Myosin 15 promoter and its use

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] This specification describes polynucleotides containing a myosin 15 (Myo15) promoter region that can be used to promote 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 containing the same. Methods of using the polynucleotides and vectors of the present invention to achieve transgene expression in hair cells for the treatment of hearing loss and / or vestibular dysfunction are also disclosed. [Background technology]

[0002] Hearing loss is a significant public health issue, with an estimated 15% of school-aged children and one in three by age 65 affected. The most common type of hearing loss is sensorineural hearing loss, which is caused by defects in inner ear cells, such as cochlear hair cells, or in 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 inner ear development and function. More than 90 such gene mutations have been identified, including autosomal recessive, autosomal dominant, and X-linked pattern mutations.

[0003] Factors that disrupt the development, survival, or integrity of cochlear hair cells, such as gene mutations, diseases or infections, ototoxic drugs, head trauma, and aging, can similarly affect vestibular hair cells and are therefore also associated with vestibular dysfunction, including vertigo, dizziness, and balance disorders. In fact, patients with mutations that disrupt the development or function of hair cells may exhibit both hearing loss and vestibular dysfunction, or only one of them. In recent years, efforts to treat hearing loss have increasingly focused on gene therapy as a possible solution; however, there are few approaches that specifically target hair cells, which are frequently involved in hearing loss and vestibular dysfunction. Novel therapies targeting hair cells are needed to treat sensorineural hearing loss or vestibular dysfunction. [Overview of the project]

[0004] The present invention provides compositions and methods for promoting the expression of a target gene as a gene that promotes or improves the function or survival of hair cells in a particular cell type. The compositions and methods described herein relate to polynucleotides that stimulate the transcription of transgenes in hair cells of the inner ear (e.g., cochlear hair cells and vestibular hair cells). The polynucleotides described herein may be operably ligated 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 ataxia).

[0005] In a first aspect, the present invention relates to a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to sequence SEQ ID NO: 1 or its functional part or derivative, including sequence SEQ ID NO: 3 and / or SEQ ID NO: 4, and sequence SEQ ID NO: 8 and / or SEQ ID NO: 9, which binds thereto (e.g., operably linked), and sequence SEQ ID NO: 2 or its functional part or derivative, including sequence SEQ ID NO: 2 and / or SEQ ID NO: 9, which bind thereto (e.g., operably linked) The present invention provides a polynucleotide comprising a linker having a second region having identity, and optionally 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.

[0006] In some embodiments, the first region includes or consists of the sequence of sequence number 1. In some embodiments, the second region includes or consists of the sequence of sequence number 2.

[0007] In some embodiments, the polynucleotide contains or consists of the sequence of SEQ ID NO: 13. In another aspect, the present invention relates to a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to sequence 2 or its functional portion or derivative, including sequence 8 and / or sequence 9, and a sequence having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to sequence 1 or its functional portion or derivative, including sequence 3 and / or sequence 4 which are linked thereto (e.g., operably linked). The present invention provides a polynucleotide comprising a second region having a unique nature, 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 includes or consists of the sequence of sequence number 2. In some embodiments, the second region includes or consists of the sequence of sequence number 1.

[0009] In some embodiments, the polynucleotide contains or consists of the sequence of SEQ ID NO: 14. 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 higher) with respect to sequence number 1 or a functional portion or derivative thereof, including the sequence of sequence number 3 and / or sequence number 4.

[0010] In some embodiments, this region includes or consists of the sequence of sequence number 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 the sequence of SEQ ID NO: 2 or a functional portion or derivative thereof, which comprises 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 foregoing aspects, the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3. In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4. In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO: 1 comprises the sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5. In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6. In some embodiments of any of the foregoing 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 foregoing aspects, the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8. In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9. In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO: 2 comprises the sequences of SEQ ID NO: 8 and SEQ ID NO: 9. In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10. In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11. In some embodiments of any of the foregoing 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 foregoing aspects, when the polynucleotide is operably linked to a transgene and introduced into a hair cell, expression of the transgene is induced. In another embodiment, the present invention provides a nucleic acid vector comprising the polynucleotide of the present invention. In some embodiments, the polynucleotide is operably ligated to a transgene. In some embodiments, the transgene comprises a nucleic acid sequence encoding a therapeutic protein. In some embodiments, the polynucleotide can direct hair cell-specific expression of a therapeutic protein derived from the nucleic acid sequence in mammalian hair cells. In some embodiments, the 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. In some embodiments, the hair cells are vestibular hair cells.

[0014] In some embodiments, the therapeutic proteins 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, and EPS. The group is selected from 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 viruses (AAVs), adenoviruses, and lentiviruses. 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 embodiment, the present invention provides compositions comprising the nucleic acid vector of the present invention. In some embodiments, the composition further comprises pharmaceutically acceptable excipients. In another embodiment, the present invention provides a method for increasing the expression of a therapeutic protein in mammalian hair cells by contacting them with a nucleic acid vector or composition of the present invention. In some embodiments, the expression of the therapeutic protein is specifically increased in hair cells.

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

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

[0019] In another aspect, the present invention provides a method for treating a subject who has or is at risk of developing hearing loss (e.g., sensorineural hearing loss) by administering an effective amount of the nucleic acid vector or composition of the present invention to the subject.

[0020] In some embodiments, the hearing loss is hereditary. 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, traumatic head injury 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 embodiment, the present invention provides a method for treating subjects who have or are at risk of developing vestibular dysfunction by administering an effective amount of the nucleic acid vector or composition of the present invention to the subject. In some embodiments, vestibular dysfunction is rotational vertigo, dizziness, or avalanche.

[0022] In another embodiment, the present invention provides a method for promoting hair cell regeneration in a subject requiring it by administering an effective amount of the nucleic acid vector or composition of the present invention to the subject. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0023] In another embodiment, the present invention provides a method for preventing or mitigating ototoxic drug-induced hair cell damage or death by administering an effective amount of the nucleic acid vector or composition of the present invention to a target. In some embodiments, the ototoxic drug is selected from the group comprising aminoglycosides (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), antitumor agents (e.g., platinum-containing chemotherapeutic agents, e.g., cisplatin, carboplatin, and oxaliplatin), ethacrine, furosemide, salicylates (e.g., aspirin, especially at high doses), and kinins. 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 for treating a subject having tinnitus by administering an effective amount of the nucleic acid vector or composition of the present invention to the subject. In some embodiments of any of the aforementioned aspects, hearing loss, vestibular dysfunction, or tinnitus is associated with the loss of hair cells (e.g., cochlear hair cells and / or vestibular hair cells).

[0025] In another embodiment, the present invention provides a method for preventing or mitigating damage or death of hair cells in a subject requiring such treatment by administering an effective amount of the nucleic acid vector or composition of the present invention to the subject. In some embodiments, the hair cells are cochlear hair cells. In some embodiments, the hair cells are vestibular hair cells.

[0026] In another embodiment, the present invention provides a method for increasing the viability of hair cells in a subject requiring such a method by administering an effective amount of the nucleic acid vector or composition of the present invention to the subject. 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 above embodiments, the hair cells are cochlear hair cells. In some embodiments of any of the above embodiments, the cochlear hair cells are inner hair cells. In some embodiments of any of the above embodiments, the cochlear hair cells are outer hair cells. In some embodiments of any of the above embodiments, the mammalian hair cells are vestibular hair cells.

[0028] In some embodiments of any of the above-described aspects, the method further includes the step of evaluating the subject's hearing before administering the nucleic acid vector or composition (e.g., evaluating hearing using standard tests such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), or otoacoustic emissions).

[0029] In some embodiments of any of the above-described aspects, the method further includes the step of evaluating the hearing of a subject after administration of a nucleic acid vector or composition (e.g., evaluating hearing using standard tests such as audiometry, ABR, ECOG, or otoacoustic emissions).

[0030] In some embodiments of any of the aforementioned aspects, the method further includes the step of evaluating the vestibular function of the subject before administering the nucleic acid vector or composition (e.g., evaluating vestibular function using standard tests such as electrooculography (ENG) or video nystagmography (VNG), postural imaging, swivel chair testing, ECOG, vestibular evoked myoencephalography (VEMP), or specialized clinical balance function tests).

[0031] In some embodiments of any of the aforementioned aspects, the method further includes the step of evaluating the vestibular function of the subject before administering the nucleic acid vector or composition (e.g., evaluating vestibular function using standard tests such as ENG or VNG, posture diagramming, swivel chair test, ECOG, VEMP, or specialized clinical balance function tests).

[0032] In some embodiments of any of the aforementioned aspects, the nucleic acid vector or composition is administered locally (for example, into the inner ear, into the perilymph or endolymph, via the oval window, round window, or horizontal semicircular canal, etc.).

[0033] In some embodiments of any of the above-described 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, 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 the number of hair cells.

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

[0035] definition As used herein, the term "approximately" means a value that is within 10% of the stated value, either above or below it.

[0036] As used herein, “administration” means providing or delivering a therapeutic agent (e.g., a nucleic acid vector containing a myosin 15 (Myo15) promoter operably linked to a transgene) to a subject by any effective route. Exemplary routes of administration are described below herein.

[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-presenting properties. Cells of a common cell type may include cells isolated from common tissues in an organism (e.g., epithelial tissue, nerve tissue, connective tissue, or muscle tissue) and / or common organs, tissue systems, blood vessels, or other structures and / or regions.

[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 perception. There are two types of cochlear hair cells: inner hair cells and outer hair cells. Damage to cochlear hair cells and gene mutations that disrupt the function of cochlear hair cells are associated with hearing loss and deafness.

[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, static charge, and stereovolume. 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 families of conserved amino acids include (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Therefore, a conserved mutation or substitution is a mutation or substitution that replaces an amino acid with a member of the same amino acid family (for example, substituting Thr with Ser, or Arg with Lys).

[0042] As used herein, the terms “effective dose,” “therapeutic effective dose,” and “sufficient dose” of the compositions, vector constructs, or viral vectors described herein refer to an amount sufficient to produce a beneficial or desirable outcome, including a clinical outcome, when administered to a subject in need, such as a mammal, including a human. Therefore, the “effective dose” or its synonyms depends on the context in which it is applied. For example, in relation 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 a response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein corresponding to such an amount will vary depending on various factors such as a given drug, pharmaceutical formulation, route of administration, type of disease or disorder, characteristics of the subject (e.g., age, sex, weight), or host of the subject being treated, but can nevertheless be routinely determined by a person skilled in the art. Also, as used herein, the “therapeutic effective dose” of the compositions, vector constructs, or viral vectors disclosed herein is the amount that produces a beneficial or desired outcome in a subject compared to a control. When active ingredients are administered in combination, it should be noted that the effective dose of the combination may or may not include the amount of each ingredient that would have been effective if administered individually. As defined herein, the therapeutic effective dose of the compositions, vector constructs, or viral vectors of this disclosure can be readily determined by routine methods known in the art to those skilled in the art. The administration plan may be adjusted to provide an optimal therapeutic response.

[0043] As used herein, the term “endogenous” means a molecule (e.g., polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., 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” means one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0045] As used herein, the term “exogenous” refers to a molecule (e.g., polypeptide, nucleic acid, or cofactor) that is not found in nature in a particular organism (e.g., 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 substances include substances supplied from an external source to an organism or a culture extracted therefrom.

[0046] As used herein, the term “hair cell-specific expression” refers primarily to the production of RNA transcripts or polypeptides in hair cells (e.g., cochlear hair cells and / or vestibular hair cells) compared 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 the expression of the transgene (e.g., RNA or protein expression) among various cell types of the inner ear (e.g., hair cells versus non-hair cells) using any standard technique (e.g., quantitative RT-PCR, immunohistochemistry, Western blot analysis, or fluorescence measurement of a reporter (e.g., GFP) operably linked to a promoter). Hair cell-specific promoters induce the expression of transgenes (e.g., RNA or protein expression) to which they are 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 of the following inner ear cell types (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more): border cells, internal phalangeal cells, internal column cells, external column cells, first row deiters cells, second row deiters cells, third row deiters cells, Hensens cells, Claudius cells, internal groove cells, external groove cells, spiral ridge cells, root cells, interdental cells, basal cells of the striae dendrite, intermediate cells of the striae dendrite, marginal cells of the striae dendrite, spiral ganglion neurons, and Schwann cells.

[0047] As used herein, the terms “increase” and “decrease” refer to adjustments that result in a greater or lesser metric of function, expression, or activity compared to the reference, respectively. For example, following administration of a composition according to the method 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 compared to the amount of the marker before administration. Generally, the metric is measured at the point in time when the effects described are obtained by the administration, for example, at least one week, one month, three months, or six months after the start of the treatment regimen.

[0048] As used herein, the term "intron" refers to a region within the coding region of a gene whose nucleotide sequence is not translated into the amino acid sequence of the corresponding protein. The term intron also refers to the corresponding region of RNA transcribed from a gene. Introns are transcribed into premRNA but are removed during processing and are not present in mature mRNA.

[0049] As used herein, the term “linker” refers to a set of nucleotides that connect two distinct regions of a polynucleotide. A linker does not interfere with the function of the two regions of the polynucleotide it connects.

[0050] As used herein, “topical” or “topical administration” means administration to a specific site of the body intended for a local effect rather than a systemic effect. Examples of topical administration include administration onto the skin, by inhalation, into joints, into the spinal cavity, into the vagina, into the vitreous cavity, into the uterus, into lesions, into lymph nodes, into tumors, into the inner ear, and into mucous membranes, in which case the administration is intended to produce a local effect rather than a systemic effect.

[0051] As used herein, the term “operatably linked” means a first molecule that can bind to a second molecule, in which case the molecules are positioned such that the first molecule influences the function of the second molecule. The term “operatably linked” includes juxtaposing two or more components (e.g., a promoter and another sequence element) so that both components function properly and at least one component can mediate a function that acts on at least one other component. The two molecules may or may not be parts of a single, uninterrupted molecule, and may or may not be adjacent. For example, if a promoter regulates the transcription of a desired transcribable polynucleotide molecule within a cell, the promoter is operatably linked to the transcribable polynucleotide molecule. In further embodiments, two parts of a transcriptional regulatory element are operatably linked to each other if they are linked in such a way that the transcriptional activation function of one part is not adversely affected by the presence of the other part. The two transcriptional regulatory elements may be operatably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid), or they may be operatably linked to each other without the presence of an intervening nucleotide.

[0052] As used herein, the term “plasmid” refers to an extrachromosomal circular double-stranded DNA molecule capable of ligating additional DNA segments. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. Certain plasmids are capable of self-replication in the host cell into which they are introduced (e.g., bacterial plasmids with bacterial origins of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Certain plasmids can induce the expression of the gene to which they are operably ligated.

[0053] As used herein, the terms “nucleic acid” and “polynucleotide,” as used interchangeably herein, refer to polymeric forms of nucleosides of any length. Generally, polynucleotides consist 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 skeletons, etc., whether or not they are found in natural nucleic acids, and such molecules may be preferred for certain applications. When this application refers to polynucleotides, it is understood that both DNA and RNA, and both single-stranded and double-stranded forms in each case (and complements of each single-stranded molecule) are provided. As used herein, “polynucleotide sequence” may refer to the sequence information (i.e., a series of letters used as abbreviations for bases) that biochemically characterizes the polynucleotide substance itself and / or a particular nucleic acid. Unless otherwise specified, the polynucleotide sequences presented herein are shown in the 5' to 3' direction.

[0054] As used herein, the terms “complementarity” or “complementary” of nucleic acids mean that, due to the orientation of their nucleic acid bases, the nucleotide sequence of one strand of nucleic acid forms hydrogen bonds with another sequence on the opposite nucleic acid strand. The complementary bases of 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 double helix, in which case all the bases of the double helix bond to complementary bases by Watson-Crick pairing. “Substantially” or “sufficiently” complementary means that the sequence of one strand is not completely and / or perfectly complementary to the sequence of the opposite strand, but sufficient bonding 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 sequences and standard mathematical calculations, or by empirically determining Tm using routine methods. 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). Below Tm, the formation of hybridization complexes is favored, while above Tm, the melting or separation of strands in the hybridization complex is favored. The Tm of nucleic acids may be estimated using known G+C content in 1M NaCl aqueous solution, for example, Tm = 81.5 + 0.41 (%G+C), but 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. Polymerase promotes the transcription of a transgene. The "percentage of amino acid sequence identity (%)" with respect to a reference polynucleotide 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 or reference polypeptide sequence, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage of sequence identity. Alignment aimed at determining the percentage of nucleic acid or amino acid sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm required to obtain the maximum alignment over the full length of the sequences being compared. For example, the value of the percentage of sequence identity may be generated using the sequence comparison computer program BLAST. As an example, the percentage of sequence identity of a given nucleic acid or amino acid sequence A with respect to a given nucleic acid or amino acid sequence B (or can be expressed as a given nucleic acid or amino acid sequence A having a specific percentage of sequence identity with respect to B) is calculated as follows: 100×(fraction X / Y) In the formula, X is the number of nucleotides or amino acids scored as identical by a sequence alignment program (e.g., BLAST) in the program 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, the ratio of sequence identity of A to B is considered to be not equal to the ratio of 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-exclusive examples of chemical modifications involving nucleic acids include modifications to base moieties, sugar moieties, phosphate moieties, phosphate-sugar backbone, or combinations thereof.

[0057] As used herein, the term “pharmaceutical composition” means a mixture comprising a therapeutic agent to be administered to a mammal, such as a human, in combination, optionally with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, for the purpose of preventing, treating, or controlling a particular disease or condition that affects or may affect a subject.

[0058] As used herein, the term “pharmaceutically acceptable” means a compound, substance, composition, and / or dosage form that is free from excessive toxicity, irritation, allergic reactions, and other problematic ailments, has a reasonable benefit-to-risk ratio, and is suitable for contact with the tissues of a subject, such as mammals (e.g., humans). Preferably, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in mammals, more specifically in humans.

[0059] As used herein, the term “sample” means a specimen separated from the 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 villi specimens, 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 may include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or assist in controlling 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 “transfer” refers to any and all of the diverse techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transferation, nucleofection, squeezeporation, sonoporation, phototransferation, magnetofection, imparefection, and the like.

[0062] As used herein, the terms “subject” and “patient” refer to animals (e.g., mammals such as humans), veterinary subjects (e.g., cats, dogs, cattle, horses, sheep, pigs, etc.), and experimental animal models of diseases (e.g., mice, rats). Subjects treated according to the methods described herein may be subjects diagnosed with hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction (e.g., vertigo, vertigo, or ataxia), or subjects at risk of developing these conditions. Diagnosis may be performed by any method or technique known in the art. Those skilled in the art will understand that subjects treated according to this disclosure may have undergone standard tests, or may have been identified as subjects at risk due to the presence of one or more risk factors associated with the disease or condition, even if they have not been tested.

[0063] As used herein, the terms “transduction” and “transduction” refer to a method of introducing a vector construct or a portion thereof into a cell. If the vector construct is contained in a viral vector, such as an AAV vector, transduction refers to the viral infection of the cell and the subsequent transfer and integration of the vector construct or a portion thereof into the cell genome.

[0064] As used herein, “treatment” and “to treat” a condition, disorder, or pathology may include: (1) preventing, delaying, or reducing the incidence and / or likelihood of developing at least one clinical or asymptomatic symptom of a condition, disorder, or pathology in a subject who is already suffering from or predisposed to a condition, disorder, or pathology but has not yet experienced or exhibited any clinical or asymptomatic symptoms; or (2) inhibiting a condition, disorder, or pathology, i.e., preventing, mitigating, or delaying the onset or recurrence of the disease or the onset of at least one clinical or asymptomatic symptom thereof; or (3) reducing the disease, i.e., regressing the condition, disorder, or pathology or at least one of its clinical or asymptomatic symptoms. The benefit to the subject being treated must be statistically significant or at least perceptible to the patient or physician.

[0065] As used herein, the term “vector” includes nucleic acid vectors, DNA vectors such as plasmids, cosmids, or artificial chromosomes, RNA vectors, viruses, or any other suitable replicons (e.g., viral vectors). Various vectors have been developed to deliver polynucleotides encoding exogenous proteins to 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 include 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. Specific vectors that can be used for the expression of transgenes described herein include vectors that include regulatory sequences such as promoter and enhancer regions that direct gene transcription. Other useful vectors for transgene expression include 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 signaling sites to direct the efficient transcription of genes incorporated into the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also include polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or noseoslysin.

[0066] As used herein, the term “vestibular hair cells” refers to a specialized group of cells in the inner ear that are involved in motion sensing 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 gene 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 drawing]

[0068] [Figure 1A] These are a series of fluorescence images of mouse cochlea transduced with an adeno-associated virus (AAV) vector expressing GFP under the control of a cytomegalovirus (CMV) promoter. AAV-CMV-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 inner ear temporal bone was harvested and decalcified in 8% EDTA for 3 days. The cochlea was dissected from the decalcified temporal bone 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 glial cells. [Figure 1B] These are a series of fluorescence images of mouse cochlea transduced with an AAV vector (SEQ ID NO: 13) expressing GFP under the control of the Myo15 promoter. AAV-Myo15-GFP virus was injected into 6-8 week old C57Bl / 6J male mice via the posterior semicircular canal. The mice were allowed to recover from surgery, euthanized, and perfused with 10% neutral buffered formalin 10 days later. The inner ear temporal bone was harvested and decalcified in 8% EDTA for 3 days. The cochlea was dissected from the decalcified temporal bone and mounted on slides for confocal image analysis. Using a hair cell-specific promoter, AAV-Myo15-GFP expression was induced 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). AAV-Myo15-GFP virus was injected into 6-8 week old C57Bl / 6J male mice via the posterior semicircular canal. The mice were allowed to recover from surgery, euthanized, and perfused with 10% neutral buffered formalin 10 days later. The temporal bone of the inner ear was harvested and decalcified in 8% EDTA for 3 days. The vestibular organs were dissected from the decalcified temporal bone and mounted on slides for image analysis. Using a hair cell-specific promoter, AAV-Myo15-GFP expression was induced only in vestibular hair cells (Figure 2). [Figure 3A] A series of fluorescence images of non-human primate cochleas demonstrate that the Myo15 promoter restricts GFP expression to hair cells in non-human primate cochleas. Figure 3A is a confocal image of a non-human primate cochlea that received local injection of AAV1-CMV-GFP through a round window membrane. Tissue was collected 28 days after injection. It shows native GFP fluorescence. GFP expression was detected in various cell types throughout the organ. [Figure 3B] Figure 3B shows a series of fluorescence images of non-human primate cochleas, demonstrating that the Myo15 promoter restricts GFP expression to hair cells in non-human primate cochleas. Confocal images of non-human primate cochleas injected with AAV1-Myo15-GFP and processed in the same manner as the cochlea in Figure 3A are shown. GFP expression was restricted to hair cells. [Figure 3C] These are a series of fluorescence images of non-human primate cochlea, showing that the Myo15 promoter restricts GFP expression to hair cells in non-human primate cochlea. Figure 3C is a magnified view of hair cells within the box region shown in Figure 3B. [Figure 4]This graph shows that the 1.6kb 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 response (ABR) was evaluated at specified weeks of age. ABR thresholds were plotted as a function of stimulation frequency for naive homozygous (white circles and light gray lines) and heterozygous (black circles and dark gray lines) Tmc1 KO mice, as well as homozygous Tmc1 KO mice injected with AAV-CMV mouse TMC1 (CMV_mTmc1; white circles and dark gray lines) or AAV-Myo15-mouse TMC1 (PdBx_mTmc1; P23, light gray line through light gray circle; P28, black circles and dark gray lines). Recovery of the ABR threshold was significantly improved in homozygous Tmc1 KO mice injected with AAV-Myo15-mouse TMC1 compared to AAV-CMV-mouse TMC1. [Modes for carrying out the invention]

[0069] This specification describes compositions and methods for inducing transgene expression, particularly in hair cells (e.g., cochlear hair cells and / or vestibular hair cells). The present invention features a polynucleotide comprising a myosin 15 (Myo15) promoter region capable of specifically expressing transgenes in cochlear hair cells. The present invention also features nucleic acid vectors having these promoters operably linked to polynucleotides encoding polypeptides. Using the compositions and methods described herein, polynucleotides encoding hair cell proteins can be expressed, particularly in cochlear hair cells and vestibular hair cells, and thus the compositions described herein can be administered to subjects (e.g., mammalian subjects, e.g., humans) 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 located in the inner ear. Cochlear hair cells are sensory cells of the auditory system and consist of two main cell types: inner hair cells, which are involved in sound perception, and outer hair cells, which are thought to amplify low frequencies. Vestibular hair cells are located in the semicircular canals and otolith organs of the inner ear and are involved in the sense of movement, which contributes to balance and spatial orientation. Hair cells are named after the cilia, which protrude from the apical surface of the cell and form bundles of hair cells. Deflection of the cilia (for example, by sound waves from cochlear hair cells or by rotation or linear acceleration of vestibular hair cells) opens mechanically-dependent ion channels, allowing hair cells to release neurotransmitters and activate nerves, thereby converting mechanical sounds or motor 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 gene mutations that disrupt their function are associated with hearing loss and deafness. Damage to vestibular hair cells and gene mutations that disrupt their function are associated with vestibular dysfunction, such as balance disorders and dizziness (e.g., vertigo). In recent years, gene therapy has attracted attention as an attractive therapeutic approach for treating hearing loss and vestibular dysfunction; however, there is no method in this field to specifically target hair cells with nucleic acid vectors used in gene therapy.

[0071] Myosin 15 Myo15 is an unconventional actin-based molecular motor that regulates the development of immobiliform hairs. Mice with mutations in Myo15 have been shown to have short immobiliform hairs, severe hearing loss, and vestibular dysfunction, and mutations in the human ortholog, Myo15A, cause DFNB3, a non-syndromic autosomal recessive hearing loss. Myo15 has been observed to localize to immobiliform hairs and is essential for their development and maintenance. The pattern of localization suggests that Myo15 may be specifically expressed in hair cells. However, the Myo15 promoter has not yet been isolated or characterized. The inventors identified evolutionarily conserved blocks within the orthologous genome sequence that may constitute the regulatory elements of the promoter and found that they are located more than 7200 base pairs (bp) upstream of the translation initiation site. This genomic region is too large to be used in combination with an adeno-associated virus (AAV) vector, which has a maximum package size of 4.7 kb, for delivering the target transgene for gene therapy.

[0072] The present invention is partly based on the discovery of an upstream region of the Myo15 translation initiation site that can be used to specifically promote the expression of a transgene 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 target gene (e.g., a gene involved in hair cell development, function, cell fate determination, regeneration, survival, or maintenance, or a gene known to be disrupted, for example, mutated, in subjects with hearing loss or vestibular dysfunction) in hair cells, thereby treating subjects with or at risk of developing hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction (e.g., vertigo, dizziness, or ataxia).

[0073] The polynucleotides of the compositions and methods described herein have nucleic acid sequences derived from a region of the Myo15 locus that can specifically express a transgene in hair cells, or variants thereof, for example, nucleic acid sequences having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to a region of the Myo15 locus that can specifically express a transgene in hair cells. These regions include the nucleic acid sequence immediately preceding the Myo15 translation initiation site and an upstream regulatory element located at least 5 kb away from the Myo15 translation initiation site. The polynucleotides of the compositions and methods described herein may optionally include a linker that operably ligates a region of the Myo15 locus that can specifically express a transgene in hair cells, or the region of the Myo15 locus can be directly ligated without an intervening linker.

[0074] In some embodiments, the polynucleotides described herein include a first region (upstream regulatory element) having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) with respect to the first non-coding exon of the Myo15 gene (a nucleic acid from -6755 to -7209 relative to the Myo15 translation initiation site, the sequence of which is described in SEQ ID NO: 1) or a region containing its functional part or derivative, and a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) with respect to the nucleic acid sequence immediately preceding the Myo15 translation initiation site (a nucleic acid from -1 to -1157 relative to the Myo15 translation initiation site, the sequence of which is described in SEQ ID NO: 2) or a functional part or derivative bound thereto (e.g., operably linked). The functional portion of SEQ ID NO: 1 may have a nucleic acid sequence from -7166 to -7091 relative to the Myo15 translation initiation site (described in SEQ ID NO: 3) and / or a nucleic acid sequence from -7077 to -6983 relative to the Myo15 translation initiation site (described in SEQ ID NO: 4). The first region may include the nucleic acid sequence of SEQ ID NO: 3 fused to the nucleic acid sequence of SEQ ID NO: 4 without the intervening nucleic acid described in SEQ ID NO: 5, or the first region may include the nucleic acid sequence of SEQ ID NO: 4 fused to the nucleic acid sequence of SEQ ID NO: 3 without the intervening nucleic acid described in SEQ ID NO: 6. Alternatively, the first region may include an endogenous intervening nucleic acid sequence (for example, the first region may have a nucleic acid sequence from -7166 to -6983 relative to the Myo15 translation initiation site, as described in SEQ ID NO: 7) or sequences of SEQ ID NO: 3 and SEQ ID NO: 4 linked by a nucleic acid linker. In a polynucleotide where the first region contains both SEQ ID NO: 3 and SEQ ID NO: 4, the two sequences may be included in any order (for example, SEQ ID NO: 3 may be linked to SEQ ID NO: 4 (e.g., preceded), or SEQ ID NO: 4 may be linked to SEQ ID NO: 3 (e.g., preceded). The functional portion of SEQ ID NO: 2 may have a nucleic acid sequence from -590 to -509 relative to the Myo15 translation initiation site (described in SEQ ID NO: 8) and / or a nucleic acid sequence from -266 to -161 relative to the Myo15 translation initiation site (described in SEQ ID NO: 9).The second region may include the nucleic acid sequence of Sequence ID No. 8 fused to the nucleic acid sequence of Sequence ID No. 9 without the intervening nucleic acid described in Sequence ID No. 10, or the second region may include the nucleic acid sequence of Sequence ID No. 9 fused to the nucleic acid sequence of Sequence ID No. 8 without the intervening nucleic acid described in Sequence ID No. 11. Alternatively, the second region may include the sequences of Sequence ID No. 8 and Sequence ID No. 9 linked by an endogenous intervening nucleic acid sequence (for example, the second region may have a nucleic acid sequence from -590 to -161 relative to the Myo15 translation start site, as described in Sequence ID No. 12) or by a nucleic acid linker. In polynucleotides in which the second region includes both Sequence ID No. 8 and Sequence ID No. 9, the two sequences may be included in any order (for example, Sequence ID No. 8 may be linked to Sequence ID No. 9 (e.g., preceded), or Sequence ID No. 9 may be linked to Sequence ID No. 8 (e.g., preceded)).

[0075] The first and second regions of a polynucleotide can be directly bound or bound by a nucleic acid linker. For example, a polynucleotide may include the sequence of Sequence ID No. 1 or its functional part or derivative (e.g., one or more of Sequence IDs No. 3 to 7, e.g., Sequence IDs No. 3 and 4) fused with the sequence of Sequence ID No. 2 or its functional part or derivative (e.g., one or more of Sequence IDs No. 8 to 12, e.g., Sequence IDs No. 8 and 9) without intervening nucleic acids. For example, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of Sequence ID No. 1 and Sequence ID No. 2 is shown in Sequence ID No. 13. Alternatively, a linker can be used to bind the sequence of Sequence ID No. 1 or its functional part or derivative (e.g., one or more of Sequence IDs No. 3 to 7, e.g., Sequence IDs No. 3 and 4) to the sequence of Sequence ID No. 2 or its functional part or derivative (e.g., one or more of Sequence IDs No. 8 to 12, e.g., Sequence IDs No. 8 and 9).

[0076] The length of the nucleic acid linker for use with the polynucleotides described herein may be about 5 kb or less (for example, 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). The nucleic acid linker that can be used with the polynucleotides described herein does not impair the ability of the polynucleotides of the present invention to induce transgene expression in hair cells.

[0077] In some embodiments, the sequence of SEQ ID NO: 1 or its functional part or derivative (e.g., one or more of SEQ ID NOs: 3-7, e.g., SEQ ID NOs: 3 and 4) is linked (e.g., operably linked) to the sequence of SEQ ID NO: 2 or its functional part or derivative (e.g., 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 its functional part or derivative (e.g., one or more of SEQ ID NOs: 8-12, e.g., SEQ ID NOs: 8 and 9) is linked to the sequence of SEQ ID NO: 1 or its functional part or derivative (e.g., one or more of SEQ ID NOs: 3-7, e.g., SEQ ID NOs: 3 and 4) (e.g., operably linked)). 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 shown in SEQ ID NO: 14. Regardless of the order, the sequences of SEQ ID NO: 1 or its functional part or derivative and the sequences of SEQ ID NO: 2 or its functional part or derivative can be linked by direct fusion or a nucleic acid linker as described above.

[0078] In some embodiments, the polynucleotides described herein include a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) with respect to the first non-coding exon of the Myo15 gene (a nucleic acid sequence from -6755 to -7209 relative to the Myo15 translation initiation site, the sequence of which is shown in SEQ ID NO: 1) or a region containing its functional portion or a derivative thereof. The functional portion of SEQ ID NO: 1 may have a nucleic acid sequence from -7166 to -7091 relative to the Myo15 translation initiation site (described in SEQ ID NO: 3) and / or a nucleic acid sequence from -7077 to -6983 relative to the Myo15 translation initiation site (described in SEQ ID NO: 4). The polynucleotide may include the nucleic acid sequence of SEQ ID NO: 3 fused to the nucleic acid sequence of SEQ ID NO: 4 without the intervening nucleic acid described in SEQ ID NO: 5, or the polynucleotide may include the nucleic acid sequence of SEQ ID NO: 4 fused to the nucleic acid sequence of SEQ ID NO: 3 without the intervening nucleic acid described in SEQ ID NO: 6. Alternatively, the polynucleotide may contain an endogenous intervening nucleic acid sequence (for example, the first region may have a nucleic acid sequence from -7166 to -6983 relative to the Myo15 translation start site, as described in SEQ ID NO: 7) or sequences of SEQ ID NO: 3 and 4 linked by a nucleic acid linker. In a polynucleotide containing both SEQ ID NO: 3 and 4, the two sequences may be included in any order (for example, SEQ ID NO: 3 may be linked to SEQ ID NO: 4 (e.g., preceded), or SEQ ID NO: 4 may be linked to SEQ ID NO: 3 (e.g., preceded).

[0079] In some embodiments, the polynucleotides described herein include a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) with respect to the nucleic acid sequence upstream of the Myo15 translation initiation site (a nucleic acid sequence from -1 to -1157 relative to the Myo15 translation initiation site, the sequence of which is shown in SEQ ID NO: 2) or its functional portion or derivative thereof. The functional portion of SEQ ID NO: 2 may have a nucleic acid sequence from -590 to -509 relative to the Myo15 translation initiation site (described in SEQ ID NO: 8) and / or a nucleic acid sequence from -266 to -161 relative to the Myo15 translation initiation site (described in SEQ ID NO: 9). The polynucleotide may include the nucleic acid sequence of SEQ ID NO: 8 fused to the nucleic acid sequence of SEQ ID NO: 9 without the intervening nucleic acid described in SEQ ID NO: 10, or the polynucleotide may include the nucleic acid sequence of SEQ ID NO: 9 fused to the nucleic acid sequence of SEQ ID NO: 8 without the intervening nucleic acid described in SEQ ID NO: 11. Alternatively, the polynucleotide may contain an endogenous intervening nucleic acid sequence (for example, the second region may have a nucleic acid sequence from -590 to -161 relative to the Myo15 translation start site, as described in SEQ ID NO: 12) or sequences of SEQ ID NO: 8 and SEQ ID NO: 9 linked by a nucleic acid linker. In a polynucleotide containing both SEQ ID NO: 8 and SEQ ID NO: 9, the two sequences may be included in any order (for example, SEQ ID NO: 8 may be linked to SEQ ID NO: 9 (e.g., preceded by it), or SEQ ID NO: 9 may be linked to SEQ ID NO: 8 (e.g., preceded by it)).

[0080] The nucleic acid sequences mentioned above 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] Further polynucleotides useful in combination 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 higher) to the nucleic acid sequences and functional portions or derivatives of the nucleic acid sequences shown in Table 2.

[0087] The aforementioned polynucleotides can be included in a nucleic acid vector and operably ligated 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, determination of hair cell fate, 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, deafness, auditory neuropathy, tinnitus, or vestibular dysfunction (e.g., vertigo, dizziness, or ataxia). A composition comprising one or more of the aforementioned polynucleotides (e.g., one or more of the polynucleotides listed in Table 2) operably ligated to a transgene encoding a therapeutic protein for the treatment of hearing loss and / or vestibular dysfunction can be administered to a subject according to the method described herein. In some embodiments, the transgenes include actin γ1 (ACTG1), phascin actin bundling protein 2, retinal (FSCN2), radixin (RDX), POU class 4 homeobox 3 (POU4F3), TRIO and F-actin binding proteins (TRIOBP), taperin (TPRN), Xin actin-binding repeat-containing protein 2 (XIRP2), Atonal BHLH transcription factor 1 (ATOH1), growth factor-independent transcription repressor 1 (GFI1), cholinergic receptor nicotinic α9 subunit (CHRNA9), calcium and integrin-binding family member 3 (CIB3), cadherin 23 (CDH23), protocadherin 15 (PCDH15), quinosylline (KNCN), pedivaquine (DFNB59), otoferrin (OTOF), MKRN2 counterchain (MKRN2OS), and LIM. Meobox protein 3 (LHX3), transmembrane channel-like protein 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 keratinization envelope 6A (LCE6A),Lipoxygenase homology domain-containing protein 1 (LOXHD1), ADP-ribosyltransferase 1 (ART1), ATPase cell membrane Ca2+ transporter 2 (ATP2B2), calcium and integrin-binding family member 2 (CIB2), calcium voltage-gated channel accessory 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 protein (LRTOMT2, LRTOMT1), USH1 protein network component harmoniin (USH1C), Extracellular leucine-rich repeat and fibronectin type III domain-containing protein 1 (ELFN1), Tetratricopeptide repeat protein 24 (TTC24), Dystrothelin (DYTN), Key Phosphate / Chordin-like protein (KCP), Coiled-coil glutamate-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), Clarine 1 (CLRN1), Clarine 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), glutarredoxine domain-containing cysteine-rich protein 2 (GRXCR2), brain-derived neurotrophic factor (BDNF), serpine 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 2α-kinase 3 (PERK), serine / threonine protein kinase / endoribonuclease IRE1 (IRE1),It encodes a protein selected from the group consisting of and binding immunoglobulin proteins (BIPs).

[0088] Expression of exogenous 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, for example, mutations in MYO7A, are also associated with vestibular dysfunction. Using the compositions and methods described herein, a nucleic acid vector containing a Myo15 promoter operably ligated to a nucleic acid sequence encoding a protein of interest, particularly in hair cells (e.g., cochlear hair cells and / or vestibular hair cells), is used to ligate a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to SEQ ID NO: 1 or its functional portion or derivative, and / or SEQ ID NO: 2 or its functional portion or derivative. In contrast, by administering a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, possibly including a linker connecting the first and second regions), the expression of a protein encoded by the target gene (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 determination, 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-coding genes in mammalian cells.

[0089] The proteins that can be expressed in connection with the compositions described herein (for example, when a transgene encoding a protein is operably linked to a polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 1 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) and / or a second region having at least 85% sequence identity to SEQ ID NO: 2 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity)) are proteins expressed in healthy hair cells (e.g., cochlear hair cells and / or vestibular hair cells, for example, proteins that play a role in the development, function, regeneration, determination of cell fate, survival, or maintenance of hair cells, or proteins that are deficient in subjects with sensorineural hearing loss or vestibular dysfunction) or other therapeutic proteins for specific purposes. 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 Examples include 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] Polynucleotides that code for the target protein One platform that can be used to bring a target protein to a therapeutically effective intracellular concentration within mammalian cells is the stable expression of the gene encoding the target protein (e.g., by integration into the nuclear or mitochondrial genome of the mammalian cell, or by episomal chain formation in the nucleus of the mammalian cell). The 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. The vector can be introduced into cells by a variety of methods, including transformation, translocation, transduction, direct uptake, particle impaction, and encapsulation of the vector into liposomes. Examples of suitable methods for translocating or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail, for example, 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 which are incorporated herein by reference.

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

[0092] For gene expression, it is crucial that polynucleotides encoding a target protein are recognized and bound by mammalian RNA polymerase. Therefore, the polynucleotide may contain sequence elements that exhibit high affinity for transcription factors, thereby recruiting RNA polymerase and promoting the assembly of transcription complexes at the transcription initiation site. Such sequence elements include, for example, mammalian promoters, whose sequences can be recognized and bound by specific transcription initiation factors and ultimately by RNA polymerase. An example of a mammalian promoter is described in Smith, et al., Mol. Sys. Biol., 3:73 (online publication), the disclosure of which is incorporated herein by reference. The 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 higher) to SEQ ID NO: 1 or its functional portion or derivative, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) to SEQ ID NO: 2 or its functional portion or derivative, and optionally comprising a linker between the first and second regions.

[0093] Once a polynucleotide encoding a target protein is incorporated into the nuclear DNA of a mammalian cell, the transcription of this polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing mammalian cells to external chemical reagents, such as agents that regulate the binding of transcription factors and / or RNA polymerase to a mammalian promoter, and thus regulate gene expression. The chemical reagents may function to promote the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example, by removing a repressor protein bound to the promoter. Alternatively, the chemical reagents may function to increase the affinity of the mammalian promoter for RNA polymerase and / or transcription factors, thereby increasing the transcription rate of genes located downstream of the promoter in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells to promote gene expression according to established protocols.

[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 containing the gene of interest so that the DNA adopts a three-dimensional orientation favorable for the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein include polynucleotides encoding the protein of interest and further include mammalian enhancer sequences. Many enhancer sequences derived from mammalian genes are currently known, examples of which include enhancers for 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 that can infect eukaryotic cells. Examples include SV40 enhancers (bp100-270) on the posterior side of the origin of replication, cytomegalovirus early promoter enhancers, posterior side of the origin of replication polyoma enhancers, and adenovirus enhancers. Further enhancer sequences that induce activation of eukaryotic gene transcription include CMV enhancers and RSV enhancers. The enhancers may be spliced, for example, at the 5' or 3' position of the gene within a vector containing the polynucleotide encoding the protein of interest. In a preferred orientation, the enhancer is positioned 5' to the promoter, and then the promoter is positioned 5' to the polynucleotide encoding the protein of interest.

[0095] Nucleic acid vectors containing the Myo15 promoter described herein may also include a Woodchuck post-transcriptional regulatory element (WPRE). WPREs act at the transcriptional level, increasing the total amount of mRNA in cells by promoting nuclear export of transcripts and / or by enhancing the efficiency of polyadenylation of nascent transcripts. Addition of WPREs to vectors 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, the nucleic acid vectors comprising the Myo15 promoter described herein include a reporter sequence, which may be useful for verifying the expression of a gene operably linked to the Myo15 promoter in cells and tissues (e.g., cochlear hair cells and / or vestibular hair cells). Examples of 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 well known in the art. When the reporter sequence is associated with a regulatory element such as the Myo15 promoter that drives their expression, it provides a signal detectable by conventional means, including enzyme assays, radiometric assays, colorimetric assays, fluorescence assays or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry. For example, if the marker sequence is the LacZ gene, the presence of the signal-containing vector can be detected by an assay for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-containing vector may be visually measured by the generation of color or light using a luminometer.

[0097] Method for delivering exogenous nucleic acids to target cells Techniques for introducing transgenes, such as transgenes operably linked to the Myo15 promoter described herein, into target cells (e.g., mammalian cells) are well known in the art. For example, by applying an electrostatic potential to the cells of interest using electroporation, mammalian cells (e.g., human target cells) can be made permeable. Mammalian cells, such as human cells, thus exposed to an external electric field, are then more readily able to take up exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, 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 technology are described in detail, for example, Distler et al., Experimental Dermatology 14:315 (2005) and US2010 / 0317114, the respective disclosures of which are incorporated herein by reference.

[0098] Another useful technique for translocation of target cells is squeeze-poration. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membranous pores formed in response to applied stress. This technique is advantageous in that it does not require a vector for the delivery of nucleic acids to cells such as human target cells. Squeeze-poration is described in detail, 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 translocation of target cells. This method involves loading nucleic acids into liposomes, often by presenting cationic functional groups, such as quaternary amines or protonated amines, toward the outside of the liposomes. This facilitates electrostatic interactions between the liposomes and cells due to the anionicity of the cell membrane, ultimately leading to the uptake of exogenous nucleic acids, for example, by direct fusion of the liposomes and the cell membrane or endocytosis of the complex. Lipofection is described in detail, for example, U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that utilize ionic interactions with the cell membrane to induce uptake of exogenous nucleic acids involve contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to confer a positive charge favorable to interaction with the cell membrane include activated dendrimers (e.g., 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 these as translocation agents is described in detail, e.g., Gulick et al., Current Protocols in Molecular Biology 40:I: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 translocate target cells in a gentle and efficient manner because this methodology utilizes the application of a magnetic field to direct nucleic acid uptake. This technique is described in detail, e.g., 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 laserfaction, also known as optical translocation, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeate the cells, allowing 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. Then, a chip equipped with these needle arrays is pressed onto cells or tissue. Cells stimulated by the nanostructures can express the delivered gene(s). Examples of this technique are described in Shalek et al., PNAS 107:1870 (2010), the disclosure of which is incorporated herein by reference.

[0102] Nucleic acids can also be delivered to target cells using magnetofection. The principle of magnetofection is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of fully biodegradable iron oxide and are coated with specific, unique cationic molecules that differ depending on the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved by colloidal aggregation and electrostatic interactions induced by salt. The magnetic particles are then concentrated on the 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 acids by target cells is sonoporation, a technique that involves using sound waves (usually ultrasonic frequencies) to modify the permeability of the cell membrane, making the cell permeable and allowing polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0104] Microvesicles represent another potential medium that can be used to modify the genome of target cells according to the methods described herein. For example, by using microvesicles induced by co-overexpression of the glycoprotein VSV-G with a genome-modifying protein such as a nuclease, the protein can be efficiently delivered to the cell, and then site-specific cleavage of endogenous polynucleotide sequences can be catalyzed, thereby preparing the cell's genome for covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also called Gesicles, for genetic modification of eukaryotic cells is, for example, described by Quinn et al., Genetic Modification of Target 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; 2015 May 13, Abstract No. 122, is described in detail.

[0105] Vectors for delivering exogenous nucleic acids to target cells In addition to achieving rapid transcription and translation, stable expression of exogenous genes in mammalian cells can be achieved by incorporating the polynucleotide containing the gene into the nuclear genome of the mammalian cell. Various vectors have been developed for delivering and incorporating 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). The expression vectors used in the compositions and methods described herein include a polynucleotide comprising a polynucleotide sequence encoding a protein of interest, and a Myo15 promoter operably linked to additional sequence elements used for the expression of these activators and / or the integration of these polynucleotide sequences into a mammalian cell genome (e.g., a polynucleotide comprising a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) to SEQ ID NO: 1 or its functional portion or derivative, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) to SEQ ID NO: 2 or its functional portion or derivative, and optionally comprising a linker between the first and second regions). Vectors that can contain a Myo15 promoter operably linked to a transgene encoding the target protein include plasmids (e.g., circular DNA molecules that can autonomously replicate in cells), cosmids (e.g., pWE vectors or sCos vectors), artificial chromosomes (e.g., human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)), and viral vectors. Specific vectors that can be used for the expression of the target protein include plasmids containing regulatory sequences such as enhancer regions that direct gene transcription.Other vectors useful for the expression of target proteins include 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, intra-sequence ribosome entry sites (IRESs), and polyadenylation signaling sites to direct the efficient transcription of the genes contained in the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also include polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or noseoslysin.

[0106] Viral vectors for nucleic acid delivery Viral genomes provide a rich source of vectors that can be used to efficiently deliver desired genes 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 incorporated into the nuclear genome of mammalian cells by universal 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 virus vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses, influenza viruses, rhabdoviruses, rabies viruses and varicella stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, variant 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 viruses. Examples of retroviruses include: avian leukemia sarcoma, avian type C virus, mammalian type C, B, and D viruses, onchoretrovirus, HTLV-BLV group, lentivirus, alpha-retrovirus, gamma-retrovirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).Other examples include mouse leukemia virus, mouse sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, 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. An rAAV vector useful for the compositions and methods described herein is a recombinant nucleic acid construct comprising (1) a Myo15 promoter as described herein (e.g., a polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 1 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) and / or a second region having at least 85% sequence identity to SEQ ID NO: 2 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity), optionally including a linker between the first and second regions), (2) a heterologous sequence to be expressed, and (3) a viral sequence that promotes the stability and expression of the heterologous gene. The viral sequence may include the AAV sequence required in cis for DNA replication and packaging into viral particles (e.g., functional ITR). In common applications, the transgene encodes a therapeutic protein that can promote hair cell development, hair cell function, hair cell regeneration, hair cell fate determination, hair cell survival, or hair cell maintenance, or, in subjects with hereditary hearing loss or vestibular dysfunction, the wild-type of a mutated hair cell protein, which may be useful in improving hearing or vestibular function in subjects with mutations associated with hearing loss, deafness, or vestibular dysfunction (e.g., dizziness, vertigo, or ataxia). Such rAAV vectors may also include a marker or reporter gene. In useful rAAV vectors, one or more AAV WT genes are deleted in whole or in part, but the functional adjacent ITR sequence is retained. The AAV ITR may be any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITR may be an AAV2 ITR.Methods of 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), and each of these disclosures is incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0108] To facilitate the introduction of polynucleotides or vectors into cells, the polynucleotides and vectors described herein (e.g., a Myo15 promoter operably linked to a transgene encoding the protein of interest) can be incorporated into rAAV virus particles. The AAV capsid protein constitutes the non-nucleic acid portion outside the virus particle and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, which are necessary for the assembly of the virus particle. Constructions of rAAV virus particles are described, for example, in US5,173,414;US5,139,941;US5,863,541;US5,869,305;US6,057,152; and US6,376,237; as well as in Rabinowitz et al., J.Virol.76:791(2002) and Bowles et al., J.Virol.77:423(2003), each of which disclosures are incorporated herein by reference as they belong to AAV vectors for gene delivery.

[0109] rAAV virus particles useful in combination with the compositions and methods described herein include virus 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. When targeting hair cells, AAV1, AAV2, AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, and PHP.B may be particularly useful. Serotypes evolved for retinal transduction 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); Harlbert et al., J.Virol.74:1524(2000); Harlbert et al., J.Virol.75:6615(2001); and Auricchio et al., Hum.Molec.Genet.10:3075(2001), and each of these disclosures is incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0110] Pseudotyped rAAV vectors are useful in combination with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) that have been pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques including the construction and use of pseudotyped rAAV virus 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 virus particles with mutations within the capsid of the virus particle can be used to more effectively infect specific cell types than non-mutated capsid virus particles. For example, a suitable AAV variant may have ligand insertion mutations to facilitate AAV targeting of specific cell types. The construction and characterization of AAV capsid variants, including insertion variants, alanine screening variants, and epitope tag variants, are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virus particles that can be used in the methods described herein include capsid hybrids produced 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 (for example, a polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 1 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) and / or a second region having at least 85% sequence identity to SEQ ID NO: 2 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity), optionally including a linker between the first and second regions) may be operably ligated to a transgene (e.g., a transgene encoding a protein of interest) and incorporated into a vehicle for administration to a patient, for example, a human patient suffering from sensorineural hearing loss and / or vestibular dysfunction. A pharmaceutical composition comprising a vector, such as a viral vector, containing the polynucleotide described herein operably ligated 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 an aqueous solution, using, for example, a physiologically acceptable carrier, excipient, or stabilizer (as incorporated herein by reference in Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013)).

[0113] A mixture of nucleic acid vectors (e.g., viral vectors) containing the polynucleotides described herein, operably linked to a therapeutic transgene (e.g., a polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 1 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) and / or a second region having at least 85% sequence identity to SEQ ID NO: 2 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity), and optionally containing a linker between the first and second regions), may be prepared in water appropriately mixed with one or more excipients, carriers, or diluents. The dispersion may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil. These formulations may contain preservatives to inhibit microbial growth under normal storage and use conditions. Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of injectable sterile solutions or dispersions (described in US5,466,468, the disclosure of which is incorporated herein by reference). Optionally, the formulation may be sterile and have sufficient fluidity for easy injection. The formulation may be stable under manufacturing and storage conditions and may be protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Adequate fluidity may be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Inhibition of microbial activity may be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it would be preferable to include an isotonic agent, such as sugar or sodium chloride.The extension of absorption of an injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0114] For example, solutions containing the pharmaceutical compositions described herein may be appropriately buffered as needed, and the liquid diluent is first 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 are known to those skilled in the art in light of this disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the intended injection site. Depending on the condition of the target being treated, some variation in dose is inevitable. For local administration to the inner ear, the composition may be formulated to contain a synthetic perilymphatic solution. An example of a synthetic perilymphatic solution contains 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl2, 1-10 mM glucose, and 2-50 mM HEPES, with a pH of approximately 6-9 and a weight osmolality of approximately 300 mOsm / kg. In any case, the person administering the drug will determine the appropriate dose for each individual patient. Furthermore, in the case of administration to humans, the formulation may meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biologics.

[0115] Treatment method The compositions described herein may be administered to subjects with sensorineural hearing loss and / or vestibular dysfunction by various routes, such as local administration to the inner ear (e.g., administration to the perilymph or endolymph via the oval window, round window, or semicircular canals (e.g., horizontal semicircular canal), e.g., administration to cochlear hair cells or vestibular hair cells), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intra-arterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most appropriate route of administration in a given case depends on the specific composition to be administered, the patient, the pharmaceutical formulation method, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. The compositions may be administered once or more times (e.g., once a year, twice a year, three times a year, every other month, or monthly).

[0116] Subjects who can be treated as described herein are those who have or are at risk of developing sensorineural hearing loss and / or vestibular dysfunction (e.g., subjects who have or are at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods described herein can be used to treat subjects who have or are at risk of developing damage to cochlear hair cells (e.g., damage related to acoustic trauma, disease or infection, head injury, ototoxic drugs, or aging), subjects who have or are at risk of developing damage to vestibular hair cells (e.g., damage related to disease or infection, head injury, ototoxic drugs, or aging), subjects who have or are at risk of developing sensorineural hearing loss, deafness, or auditory neuropathy, subjects who have or are at risk of developing vestibular dysfunction (e.g., vertigo, vertigo, or ataxia), subjects who have tinnitus (e.g., tinnitus alone, or tinnitus associated with sensorineural hearing loss or vestibular dysfunction), subjects who have gene mutations associated with hearing loss and / or vestibular dysfunction, or subjects who have a family history of hereditary hearing loss, deafness, 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 a step of screening the subject for gene mutations known to be associated with hearing loss or vestibular dysfunction prior to treatment or administration with the compositions described herein. The subject can be screened for gene mutations using standard methods known to those skilled in the art (e.g., genetic testing). The methods described herein may also include a step of evaluating the auditory function and / or vestibular function of the subject prior to treatment or administration with the compositions described herein. Hearing can be evaluated using standard tests such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions.Vestibular function may be evaluated using standard tests such as eye movement tests (e.g., electrooculography (ENG) or videonythragography (VNG)), postural tests, swivel chair tests, ECOG, vestibular evoked myoencephalography (VEMP), and specialized clinical balance function 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 prophylactic treatment to patients at risk of developing hearing loss and / or vestibular dysfunction, for example, patients with a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), patients with gene mutations associated with hearing loss or vestibular dysfunction who have not yet shown hearing impairment or vestibular dysfunction, or patients exposed to risk factors for acquired hearing loss (e.g., disease or infection, head injury, ototoxic drugs, or aging) or vestibular dysfunction (e.g., acoustic trauma, disease or infection, head injury, ototoxic drugs, or aging).

[0117] The compositions and methods described herein can be used to promote or induce the regeneration of hair cells in a subject (e.g., regeneration of cochlear hair cells and / or vestibular hair cells). Subjects who may benefit from compositions that promote or induce hair cell regeneration include subjects 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 injury), disease or infection, ototoxic drugs, or aging), and subjects 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 injury), disease or infection, ototoxic drugs, or aging), or subjects with a reduced number of hair cells due to genetic mutation or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase the survival of hair cells (for example, to increase the survival rate of damaged hair cells, to promote the repair of damaged hair cells, or to preserve hair cells in subjects at risk of hair cell loss (for example, hair cell loss due to aging, exposure to loud noises, disease or infection, head injury or ototoxic drugs)).

[0118] The compositions and methods described herein can also be used to prevent or mitigate ototoxic drug-induced hair cell damage or cell death (e.g., damage or cell death of cochlear hair cells and / or vestibular hair cells) in subjects treated with ototoxic drugs, or subjects currently being treated with or scheduled to be treated with ototoxic drugs. Ototoxic drugs are toxic to inner ear cells and may cause sensorineural hearing loss, vestibular dysfunction (e.g., vertigo, dizziness, or ataxia), tinnitus, or a combination of these symptoms. Drugs that have been shown to be ototoxic include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), biomycin, antitumor agents (e.g., platinum-containing chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacric acid and furosemide), salicylates (e.g., particularly high doses of aspirin), and kinins. In some embodiments, the methods described herein prevent or mitigate hair cell damage or cell death associated with acoustic trauma, disease or infection, head trauma, or aging.

[0119] A transgene operably linked to the Myo15 promoter for the treatment of a subject, as 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 higher) to SEQ ID NO: 1 or its functional portion or derivative, and / or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) to SEQ ID NO: 2 or its functional portion or derivative, may 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 determination, regeneration, survival, or maintenance, or a protein that is deficient in subjects with sensorineural hearing loss and / or vestibular dysfunction) or a therapeutic protein for another purpose. The introduced gene may be selected based on the cause of the subject's hearing loss or vestibular dysfunction (for example, if the subject's hearing loss or vestibular dysfunction is associated with a specific gene mutation, the introduced gene may be the wild type of the mutated gene in the subject, or if the subject has hearing loss associated with hair cell loss, the introduced gene 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 transgenes operably linked to the Myo15 promoter for the treatment of the target, 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, and ATP2B2. Examples include 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] Therapy may involve the administration of a composition comprising a nucleic acid vector (e.g., an AAV virus vector) containing the Myo15 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, are within the scope of the art. The unit dose does not need to be administered as a single injection, but may involve continuous infusion over a set period of time. Administration may be carried out using a syringe pump to control the infusion rate in order to minimize damage to the inner ear (e.g., the cochlea). If the nucleic acid vector is an AAV vector (e.g., 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 is, for example, approximately 1 × 10⁻¹⁶. 10 Vector genome (VG) ~ 1 × 10⁻¹⁴ 15 VG (for example, 1 x 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 patients in doses of 1 μL to 200 μL (for example, 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 the expression of therapeutic proteins encoded by transgenes, increase the function of therapeutic proteins encoded by transgenes, prevent or reduce hair cell damage, prevent or reduce hair cell death (e.g., ototoxic drug-induced hair cell death, age-related hair cell death, or noise-related hair cell death (e.g., acoustic trauma)), 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 also be assessed using standard audiometry (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 pre-treatment hearing measurements. Vestibular function may also be assessed using standard tests for balance and rotational vertigo (e.g., eye movement tests (e.g., ENG or VNG), posture tests, swivel chair tests, 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 pre-treatment measurements. 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 amounts sufficient to delay or prevent the onset or progression of sensorineural hearing loss and / or vestibular dysfunction (for example, in subjects who have a gene mutation associated with hearing loss or vestibular dysfunction, a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), or who are exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, acoustic trauma, or infection) but do not exhibit hearing loss or vestibular dysfunction (e.g., vertigo, dizziness, or ataxia), or in subjects exhibiting mild to moderate hearing loss or vestibular dysfunction). The expression of the therapeutic protein encoded by the transgene operably linked to the Myo15 promoter in the nucleic acid vector administered to the target may be evaluated using immunohistochemistry, Western blotting, quantitative real-time PCR, or other methods known in the art for detecting proteins 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 the expression before administration of the composition described herein. The number of hair cells, hair cell function, or the function of the therapeutic protein encoded by the nucleic acid vector administered to the subject may be indirectly assessed based on hearing tests or vestibular function tests, and the number of hair cells, hair cell function, or therapeutic protein function may increase 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 the compositions 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 normally observed in untreated subjects. These effects may occur within, for example, one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or twenty-five weeks, or longer, after administration of the compositions described herein.Depending on the dosage and route of administration used for treatment, the patient may be evaluated one, two, three, four, five, six months, or more after administration of the composition. Depending on the evaluation results, the patient may be given additional treatment.

[0122] kit The compositions described herein can be provided in kits for use in the treatment of sensorineural hearing loss or vestibular dysfunction. The compositions may include a polynucleotide described herein (e.g., a polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 1 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) and / or a second region having at least 85% sequence identity to SEQ ID NO: 2 or its functional portion or derivative (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity), optionally comprising a linker between the first and second regions), a nucleic acid vector comprising such a polynucleotide, and a nucleic acid vector comprising the polynucleotide described herein operably ligated 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). Nucleic acid vectors may be packaged in AAV virus capsids (e.g., AAV1, AAV2, AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B). The kit may further include instructions for the kit user, e.g., a physician, to carry out the methods described herein. The kit may optionally include syringes or other devices for administering the composition. [Examples]

[0123] The following examples are provided to those skilled in the art to illustrate how the compositions and methods described herein may be used, manufactured, and evaluated, and are intended purely to illustrate the present invention and not to limit the scope of what the inventors consider to be their invention.

[0124] Example 1. Generation of Myo15 promoter First, the evolutionarily conserved region of the vertebrate Myo15 promoter was identified in UCSC Genome Identification was performed using Browser (genome.ucsc.edu). The region immediately upstream of the Myo15 translation start site (-1 to -1157, SEQ ID NO: 1) and the upstream region containing non-coding exon 1 of the Myo15 gene (-6755 to -7209, SEQ ID NO: 2) were identified using de The genes were synthesized via novo gene synthesis and joined together as a single DNA fragment (SEQ ID NO: 13). The total size of the truncated Myo15 promoter is 1611 bp, but the entire genomic region exceeds 7000 bp.

[0125] In experiments evaluating the tropism (cell targeting) and the degree and duration of transgene expression using the Myo15 promoter compared to the cytomegalovirus (CMV) promoter in mouse cochles, selective expression in cochlear hair cells was obtained with the Myo15 promoter, but not with the CMV promoter. AAV constructs were created using Myo15, which drives the expression of Aequorea coerulescens green fluorescent protein (AcGFP), and the progression of gene expression was analyzed compared to a matching standard AAV construct using CMV. Transgene expression was evaluated in experiments where the virus was delivered to mouse cochles in neonatal mice, adult mice, and ex vivo in 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. The mice were allowed to recover from surgery, euthanized, and perfused with 10% neutral buffered formalin 10 days later. The inner ear temporal bone was 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 expression was induced only in vestibular hair cells (Figure 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 generated promoters include: 1) Sequence ID 2 fused with Sequence ID 3; 2) Sequence ID 2 fused with Sequence ID 4; 3) Sequence ID 2 fused with a fusion of Sequence ID 3 and Sequence ID 4 (e.g., Sequence ID 5, 6, or 7); 4) Sequence ID 9 fused with Sequence ID 8 (for example, Sequence ID 10, 11, or 12). 5) A fusion of sequence number 8 and sequence number 9, fused with sequence number 1 (e.g., sequence number 10, 11, or 12); 6) A fusion of sequence number 8 and sequence number 9, fused with sequence number 3 (e.g., sequence number 10, 11, or 12); 7) A fusion of sequence number 8 and sequence number 9, fused with sequence number 4 (for example, sequence number 10, 11, or 12); 8) A fusion of SEQ ID NO: 3 and SEQ ID NO: 4 (e.g., SEQ ID NO: 5, 6, or 7) with a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12); 9) A fusion of sequence number 3 and sequence number 4 (e.g., sequence numbers 5, 6, or 7); 11) Sequence ID 1; and 12) Sequence ID 2.

[0128] The promoter construct is packaged into an AAV serotype (e.g., AAV1, AAV2, AAV6, AAV9, Anc80, or Anc80L65) that can be transduced into hair cells.

[0129] A viral promoter construct is used to infect organ-type cochlear explants. After incubation with the virus for 48 hours, the grafts 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 with a Myo15 promoter to a subject with sensorineural hearing loss. A person skilled in the art can treat a patient, for example, a human patient with sensorineural hearing loss, thereby improving or restoring their hearing, according to the methods disclosed herein. For this purpose, a person skilled in the art may operably link a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to 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) and / or SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., one of SEQ ID NOs: 8-12) to a transgene encoding a therapeutic protein. As described above, a composition containing an AAV vector (e.g., AAV1, AAV2, AAV6, AAV9, Anc80, Anc80L65, DJ / 9, 7m8, or PHP.B) having a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) to sequence numbers 8 and 9), and optionally having a polynucleotide containing a linker between the first and second regions, can be administered to a human patient. For example, the polynucleotide operably linked to the transgene encoding the therapeutic protein may be sequence number 13. The composition containing the AAV vector may be administered to the 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, those skilled in the art can monitor the expression of the therapeutic protein encoded by the transgene and the patient's improvement in response to the treatment in various ways. For example, a physician can monitor the patient's hearing after administration of the composition by performing standard tests such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions. If improvement in the patient's hearing is found in one or more tests after administration of the composition compared to the audiometry results before administration of the composition, it indicates that the patient is responding favorably to the 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. 30 microliters of AAV1-CMV-GFP or AAV1-Myo15-GFP were injected into the cochlea at 15 μl / min through a round window membrane. Four weeks after AAV injection, the animals were euthanized, the cochlea were collected and processed as surface preparations, and transgene expression was examined without antibody enhancement. AAV1 was highly infective. Under the ubiquitous CMV promoter, GFP was expressed in hair cells, supporting cells, and fibrous cells of the lateral wall of rhesus monkey cochlea (Figure 3A). In contrast, the 1.6 kb Myo15 promoter restricted GFP transgene expression to hair cells in cynomolgus monkey cochlea (Figures 3B-3C).

[0133] Example 5. The Myo15 promoter enhances the biological efficacy of AAV mouse Tmc1 in Tmc1 knockout mice compared to the ubiquitous promoter. Tmc1 knockout (KO) mice were anesthetized with isoflurane, shaved, and povidone-iodine was applied to the skin. An incision was made below the left ear, over the cheek muscle, and behind the ear. The skin was separated, the muscle was cut, and the area was washed to expose the posterior semicircular canal. A small hole was made in the semicircular canal using a drill bit, and the bone was held dry using a thin cotton swab. A polyamide / polyethylene tube was inserted into the hole and sealed with bone glue. Using a micropump 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. After delivering 1 μl, 5 minutes were allowed to pass to allow the liquid to reach the apex of the cochlea and to prevent backflow and leakage. The tube was bent and cut near the bone. The muscle was returned to its original position, and the skin was reattached. Mice were given 0.01 cc of meloxicam before recovery under a heating lamp. Animals were checked for signs of pain or infection for 5 days postoperatively. Animals were anesthetized with ketamine and xylazine from 21 to 28 days postoperatively, and auditory brainstem response (ABR) was measured. As shown in Figure 4, homozygous Tmc1 KO mice injected with AAV-Myo15-mouse TMC1 showed significantly improved recovery of the ABR threshold compared to AAV-CMV-mouse TMC1.

[0134] Other Embodiments Various modifications and variations of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in relation to specific embodiments, it should be understood that the claimed invention should not be limited beyond such specific embodiments. In fact, various modifications of the form described for carrying out the invention, which will be apparent to those skilled in the art, are intended to be within the scope of the invention. Other embodiments are found in the claims. (Note) The technical concepts that can be understood from the above embodiments and modified examples are described below. [Item 1] A polynucleotide comprising a first region having at least 85% sequence identity with respect to SEQ ID NO: 1 or its functional portion or derivative, including the sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4, and a second region having at least 85% sequence identity with respect to [Item 2] The polynucleotide according to item 1, wherein the first region includes or consists of the sequence of sequence number 1. [Item 3] The polynucleotide according to item 1, wherein the functional portion of sequence number 1 includes the sequence of sequence number 3. [Item 4] The polynucleotide described in item 1, wherein the functional portion of sequence number 1 includes the sequence of sequence number 4. [Item 5] The polynucleotide according to item 1, wherein the functional portion of sequence number 1 includes the sequence of sequence number 3 and the sequence of sequence number 4. [Item 6] The polynucleotide according to item 5, wherein the functional portion of sequence number 1 includes the sequence of sequence number 5. [Item 7] The polynucleotide according to item 5, wherein the functional portion of sequence number 1 includes the sequence of sequence number 6. [Item 8] The polynucleotide according to item 5, wherein the functional portion of sequence number 1 includes the sequence of sequence number 7. [Item 9] The polynucleotide according to any one of items 1 to 8, wherein the second region contains or consists of the sequence of sequence number 2. [Item 10] The polynucleotide according to any one of items 1 to 8, wherein the functional portion of the sequence number 2 includes the sequence of sequence number 8. [Item 11] The polynucleotide according to any one of items 1 to 8, wherein the functional portion of the sequence number 2 includes the sequence of sequence number 9. [Item 12] The polynucleotide according to any one of items 1 to 8, wherein the functional portion of SEQ ID NO: 2 includes the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9. [Item 13] The polynucleotide described in item 12, wherein the functional portion of sequence number 2 includes the sequence of sequence number 10. [Item 14] The polynucleotide described in item 12, wherein the functional portion of sequence number 2 includes the sequence of sequence number 11. [Item 15] The polynucleotide according to item 12, wherein the functional portion of sequence number 2 includes the sequence of sequence number 12. [Item 16] The polynucleotide according to item 1, wherein the polynucleotide contains or consists of the sequence of sequence number 13. [Item 17] A polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 2 or its functional portion or derivative, 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 its functional portion or derivative, including the sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4 operably linked thereto, and optionally having a linker between the first and second regions containing 1 to 100 nucleotides. [Item 18] The polynucleotide according to item 17, wherein the first region includes or consists of the sequence of sequence number 2. [Item 19] The polynucleotide according to item 17, wherein the functional portion of sequence number 2 includes the sequence of sequence number 8. [Item 20] The polynucleotide according to item 17, wherein the functional portion of sequence number 2 includes the sequence of sequence number 9. [Item 21] The polynucleotide according to item 17, wherein the functional portion of SEQ ID NO: 2 includes the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9. [Item 22] The polynucleotide according to item 21, wherein the functional portion of sequence number 2 includes the sequence of sequence number 10. [Item 23] The polynucleotide according to item 21, wherein the functional portion of sequence number 2 includes the sequence of sequence number 11. [Item 24] The polynucleotide according to item 21, wherein the functional portion of sequence number 2 includes the sequence of sequence number 12. [Item 25] The polynucleotide according to any one of items 17 to 24, wherein the second region contains or consists of the sequence of sequence number 1. [Item 26] The polynucleotide according to any one of items 17 to 24, wherein the functional portion of SEQ ID NO: 1 includes the sequence of SEQ ID NO: 3. [Item 27] The polynucleotide according to any one of items 17 to 24, wherein the functional portion of SEQ ID NO: 1 includes the sequence of SEQ ID NO: 4. [Item 28] The polynucleotide according to any one of items 17 to 24, wherein the functional portion of SEQ ID NO: 1 includes the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4. [Item 29] The polynucleotide according to item 28, wherein the functional portion of sequence number 1 includes the sequence of sequence number 5. [Item 30] The polynucleotide according to item 28, wherein the functional portion of sequence number 1 includes the sequence of sequence number 6. [Item 31] The polynucleotide according to item 28, wherein the functional portion of SEQ ID NO: 1 includes the sequence of SEQ ID NO: 7. [Item 32] The polynucleotide according to item 17, wherein the polynucleotide contains or consists of the sequence of sequence number 14. [Item 33] A polynucleotide comprising a region having at least 85% sequence identity with respect to sequence number 1 or its functional portion or derivative, including the sequence of sequence number 3 and / or sequence number 4. [Item 34] The polynucleotide according to item 33, wherein the region includes or consists of the sequence of sequence number 1. [Item 35] The polynucleotide according to item 33, wherein the functional portion of sequence number 1 includes the sequence of sequence number 3. [Item 36] The polynucleotide according to item 33, wherein the functional portion of sequence number 1 includes the sequence of sequence number 4. [Item 37] The polynucleotide according to item 33, wherein the functional portion of SEQ ID NO: 1 includes the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4. [Item 38] The polynucleotide according to item 37, wherein the functional portion of sequence number 1 includes the sequence of sequence number 5. [Item 39] The polynucleotide according to item 37, wherein the functional portion of SEQ ID NO: 1 includes the sequence of SEQ ID NO: 6. [Item 40] The polynucleotide according to item 37, wherein the functional portion of SEQ ID NO: 1 includes the sequence of SEQ ID NO: 7. [Item 41] A polynucleotide comprising a region having at least 85% sequence identity with respect to sequence number 2 or its functional portion or derivative, including the sequence of sequence number 8 and / or sequence number 9. [Item 42] The polynucleotide according to item 41, wherein the region includes or consists of the sequence of sequence number 2. [Item 43] The polynucleotide according to item 41, wherein the functional portion of sequence number 2 includes the sequence of sequence number 8. [Item 44] The polynucleotide according to item 41, wherein the functional portion of sequence number 2 includes the sequence of sequence number 9. [Item 45] The polynucleotide according to item 41, wherein the functional portion of SEQ ID NO: 2 includes the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9. [Item 46] The polynucleotide according to item 45, wherein the functional portion of sequence number 2 includes the sequence of sequence number 10. [Item 47] The polynucleotide according to item 45, wherein the functional portion of Sequence ID No. 2 includes the sequence of Sequence ID No. 11. [Item 48] The polynucleotide according to item 45, wherein the functional portion of sequence number 2 includes the sequence of sequence number 12. [Item 49] The polynucleotide described in any one of items 1 to 48, wherein the polynucleotide is operably linked to the transgene and, when introduced into hair cells, induces the expression of the transgene. [Item 50] A nucleic acid vector comprising a polynucleotide as described in any one of items 1 through 49. [Item 51] The nucleic acid vector according to item 50, wherein the polynucleotide is operably linked to the introduced gene. [Item 52] The nucleic acid vector according to item 51, wherein the introduced gene contains a nucleic acid sequence encoding a therapeutic protein. [Item 53] The nucleic acid vector according to item 52, wherein the polynucleotide can direct the hair cell-specific expression of the therapeutic protein derived from the nucleic acid sequence in mammalian hair cells. [Item 54] The nucleic acid vector described in item 53, wherein the hair cells are cochlear hair cells. [Item 55] The nucleic acid vector according to item 54, wherein the cochlear hair cells are inner hair cells and / or outer hair cells. [Item 56] The nucleic acid vector described in item 53, wherein the hair cells are vestibular hair cells. [Item 57] The aforementioned therapeutic proteins are 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, ESPN A nucleic acid vector 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, as described in any one of items 52 to 56. [Item 58] The nucleic acid vector according to any one of items 50 to 57, wherein the nucleic acid vector is a plasmid, cosmid, artificial chromosome, or viral vector. [Item 59] The nucleic acid vector described in 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] The nucleic acid vector described in item 59, wherein the viral vector is an AAV vector. [Item 61] The nucleic acid vector described in 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] A composition comprising a nucleic acid vector as described in any one of items 50 to 61. [Item 63] The composition according to item 62, further comprising pharmaceutically acceptable excipients. [Item 64] A method for increasing the expression of a therapeutic protein in mammalian hair cells, comprising contacting the mammalian hair cells with a nucleic acid vector according to any one of items 50 to 61 or a composition according to item 62 or 63. [Item 65] The method according to item 64, wherein the expression of the therapeutic protein is specifically increased in hair cells. [Item 66] The method according to item 64 or 65, wherein the mammalian hair cells are human hair cells. [Item 67] The method according to any one of items 64 to 66, wherein the mammalian hair cells are cochlear hair cells. [Item 68] The method according to item 67, wherein the cochlear hair cells are inner hair cells. [Item 69] The method according to item 67, wherein the cochlear hair cells are outer hair cells. [Item 70] The method according to any one of items 64 to 66, wherein the mammalian hair cells are vestibular hair cells. [Item 71] The method according to any one of items 64 to 70, wherein the expression of the therapeutic protein is not substantially increased in inner ear cells other than hair cells. [Item 72] A method for treating a subject who has hearing loss or is at risk of developing hearing loss, comprising administering to the subject an effective amount of a nucleic acid vector described in any one of items 50 to 61 or a composition described in item 62 or 63. [Item 73] The method described in item 72, wherein the hearing loss is hereditary hearing loss. [Item 74] The method according to item 73, wherein the hereditary hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss. [Item 75] The method described in item 72, wherein the hearing loss is acquired hearing loss. [Item 76] The method according to item 75, wherein the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease or infection-related hearing loss, traumatic head hearing loss, or ototoxic drug-induced hearing loss. [Item 77] A method for treating a subject having or at risk of developing vestibular dysfunction, comprising administering to the subject an effective amount of a nucleic acid vector described in any one of items 50 to 61 or a composition described in item 62 or 63. [Item 78] The method according to item 77, wherein the vestibular dysfunction is rotational vertigo, dizziness, or avalia. [Item 79] A method for promoting hair cell regeneration in a subject requiring such regeneration, comprising administering to the subject an effective amount of a nucleic acid vector described in any one of items 50 to 61 or a composition described in item 62 or 63. [Item 80] The method according to item 79, wherein the hair cells are cochlear hair cells. [Item 81] The method according to item 79, wherein the hair cells are vestibular hair cells. [Item 82] A method for preventing or mitigating ototoxic drug-induced hair cell damage or cell death, comprising administering an effective amount of a nucleic acid vector described in any one of items 50 to 61 or a composition described in item 62 or 63 to the subject. [Item 83] The method according to item 76 or 82, wherein the ototoxic agent is selected from the group consisting of aminoglycosides, antitumor agents, ethacric acid, furosemide, salicylates, and quinine. [Item 84] A method for treating a subject having tinnitus, comprising administering an effective amount of a nucleic acid vector described in any one of items 50 to 61 or a composition described in item 62 or 63 to the subject. [Item 85] A method for preventing or mitigating damage or cell death of hair cells in a subject requiring such treatment, comprising administering to the subject an effective amount of a nucleic acid vector described in any one of items 50 to 61 or a composition described in item 62 or 63. [Item 86] A method for increasing the viability of hair cells in a subject requiring such viability, comprising administering to the subject an effective amount of a nucleic acid vector described in any one of items 50 to 61 or a composition described in item 62 or 63. [Item 87] The method according to any one of items 72-76, 79, 80, and 82-86, further comprising evaluating the hearing of the subject before administering the nucleic acid vector or composition. [Item 88] The method according to any one of items 72-76, 79, 80, and 82-87, further comprising evaluating the hearing of the subject after administering the nucleic acid vector or composition. [Item 89] The method according to any one of items 77-79 and 81-88, further comprising evaluating the vestibular function of the subject before administering the nucleic acid vector or composition. [Item 90] The method according to any one of items 77-79, 81-89, further comprising evaluating the vestibular function of the subject before administering the nucleic acid vector or composition. [Item 91] The method according to any one of items 72 to 90, comprising administering the nucleic acid vector or composition topically. [Item 92] The method according to 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 the number of hair cells. [Item 93] The method described in any one of items 72 to 92, wherein the subject is a human. [Item 94] A kit comprising a nucleic acid vector as described in any one of items 50-61 or a composition as described in item 62 or 63.

Claims

[Claim 1] The invention described herein.

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