent can function to promote the binding of RNA polymerase and / or transcription factors to a mammalian promoter, for example, by removing promoter-bound repressor proteins. Alternatively, the chemical reagent can function to increase the affinity of a mammalian promoter for RNA polymerase and / or transcription factors, thereby increasing the transcription rate of a gene located downstream of the promoter in its presence. 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.

[0094] Other DNA sequence elements that may 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 that favors the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein include polynucleotides encoding proteins of interest and also include mammalian enhancer sequences. Many enhancer sequences derived from mammalian genes are currently known, including enhancers from genes encoding mammalian globin, elastase, albumin, α-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include enhancers 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 CMV enhancers and RSV enhancers.The enhancer may be spliced into the vector containing the polynucleotide encoding the protein of interest, for example, at the 5' or 3' position of the gene.In a preferred orientation, the enhancer is located 5' from the promoter, and then the promoter is located 5' from the polynucleotide encoding the protein of interest.

[0095] Nucleic acid vectors containing the Myo15 promoter described herein may also contain a woodchuck post-transcriptional regulatory element (WPRE). The WPRE acts at the transcriptional level, increasing the total amount of mRNA in the cell by facilitating nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of nascent transcripts. The 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.

[0096] In some embodiments, nucleic acid vectors comprising the Myo15 promoter described herein contain a reporter sequence, which may be useful, for example, for verifying expression of a gene operably linked to the Myo15 promoter in cells and tissues (e.g., cochlear hair cells and / or vestibular hair cells). Reporter sequences that may 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 other reporters known in the art. When associated with regulatory elements, such as the Myo15 promoter, that drive their expression, reporter sequences provide a signal 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 assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry. For example, if the marker sequence is the LacZ gene, the presence of the vector incorporating the signal is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the presence of the vector incorporating the signal can be visually measured by color or light production in a luminometer.

[0097] Methods for delivering exogenous nucleic acids to 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, exposed to an external electric field in this manner are then susceptible 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 carrying out this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0098] Another useful technique for transfection of target cells is squeezeporation.This technique induces rapid mechanical deformation of cells to stimulate the uptake of foreign DNA through membrane pores formed in response to applied stress.This technique is advantageous in that it does not require vectors to deliver nucleic acid to cells, such as human target cells.Squeezeporation 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.

[0099] Lipofection is another technique useful for transfecting target cells. This method involves loading nucleic acids into liposomes, which often present cationic functional groups, such as quaternary amines or protonated amines, on the exterior of the liposomes. This facilitates electrostatic interactions between the liposomes and cells due to the anionic nature of the cell membrane, ultimately leading to the uptake of the exogenous nucleic acid, for example, by direct fusion of the liposome with the cell membrane or endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. 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 acids involves contacting cells with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that can be associated with polynucleotides to confer a positive charge favoring interaction with cell membranes include activated dendrimers (e.g., as described 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 in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this methodology utilizes the application of a magnetic field to direct the uptake of nucleic acids. This technology is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0100] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, also known as optical transfection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to, and in some cases superior to, electroporation.

[0101] Imparefection is another technique that can be used to deliver genetic material to target cells. This technique relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-shaped nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing genes intended for intracellular delivery is attached to the surface of the nanostructures. A chip with an array of these needles is then pressed against cells or tissues. Cells stimulated 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.

[0102] 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, unique cationic 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 on target cells under 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.

[0103] Another useful tool for inducing the uptake of exogenous nucleic acid by target cells is sonoporation, which is a technique that involves using sound waves (usually ultrasonic frequencies) to modify the permeability of cell membranes, making cells permeable and allowing polynucleotides 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.

[0104] 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 derived by co-overexpression of the glycoprotein VSV-G and a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins to cells and subsequently catalyze site-specific cleavage of endogenous polynucleotide sequences, thereby preparing the genome of the cell for covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as gesicles, to genetically modify eukaryotic cells is described, for example, in Quinn et al., Genetic Modification of Target Cells. This is described in detail in Cells by Direct Delivery of Active Protein [abstract] In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; May 13, 2015, Abstract No. 122.

[0105] Vectors for delivering 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. Various vectors have been developed for delivering and integrating polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of 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 for use in the compositions and methods described herein comprise a Myo15 promoter (e.g., a polynucleotide 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 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 comprising a linker between the first and second regions) operably linked to a polynucleotide sequence encoding a protein of interest and to additional sequence elements used, for example, for expression of these agents and / or integration of these polynucleotide sequences into a mammalian cell genome. 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 in cells), cosmids (e.g., pWE vectors 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. Particular vectors that can be used to express a protein of interest include plasmids containing regulatory sequences, such as enhancer regions, that direct gene transcription.Other vectors useful for expressing proteins of interest contain polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried by the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0106] 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 genomes 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 such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specific transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., Retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, as well as 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, mutant 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, avian C virus, mammalian C, B, and D viruses, 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 viruses, murine sarcoma viruses, mouse mammary tumor viruses, bovine leukemia viruses, feline leukemia viruses, feline sarcoma viruses, avian leukemia viruses, human T-cell leukemia viruses, baboon endogenous viruses, gibbon leukemia viruses, Mason-Pfizer monkey viruses, simian immunodeficiency viruses, simian sarcoma viruses, Rous sarcoma viruses, and lentiviruses. 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 pertains to viral vectors for use in gene therapy.

[0107] 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 viral particles 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 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 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 comprising 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. The viral sequences may include AAV sequences required in cis for DNA replication and packaging into viral particles (e.g., functional ITRs). In general applications, the transgene encodes a therapeutic protein capable of promoting hair cell development, hair cell function, hair cell regeneration, hair cell fate specification, hair cell survival, or hair cell maintenance, or the wild-type form of a hair cell protein that is mutated in subjects with inherited forms of 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 also contain a marker or reporter gene. Useful rAAV vectors have one or more AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. AAV ITRs may be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs may 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.

[0108] To facilitate the introduction of a polynucleotide or vector into a cell, 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 viral particles. The capsid protein of AAV constitutes the external, non-nucleic acid portion of the viral particle and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for viral particle assembly. Construction of rAAV viral particles 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 pertain to AAV vectors for gene delivery.

[0109] rAAV viral particles useful in combination with the compositions and methods described herein include viral particles 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, 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 pertain to AAV vectors for gene delivery.

[0110] Useful in combination with the compositions and methods described herein are pseudotyped rAAV vectors. 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 for constructing and using pseudotyped rAAV viral particles 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).

[0111] AAV viral particles with mutations in the viral particle capsid can be used to infect specific cell types more effectively than non-mutated capsid viral particles. 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 viral particles that can be used in the methods described herein include capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0112] Pharmaceutical Composition A polynucleotide described herein (e.g., a polynucleotide 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 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 comprising a linker between the first and second regions) may be operably linked to a transgene (e.g., a transgene encoding a protein of interest) and incorporated into a vehicle for administration to a patient, e.g., a human patient suffering from sensorineural hearing loss and / or vestibular dysfunction. Pharmaceutical compositions comprising a vector, such as a viral vector, comprising 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).

[0113] Mixtures of nucleic acid vectors (e.g., viral vectors) comprising a polynucleotide described herein operably linked to a therapeutic transgene (e.g., a polynucleotide 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 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 comprising a linker between the first and second regions) may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. These formulations may contain a preservative to prevent the growth of microorganisms under ordinary conditions of storage and use. 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 (described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In any case, the formulation may be sterile and may have fluidity 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 may 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 dispersions, and by the use of surfactants. Prevention of the action of microorganisms may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. 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.

[0114] 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. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art in light of the present disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous fluid or injected at the intended injection site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition may be formulated to contain a synthetic perilymph solution. 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 HEPES, with a pH of about 6-9 and an osmolality of about 300 mOsm / kg. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0115] Treatment method The compositions described herein may be administered to a subject with sensorineural hearing loss and / or vestibular dysfunction by a variety of routes, including, for example, local administration to the inner ear (e.g., administration to the perilymph or endolymph via the oval window, round window, or semicircular canal (e.g., the horizontal semicircular canal), e.g., administration to cochlear hair cells or vestibular hair cells), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most appropriate route of administration in a given case depends on the particular composition administered, the patient, the pharmaceutical formulation, the method of administration (e.g., time 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., annually, twice a year, three times a year, every other month, or monthly).

[0116] Subjects that 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. In some embodiments, the subject has hearing loss and / or vestibular dysfunction associated with or resulting from the 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. Standard methods known to those skilled in the art (e.g., genetic testing) can be used to screen the subject for genetic mutations. The methods described herein may also include assessing the subject's auditory and / or vestibular function 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 may be assessed using standard tests such as eye movement tests (e.g., electronystagmography (ENG) or videonystagmography (VNG)), posturography, rotary chair testing, ECOG, vestibular-evoked myogenic potentials (VEMPs), and specialized clinical balance tests, as described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010). The compositions and methods described herein may also be administered as a prophylactic treatment to patients at risk of developing hearing loss and / or vestibular dysfunction, such as patients with a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), patients with a genetic mutation associated with hearing loss or vestibular dysfunction who 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).

[0117] 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 may 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 abnormal hair cells (e.g., hair cells that do not function properly compared to normal hair cells), damaged hair cells (e.g., hair cell damage associated with trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or subjects with reduced hair cell numbers due to 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 the survival rate of damaged hair cells, promote the repair of damaged hair cells, or preserve hair cells in subjects at risk of hair cell loss (e.g., hair cell loss due to aging, exposure to loud noise, disease or infection, head trauma, or ototoxic drugs)).

[0118] The compositions and methods described herein can also be used to prevent or reduce ototoxic drug-induced hair cell damage or cell death (e.g., cochlear and / or vestibular hair cell damage or cell death) in subjects who have been treated with an ototoxic drug, or who are currently undergoing or will begin treatment with an ototoxic drug. Ototoxic drugs are toxic to inner ear cells 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 chemotherapy agents such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, especially in high doses), and quinines. In some embodiments, the methods described herein prevent or reduce hair cell damage or cell death associated with acoustic trauma, disease or infection, head trauma, or aging.

[0119] A transgene operably linked to the Myol5 promoter for treating a subject described herein (e.g., a polynucleotide 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 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 a protein that is deficient in a subject with sensorineural hearing loss and / or vestibular dysfunction) or another therapeutic protein of interest. The transgene may 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 version of the gene that is mutated in the subject, or if the subject has hearing loss associated with hair cell loss, the transgene may 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 can be expressed by a transgene operably linked to the Myo15 promoter for the treatment of a subject as 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, NT3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

[0120] Treatment may involve the administration of a composition comprising a nucleic acid vector (e.g., an AAV viral vector) comprising 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 may comprise a continuous infusion over a set period of time. Administration may be performed using a syringe pump to control the rate of infusion to minimize damage to the inner ear (e.g., the cochlea). When the nucleic acid vector is an AAV vector (e.g., an AAV1, AAV2, 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, for example, about 1 × 10 10 Vector genome (VG) ~ 1 x 10 15 VG (e.g., 1 × 1010 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 12 VG、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 15VG) may be administered to a patient in a dose 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).

[0121] 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 the function of a therapeutic protein encoded by a transgene, prevent or reduce hair cell damage, prevent or reduce hair cell 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 the number of hair cells (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 may be assessed using standard tests of balance and vertigo (e.g., oculomotor tests (e.g., ENG or VNG), posturography, rotary chair testing, ECOG, VEMP, and specialized clinical balance tests) 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 measurements obtained before treatment. 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 delay or prevent the onset or progression of sensorineural hearing loss and / or vestibular dysfunction (e.g., in a subject who has a genetic mutation associated with hearing loss or vestibular dysfunction, has a family history of hearing loss or vestibular dysfunction (e.g., genetic hearing loss or vestibular dysfunction), or has been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, acoustic trauma, or infection), but who does not exhibit hearing loss or vestibular dysfunction (e.g., vertigo, dizziness, or disequilibrium), or in a subject who exhibits 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 number of hair cells, hair cell function, or function of a therapeutic protein encoded by a nucleic acid vector administered to a subject may be indirectly assessed based on a hearing test or vestibular function test, 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 the number of hair cells, hair cell function, or therapeutic protein function before administration of a composition described herein. Hair cell damage or cell death may 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 cell death typically observed in untreated subjects. These effects may occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more after administration of the compositions described herein.Depending on the dose and route of administration used for the treatment, the patient may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more after administration of the composition, and depending on the results of the evaluation, the patient may be subjected to additional treatment.

[0122] kit The compositions described herein can be provided in a kit for use in treating sensorineural hearing loss or vestibular dysfunction. The composition can include a polynucleotide described herein (e.g., a polynucleotide 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 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 comprising a linker between the first and second regions), a nucleic acid vector comprising such a polynucleotide, and a nucleic acid vector comprising 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, AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B). The kit may further include a package insert instructing a user of the kit, e.g., a physician, to practice the methods described herein. The kit may optionally include a syringe or other device for administering the composition. [Example]

[0123] The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may 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.

[0124] Example 1. Generation of the Myo15 promoter First, we identified the evolutionarily conserved region of the vertebrate Myo15 promoter from UCSC Genome The region immediately upstream of the Myo15 translation initiation site (-1 to -1157, SEQ ID NO: 1) and the upstream region including non-coding exon 1 of the Myo15 gene (-6755 to -7209, SEQ ID NO: 2) were identified using the genome browser (genome.ucsc.edu). It was synthesized by novo gene synthesis and spliced together as a single DNA fragment (SEQ ID NO: 13). The total size of the truncated Myo15 promoter is 1611 bp, while the entire genomic region is over 7000 bp.

[0125] Experiments evaluating the tropism (cell type targeting) and extent and duration of transgene expression by the Myo15 promoter compared with the cytomegalovirus (CMV) promoter in the mouse cochlea demonstrated selective expression in cochlear hair cells with the Myo15 promoter, but not with the CMV promoter. AAV constructs were created using Myo15 to drive expression of Aequorea coerulescens green fluorescent protein (AcGFP), and the progression of gene expression was analyzed compared with a matched standard AAV construct using CMV. Transgene expression was assessed in experiments in which the virus was delivered to the mouse cochlea in neonatal and adult mice, and in ex vivo cochlear explants.

[0126] To evaluate transgene expression, AAV-Myo15-GFP virus was injected into 6-8 week-old C57Bl / 6J male mice via the posterior semicircular canal. Mice were allowed to recover from surgery, euthanized, and perfused with 10% neutral 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 image analysis. 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). In the vestibular system, AAV-Myo15-GFP induced expression only in vestibular hair cells (Fig. 2).

[0127] 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:2 fused with SEQ ID NO:3; 2) SEQ ID NO:2 fused with SEQ ID NO:4; 3) SEQ ID NO:2 fused to a fusion of SEQ ID NO:3 and SEQ ID NO:4 (e.g., SEQ ID NO:5, 6, or 7); 4) SEQ ID NO:9 fused to SEQ ID NO:8 (e.g., SEQ ID NO:10, 11, or 12). 5) a fusion of SEQ ID NO:8 and SEQ ID NO:9 fused to SEQ ID NO:1 (e.g., SEQ ID NO:10, 11, or 12); 6) a fusion of SEQ ID NO:8 and SEQ ID NO:9 fused to SEQ ID NO:3 (e.g., SEQ ID NO:10, 11, or 12); 7) a fusion of SEQ ID NO:8 and SEQ ID NO:9 fused to SEQ ID NO:4 (e.g., SEQ ID NO:10, 11, or 12); 8) a fusion of SEQ ID NO:8 and SEQ ID NO:9 (e.g., SEQ ID NO:10, 11, or 12) fused to a fusion of SEQ ID NO:3 and SEQ ID NO:4 (e.g., SEQ ID NO:5, 6, or 7); 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; and 12) Sequence number 2.

[0128] Package the promoter construct into an AAV serotype capable of transducing hair cells (e.g., AAV1, AAV2, AAV6, AAV9, Anc80, or Anc80L65).

[0129] 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.

[0130] Example 3. Administration of a composition containing a nucleic acid vector comprising 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, e.g., a human patient, with sensorineural hearing loss, thereby improving or restoring hearing. To this end, one skilled in the art can operably link 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 SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., any one of SEQ ID NOs: 8-12), operably linked to a transgene encoding a therapeutic protein. A composition containing an AAV vector (e.g., AAV1, AAV2, AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B) having a polynucleotide comprising a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the first and second regions (e.g., SEQ ID NOS: 8 and 9), and optionally including a linker between the first and second regions, can be administered to a human patient. For example, the polynucleotide operably linked to a transgene encoding a therapeutic protein can be SEQ ID NO: 13. A composition containing an 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.

[0131] After administering the composition to a patient, one 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 in various ways. 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. If one or more tests after administering the composition show improvement in the patient's hearing compared to the hearing test results before administering the composition, this indicates that the patient is responding favorably to treatment. Subsequent doses can be determined and administered as needed.

[0132] Example 4. Specificity of the Myo15 promoter in non-human primates The specificity of the Myo15 promoter was tested in non-human primates. Thirty microliters of AAV1-CMV-GFP or AAV1-Myo15-GFP was injected into the cochlea through the round window membrane at 15 μl / min. Four weeks after AAV injection, animals were sacrificed, 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 3A). In contrast, the 1.6 kb Myo15 promoter restricted GFP transgene expression to hair cells in the cynomolgus monkey cochlea (Figures 3B-3C).

[0133] Example 5. The Myo15 promoter enhances the biological efficacy of AAV-mice Tmc1 in Tmc1 knockout mice compared to the ubiquitous promoter Tmc1 knockout (KO) mice were anesthetized with isoflurane, their hair was clipped, and povidone-iodine was applied to the skin. An incision was made below the left ear, extending from above the cheek muscle to behind the ear. The skin was separated, the muscle torn, and cleaned to expose the posterior semicircular canal. A small hole was drilled into the semicircular canal using a drill bit, and the bone was held dry with a thin cotton swab. A polyamide / polyethylene tube 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 to allow the liquid to reach the apex of the cochlea and to prevent backflow and leakage. The tube was bent and cut close to the bone. The muscle was replaced and the skin was reattached. Mice were given 0.01 cc of meloxicam before recovery 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 4, ABR threshold recovery was significantly improved in homozygous Tmc1 KO mice injected with AAV-Myo15-mouseTMC1 compared with AAV-CMV-mouseTMC1.

[0134] Other embodiments Various modifications and variations of the invention described herein 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 unnecessarily 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 claims. (Addendum) The technical concepts that can be understood from the above-described embodiments and modifications will be described below. [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 having 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] 9. The polynucleotide according to any one of items 1 to 8, wherein the second region comprises or consists of the sequence of SEQ ID NO:2. [Item 10] 9. The polynucleotide according to any one of items 1 to 8, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8. [Item 11] 9. The polynucleotide according to any one of items 1 to 8, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9. [Item 12] 9. The polynucleotide according to any one of items 1 to 8, 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 13] Item 13. The polynucleotide according to item 12, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10. [Item 14] Item 13. The polynucleotide according to item 12, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11. [Item 15] Item 13. The polynucleotide according to item 12, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12. [Item 16] 2. The polynucleotide according to item 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 13. [Item 17] A polynucleotide comprising a first 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, and a second 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, operably linked thereto, and optionally having a linker comprising 1 to 100 nucleotides between the first region and the second region. [Item 18] 18. The polynucleotide of item 17, wherein the first region comprises or consists of the sequence of SEQ ID NO:2. [Item 19] 18. The polynucleotide of item 17, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8. [Item 20] Item 18. The polynucleotide of item 17, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9. [Item 21] Item 18. The polynucleotide according to Item 17, 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 22] 22. The polynucleotide according to item 21, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10. [Item 23] 22. The polynucleotide according to item 21, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11. [Item 24] 22. The polynucleotide according to item 21, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12. [Item 25] 25. The polynucleotide according to any one of Items 17 to 24, wherein the second region comprises or consists of the sequence of SEQ ID NO: 1. [Item 26] 25. The polynucleotide according to any one of Items 17 to 24, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3. [Item 27] 25. The polynucleotide according to any one of Items 17 to 24, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4. [Item 28] 25. The polynucleotide according to any one of Items 17 to 24, 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 29] 29. The polynucleotide according to item 28, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5. [Item 30] 29. The polynucleotide according to item 28, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6. [Item 31] 29. The polynucleotide according to item 28, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 7. [Item 32] 18. The polynucleotide according to item 17, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 14. [Item 33] 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 34] 34. The polynucleotide according to item 33, wherein the region comprises or consists of the sequence of SEQ ID NO: 1. [Item 35] 34. The polynucleotide according to item 33, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3. [Item 36] 34. The polynucleotide according to item 33, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4. [Item 37] 34. The polynucleotide according to item 33, 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 38] 38. The polynucleotide according to item 37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5. [Item 39] 38. The polynucleotide according to item 37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6. [Item 40] 38. The polynucleotide according to item 37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 7. [Item 41] 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 42] 42. The polynucleotide according to item 41, wherein the region comprises or consists of the sequence of SEQ ID NO:2. [Item 43] 42. The polynucleotide of item 41, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8. [Item 44] 42. The polynucleotide of item 41, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9. [Item 45] 42. The polynucleotide according to item 41, 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 46] 46. The polynucleotide according to item 45, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10. [Item 47] 46. The polynucleotide according to item 45, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11. [Item 48] 46. The polynucleotide according to item 45, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12. [Item 49] 49. The polynucleotide of any one of items 1 to 48, wherein the polynucleotide induces expression of a transgene when operably linked to the transgene and introduced into a hair cell. [Item 50] 50. A nucleic acid vector comprising the polynucleotide according to any one of items 1 to 49. [Item 51] 51. The nucleic acid vector of item 50, wherein the polynucleotide is operably linked to a transgene. [Item 52] 52. The nucleic acid vector of item 51, wherein the transgene comprises a nucleic acid sequence encoding a therapeutic protein. [Item 53] 53. The nucleic acid vector of claim 52, wherein the polynucleotide is capable of directing hair cell-specific expression of the therapeutic protein derived from the nucleic acid sequence in mammalian hair cells. [Item 54] 54. The nucleic acid vector of item 53, wherein the hair cells are cochlear hair cells. [Item 55] 55. The nucleic acid vector of item 54, wherein the cochlear hair cells are inner hair cells and / or outer hair cells. [Item 56] 54. The nucleic acid vector of item 53, wherein the hair cells are vestibular hair cells. [Item 57] 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 ESPN. 57. The nucleic acid vector of any one of Items 52 to 56, wherein the nucleic acid vector is selected from the group consisting of L, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NT3, CLRN1, CLRN2, SKOR1, TCTEX1D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP. [Item 58] 58. The nucleic acid vector according to any one of items 50 to 57, wherein the nucleic acid vector is a plasmid, a cosmid, an artificial chromosome, or a viral vector. [Item 59] 59. The nucleic acid vector of item 58, wherein the nucleic acid vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), adenovirus, and lentivirus. [Item 60] 60. The nucleic acid vector of item 59, wherein the viral vector is an AAV vector. [Item 61] 61. The nucleic acid vector of item 60, wherein the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, 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 62] 62. A composition comprising the nucleic acid vector according to any one of items 50 to 61. [Item 63] 63. The composition of claim 62, further comprising a pharmaceutically acceptable excipient. [Item 64] 64. A method for increasing expression of a therapeutic protein in mammalian hair cells, comprising contacting the mammalian hair cells with the nucleic acid vector of any one of Items 50 to 61 or the composition of Items 62 or 63. [Item 65] 65. The method of claim 64, wherein expression of the therapeutic protein is increased specifically in hair cells. [Item 66] 66. The method of item 64 or 65, wherein the mammalian hair cells are human hair cells. [Item 67] 67. The method of any one of items 64 to 66, wherein the mammalian hair cells are cochlear hair cells. [Item 68] Item 68. The method of item 67, wherein the cochlear hair cells are inner hair cells. [Item 69] Item 68. The method of item 67, wherein the cochlear hair cells are outer hair cells. [Item 70] 67. The method of any one of items 64 to 66, wherein the mammalian hair cells are vestibular hair cells. [Item 71] 71. The method of any one of items 64 to 70, wherein expression of the therapeutic protein is not substantially increased in inner ear cells that are not hair cells. [Item 72] 64. A method for treating a subject having or at risk of developing hearing loss, comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 50 to 61 or the composition of item 62 or 63. [Item 73] 73. The method of item 72, wherein the hearing loss is hereditary hearing loss. [Item 74] 74. The method of claim 73, wherein the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss. [Item 75] 73. The method of claim 72, wherein the hearing loss is acquired hearing loss. [Item 76] 76. The method of item 75, wherein the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease- or infection-related hearing loss, head trauma-induced hearing loss, or ototoxic drug-induced hearing loss. [Item 77] 62. A method for treating a subject having or at risk of developing vestibular dysfunction, comprising administering to the subject an effective amount of the nucleic acid vector of any one of Items 50 to 61 or the composition of Items 62 or 63. [Item 78] 78. The method of claim 77, wherein the vestibular dysfunction is vertigo, dizziness, or imbalance. [Item 79] 62. A method for promoting hair cell regeneration in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 50 to 61 or the composition of item 62 or 63. [Item 80] 80. The method of claim 79, wherein the hair cells are cochlear hair cells. [Item 81] 80. The method of claim 79, wherein the hair cells are vestibular hair cells. [Item 82] 62. A method for preventing or reducing ototoxic drug-induced hair cell damage or cell death, comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 50 to 61 or the composition of item 62 or 63. [Item 83] 83. The method of claim 76 or 82, wherein the ototoxic agent is selected from the group consisting of aminoglycosides, antineoplastic agents, ethacrynic acid, furosemide, salicylates, and quinine. [Item 84] 62. A method for treating a subject having tinnitus, comprising administering to the subject an effective amount of the nucleic acid vector according to any one of items 50 to 61 or the composition according to item 62 or 63. [Item 85] 62. A method for preventing or reducing hair cell damage or cell death in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 50 to 61 or the composition of item 62 or 63. [Item 86] 62. A method for increasing hair cell viability in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of items 50 to 61 or the composition of item 62 or 63. [Item 87] 87. The method of any one of items 72-76, 79, 80, and 82-86, further comprising assessing the hearing of the subject prior to administering the nucleic acid vector or composition. [Item 88] 88. The method of any one of items 72-76, 79, 80, and 82-87, further comprising assessing the hearing of the subject after administering the nucleic acid vector or composition. [Item 89] 89. The method of any one of items 77 to 79 and 81 to 88, further comprising assessing vestibular function of the subject prior to administering the nucleic acid vector or composition. [Item 90] 90. The method of any one of items 77 to 79, 81 to 89, further comprising assessing vestibular function of the subject prior to administering the nucleic acid vector or composition. [Item 91] 91. The method of any one of items 72 to 90, wherein the nucleic acid vector or composition is administered locally. [Item 92] 92. The method of any one of items 72 to 91, 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, delay the progression of hearing loss, delay 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 93] 93. The method according to any one of items 72 to 92, wherein the subject is a human. [Item 94] A kit comprising the nucleic acid vector according to any one of Items 50 to 61 or the composition according to Item 62 or 63.

Claims

[Claim 1] The invention described herein.

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