SLC26A4 regulatory element and application thereof

Abstract

The present disclosure provides SL26A4 enhancers and SLC26A4 promoters, as well as vectors containing the same, that can increase gene expression in cells expressing SLC26A4, such as interdental cells, root cells, helicoid cells, and vestibular supporting cells. The SLC26A4 enhancers and SLC26A4 promoters described herein can be operably linked to polynucleotides encoding expression products, such as transgenes, and are used to treat subjects suffering from or at risk of developing hearing loss or vestibular dysfunction.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on July 31, 2023, named 51124-098WO3_Sequence_Listing_7_31_23, and is 33,576 bytes in size. Background Art

[0003] Hearing loss is the most common human sensory deficit, affecting nearly 15% of school-age children and one-third of people reaching age 65. In the United States, congenital hearing loss occurs in approximately one in every 500 births each year. Approximately 80% of congenital cases are due to mutations in genes essential for hearing. One of these essential hearing genes is solute carrier family 26, member 4 (SLC26A4), which encodes Pendrin, a 780 amino acid member of the solute carrier (SLC) family 26. In patients with SLC26A4 mutations, Pendrin function may be lost or disrupted, and these patients may develop pre- or post-linguistic nonsyndromic hearing loss, or may be born with hearing loss that progresses to profound deafness over time. Pan is expressed in specialized epithelial cells of the inner ear (cochlea, vestibular labyrinth and endolymphatic sac and non-sensory epithelial cells of the saccule, utricle and ampulla), thyroid (thyrocytes), kidney (B-type intercalated cells of the renal collecting duct), airways, mammary glands, salivary ducts and apical membranes of the liver. Within the inner ear, Pan regulates pH and fluid absorption by exchanging chloride and bicarbonate anions between the epithelium and the endolymphatic fluid compartment. There is currently no curative therapy for this population. Therefore, a therapeutic agent is needed to restore and / or rescue the progression of hearing loss in patients with these mutations. Summary of the invention

[0004] The present invention provides compositions and methods for promoting expression of a gene of interest (such as a gene endogenously expressed in a cell expressing SLC26A4) in a specific cell type. The present invention features a SLC26A4 promoter and enhancer that can be operably linked to a polynucleotide that can be transcribed to produce an expression product (e.g., a protein or an RNA molecule such as an inhibitory RNA molecule) to induce expression of the expression product in a cell expressing SLC26A4. The SLC26A4 promoter and enhancer can be incorporated into a nucleic acid vector and administered to a subject (such as a human subject) to treat or prevent hearing loss (e.g., sensorineural hearing loss, such as panin-associated hearing loss), Meniere's disease (e.g., hearing loss associated with Meniere's disease, tinnitus, or vestibular dysfunction), and / or vestibular dysfunction (e.g., panin-associated vestibular dysfunction or vestibular dysfunction associated with vestibular hair cell damage or loss).

[0005] In a first aspect, the invention provides a polynucleotide comprising an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3, wherein the enhancer is operably linked to a promoter, wherein the enhancer in the polynucleotide is less than 3 kilobases (kb) from the promoter. In some embodiments, the distance between the enhancer and the promoter in the polynucleotide is less than 2 kb. In some embodiments, the distance between the enhancer and the promoter in the polynucleotide is less than 1 kb. In some embodiments, the distance between the enhancer and the promoter in the polynucleotide is less than 0.5 kb. In some embodiments, the distance between the enhancer and the promoter in the polynucleotide is less than 100 bases.

[0006] In another aspect, the present invention provides a nucleic acid vector comprising the polynucleotide of the first aspect.

[0007] In another aspect, the present invention provides a nucleic acid vector comprising a polynucleotide comprising an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0008] In some embodiments of any of the aforementioned aspects, the enhancer is operably connected to the promoter. In some embodiments, the promoter is a minimal promoter, a core promoter or a constitutive promoter. In some embodiments, the promoter is a CAG promoter, a CBA promoter, a smCBA promoter, a CASI promoter, a dihydrofolate reductase (DHFR) promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, a HSV promoter or a SV40 promoter. In some embodiments, the promoter is a minimal β-globin promoter, a CMVmini promoter, a minCMV promoter, a CMV-TATA+INR promoter, a min CMV-T6 promoter, a minimal HSV ICP0 promoter, a truncated HSV ICP0 promoter or a SV40 minimal promoter. In some embodiments, the promoter is a minimal promoter. In some embodiments, the promoter is a mammalian SLC26A4 promoter. In some embodiments, the SLC26A4 promoter is a human or mouse SLC26A4 promoter. In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 1. In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NO: 12-16. In some embodiments, the SLC26A4 enhancer has a sequence of any one of SEQ ID NOs: 12-16. In some embodiments, the SLC26A4 promoter has a sequence of SEQ ID NO: 12. In some embodiments, the SLC26A4 promoter has a sequence of SEQ ID NO: 13. In some embodiments, the SLC26A4 promoter has a sequence of SEQ ID NO: 14. In some embodiments, the SLC26A4 promoter has a sequence of SEQ ID NO: 15. In some embodiments, the SLC26A4 promoter has a sequence of SEQ ID NO: 16.In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 17.

[0009] In another aspect, the present invention provides a nucleic acid vector containing a polynucleotide comprising a SLC26A4 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 1.

[0010] In another aspect, the invention provides a polynucleotide comprising a SLC26A4 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1, the SLC26A4 promoter being operably linked to a polynucleotide that can be transcribed to produce an expression product. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 1.

[0011] In another aspect, the present invention provides a nucleic acid vector comprising a polynucleotide comprising a SLC26A4 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 17.

[0012] In another aspect, the invention provides a polynucleotide comprising a SLC26A4 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17, the SLC26A4 promoter being operably linked to a polynucleotide that can be transcribed to produce an expression product. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 17.

[0013] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter is operably linked to an enhancer. In some embodiments, the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) or has at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0014] In some embodiments of any of the preceding aspects, the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2. In some embodiments of any of the preceding aspects, the enhancer has the sequence of SEQ ID NO: 2.

[0015] In some embodiments of any of the preceding aspects, the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 3. In some embodiments of any of the preceding aspects, the enhancer has the sequence of SEQ ID NO: 3.

[0016] In some embodiments of any of the aforementioned aspects, the enhancer is located 5' of a promoter in the polynucleotide. In some embodiments of any of the aforementioned aspects, the enhancer is located 3' of a promoter in the polynucleotide. In some embodiments of any of the aforementioned aspects, the enhancer is fused directly to the promoter. In some embodiments of any of the aforementioned aspects, the enhancer is linked to the promoter via a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleic acids).

[0017] In some embodiments of any of the foregoing aspects, the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product. In some embodiments of any of the foregoing aspects, the expression product is a heterologous expression product. In some embodiments of any of the foregoing aspects, the expression product is an expression product that is endogenously expressed in cells expressing SLC26A4. In some embodiments, the expression product is an expression product that is endogenously expressed in inner ear cells expressing SLC26A4. In some embodiments, the expression product is an expression product that is endogenously expressed in interdental cells, spiral protuberance cells, cochlear root cells, and / or vestibular supporting cells (e.g., expressed in at least one of these cell types). In some embodiments of any of the foregoing aspects, the expression product is a pan protein (e.g., a mammalian pan protein). In some embodiments, the pan protein (e.g., a mammalian pan protein) is a wild-type isoform that is endogenously expressed in the inner ear of a mammal. In some embodiments, the pan protein has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the pan protein has the sequence of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments of any of the foregoing aspects, the expression product is Atoh1 (e.g., mammalian Atoh1). In some embodiments, Atoh1 (e.g., mammalian Atoh1) is a wild-type isoform endogenously expressed in the inner ear of a mammal. In some embodiments, Atoh1 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) with SEQ ID NO: 8 or SEQ ID NO: 10. In some embodiments of any of the foregoing aspects, the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease, such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN) or a guide RNA (gRNA)) or a microRNA.

[0018] In some embodiments of any of the foregoing aspects, the polynucleotide contains an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2 and an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 3. In some embodiments, the polynucleotide contains an enhancer having the sequence of SEQ ID NO: 2 and an enhancer having the sequence of SEQ ID NO: 3. In some embodiments of any of the aforementioned aspects, the polynucleotide contains, in 5' to 3' order, an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a promoter having a sequence of SEQ ID NO: 17. In some more specific embodiments of any of the aforementioned aspects, the polynucleotide contains a sequence of SEQ ID NO: 18.

[0019] In some embodiments of any of the preceding aspects, the polynucleotide contains two or more copies of an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or two or more copies of an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3 (e.g., two or more copies of one or both enhancers). In some embodiments, the polynucleotide contains two or more copies of an enhancer having a sequence of SEQ ID NO: 2 and / or SEQ ID NO: 3 (eg, two or more copies of one or both enhancers).

[0020] In some embodiments of any of the aforementioned aspects, the nucleic acid vector is a viral vector, a plasmid, a cosmid or an artificial chromosome. In some embodiments, the nucleic acid vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV), an adenovirus or a lentivirus. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector has AAV1, AAV2, AAV2quad (YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB or PHP.S capsid. In some embodiments, the AAV vector has an AAV1 capsid. In some embodiments, the AAV vector has an AAV2 capsid. In some embodiments, the AAV vector has an AAV2quad (YF) capsid. In some embodiments, the AAV vector has an AAV6 capsid. In some embodiments, the AAV vector has an AAV8 capsid. In some embodiments, the AAV vector has an AAV9 capsid. In some embodiments, the AAV vector has an Anc80 capsid. In some embodiments, the AAV vector has an Anc80L65 capsid. In some embodiments, the AAV vector has a DJ capsid. In some embodiments, the AAV vector has a DJ / 9 capsid. In some embodiments, the AAV vector has a 7m8 capsid. In some embodiments, the AAV vector has a PHP.B capsid. In some embodiments, the AAV vector has a PHP.S capsid. In some embodiments, the AAV vector has a PHP.eB capsid. In some embodiments, the AAV vector has an AAV3 capsid. In some embodiments, the AAV vector has an AAV4 capsid. In some embodiments, the AAV vector has an AAV5 capsid. In some embodiments, the AAV vector has an AAV7 capsid.

[0021] In another aspect, the present invention provides a composition comprising the nucleic acid vector of any one of the aforementioned aspects and embodiments. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0022] In another aspect, the invention provides a cell comprising a polynucleotide or nucleic acid vector of any of the foregoing aspects and embodiments. In some embodiments, the cell is a cell expressing SLC26A4. In some embodiments, the cell is an inner ear cell expressing SLC26A4. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell is an interdental cell, a spiral eminence cell, a cochlear root cell, or a vestibular supporting cell.

[0023] On the other hand, the present invention provides a method of expressing an expression product in a cell, the method comprising the step of contacting the cell with a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the cell is an inner ear cell. In some embodiments, the cell is a cell expressing SLC26A4. In some embodiments, the cell is an inner ear cell expressing SLC26A4. In some embodiments, the inner ear cell expressing SLC26A4 is an interdental cell, a spiral protuberance cell, a cochlear root cell, or a vestibular supporting cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the contact is performed in a subject (e.g., in vivo).

[0024] In another aspect, the invention provides a method of treating a subject suffering from or at risk of developing hearing loss (e.g., sensorineural hearing loss), the method comprising administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the hearing loss is pan protein-related hearing loss. In some embodiments, the expression product is pan protein. In some embodiments, the hearing loss associated with pan protein is hearing loss associated with Pendred syndrome or DFNB4.

[0025] In another aspect, the present invention provides a method of treating hearing loss associated with Meniere's disease in a subject in need thereof, the method comprising the step of administering to the inner ear of the subject an effective amount of a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the expression product is pan protein.

[0026] In another aspect, the present invention provides a method of treating tinnitus associated with Meniere's disease in a subject in need thereof, the method comprising the step of administering to the inner ear of the subject an effective amount of a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the expression product is pan protein.

[0027] In another aspect, the present invention provides a method for treating vestibular dysfunction associated with Meniere's disease in a subject in need thereof, the method comprising administering to the inner ear of the subject an effective amount of a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the expression product is pan protein or Atoh1. In some embodiments, the vestibular dysfunction is vertigo.

[0028] On the other hand, the present invention provides a method for treating a subject suffering from or at risk of developing vestibular dysfunction, the method comprising the step of administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the vestibular dysfunction is a vestibular dysfunction associated with Pan protein. In some embodiments, the expression product is Pan protein. In some embodiments, the vestibular dysfunction associated with Pan protein is a vestibular dysfunction associated with Pandred syndrome or DFNB4. In some embodiments, the expression product is Pan protein or Atoh1. In some embodiments, the vestibular dysfunction is vertigo, dizziness, imbalance (e.g., loss of balance or balance disorder), oscillopsia, or bilateral vestibular lesions. In some embodiments, vestibular dysfunction is associated with damage or loss of vestibular hair cells. In some embodiments, the damage or loss of vestibular hair cells is related to age (i.e., the vestibular dysfunction is age-related vestibular dysfunction), exposure to ototoxic (e.g., vestibular toxic) drugs (i.e., the vestibular dysfunction is ototoxic drug-induced vestibular dysfunction), disease or infection (i.e., the vestibular dysfunction is disease or infection-related vestibular dysfunction), or head trauma (i.e., the vestibular dysfunction is head trauma-related vestibular dysfunction). In some embodiments, the ototoxic drug is an aminoglycoside (aminoglycoside antibiotics such as gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin, or netilmicin), puromycin, an antineoplastic drug (e.g., a platinum-containing chemotherapeutic agent such as cisplatin, carboplatin, or oxaliplatin, or another chemotherapeutic agent such as nitrogen mustard or vincristine), a loop diuretic (e.g., ethacrynic acid or furosemide), a salicylate, or quinine.

[0029] On the other hand, the present invention provides a method of inducing or increasing vestibular hair cell regeneration (i.e., inducing or increasing the differentiation of vestibular supporting cells into vestibular hair cells), the method comprising the step of contacting the vestibular supporting cells with a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the expression product is Atoh1. In some embodiments, the contact is performed in vivo (e.g., in a subject). In some embodiments, the subject suffers from vestibular dysfunction or is at risk of developing vestibular dysfunction.

[0030] On the other hand, the present invention provides a method for inducing or increasing the maturation of vestibular hair cells (e.g., regenerated vestibular hair cells), the method comprising the step of contacting the vestibular supporting cells with a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the expression product is Atoh1. In some embodiments, the contact is performed in vivo (e.g., in a subject). In some embodiments, the subject suffers from vestibular dysfunction or is at risk of developing vestibular dysfunction.

[0031] In another aspect, the present invention provides a method for improving the function of a cell expressing SLC26A4, the method comprising the step of contacting the cell expressing SLC26A4 with a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the contact is performed in vivo (e.g., in a subject). In some embodiments, the subject suffers from or is at risk of developing hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction.

[0032] In some embodiments of any of the foregoing aspects, the method further comprises evaluating the subject's hearing prior to administering the nucleic acid vector or composition.

[0033] In some embodiments of any of the foregoing aspects, the method further comprises evaluating the hearing of the subject after administering the nucleic acid vector or composition.

[0034] In some embodiments of any of the foregoing aspects, the method further comprises evaluating the subject's vestibular function prior to administering the nucleic acid vector or composition.

[0035] In some embodiments of any of the foregoing aspects, the method further comprises evaluating the subject's vestibular function after administering the nucleic acid vector or composition.

[0036] In some embodiments of any of the foregoing aspects, the nucleic acid vector or composition is topically applied. In some embodiments, the nucleic acid vector or composition is applied to the inner ear. In some embodiments, the nucleic acid vector or composition is applied to the middle ear. In some embodiments, the nucleic acid vector or composition is applied via the tympanic cavity or intratympanic cavity. In some embodiments, the nucleic acid vector or composition is applied to the perilymph. In some embodiments, the nucleic acid vector or composition is applied to the endolymph. In some embodiments, the nucleic acid vector or composition is applied to the oval window or is applied through the oval window. In some embodiments, the nucleic acid vector or composition is applied to the round window or is applied through the round window. In some embodiments, the nucleic acid vector or composition is applied to the semicircular canals.

[0037] In some embodiments of any of the foregoing aspects, the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, delay the development of hearing loss, slow the progression of hearing loss, improve hearing, increase or induce expression of an expression product in a cell expressing SLC26A4, reduce tinnitus, improve vestibular function, reduce vertigo, improve balance, increase the number of vestibular hair cells, inhibit or slow the progression of vestibular dysfunction, reduce ear fullness, increase vestibular hair cell regeneration, induce or increase differentiation of vestibular supporting cells into vestibular hair cells, increase or induce maturation of vestibular hair cells (e.g., maturation of regenerated vestibular hair cells), or improve the function of cells expressing SLC26A4 (e.g., inner ear cells expressing SLC26A4).

[0038] In some embodiments of any of the foregoing aspects, the subject is a human subject.

[0039] In another aspect, the present invention provides a kit comprising the polynucleotide, nucleic acid vector or composition of any one of the preceding aspects and embodiments.

[0040] definition

[0041] As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0042] As used herein, "administering" refers to providing or administering a therapeutic agent (eg, a nucleic acid vector containing a SLC26A4 enhancer and / or a SLC26A4 promoter) to a subject by any effective route. Exemplary administration routes are described below.

[0043] As used herein, the phrase "administering to the inner ear" refers to providing or administering a therapeutic agent described herein to a subject by any route that allows transduction of inner ear cells. Exemplary routes of administration to the inner ear include administration into the perilymph or endolymph, such as administration to or through the oval window, round window, or semicircular canal (e.g., the transverse canal), or by transtympanic or intratympanic injection, for example, administration to inner ear cells expressing SLC26A4.

[0044] As used herein, the term "cell type" refers to a group of cells that share a phenotype that is statistically separable based on gene expression data. For example, cells of common cell types may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of common cell types may include those separated from common tissues (e.g., epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those separated from other structures and / or regions of common organs, tissue systems, blood vessels, or organisms.

[0045] As used herein, the terms "conservative mutation", "conservative substitution" and "conservative amino acid substitution" refer to one or more amino acids being substituted into one or more different amino acids that exhibit similar physicochemical properties (such as polarity, electrostatic charge and steric bulk). These properties for each of the twenty naturally occurring amino acids are summarized in Table 1 below.

[0046] Table 1. Representative physicochemical properties of naturally occurring amino acids

[0047]

[0048] It will be appreciated from this table that the conservative amino acid family includes (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Thus, a conservative mutation or substitution is one in which an amino acid is substituted for a member of the same amino acid family (e.g., Ser for Thr or Lys for Arg).

[0049] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein refer to an amount sufficient to achieve a beneficial or desired outcome (including a clinical outcome) when administered to a subject, including a mammal (e.g., a human), and thus, an "effective amount" or its synonyms depend on the context in which it is applied. For example, in the case of treating hearing loss (e.g., hearing loss associated with DFNB4 or Pendrede syndrome), it is an amount of the composition, vector construct, or viral vector sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein that would correspond to such an amount will vary depending on various factors, such as a given dose, pharmaceutical formulation, route of administration, type of disease or condition, the subject being treated (e.g., age, sex, weight), or the identity of the host, etc., but can still be routinely determined by one skilled in the art. Likewise, as used herein, a "therapeutically effective amount" of a composition, vector construct, or viral vector disclosed herein is an amount that produces a beneficial or desired outcome in a subject compared to a control. As defined herein, a therapeutically effective amount of a composition, vector construct or viral vector of the present disclosure can be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimens may be adjusted to provide the optimal therapeutic response.

[0050] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally present in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell, e.g., a human cochlear supporting cell).

[0051] As used herein, the term "expression" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein. The term "expression product" refers to a protein or RNA molecule produced by any of these events.

[0052] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, a nucleic acid, or a cofactor) that does not naturally occur in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell, e.g., a human cochlear supporting cell). Exogenous materials include those materials that are provided to an organism by an external source or to culture material extracted from the organism.

[0053] As used herein, the term "heterologous" refers to a combination of elements that do not occur naturally. For example, a heterologous transgene refers to a transgene that is not naturally expressed by the promoter to which it is operably linked.

[0054] As used herein, the terms "increase" and "decrease" refer to the regulation of a function, expression or activity that results in a greater or lesser amount, respectively, relative to a reference. For example, after the composition is administered in the methods described herein, the amount of a marker of a metric (e.g., transgenic expression level or auditory brainstem response) as described herein may be increased or decreased in a subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more relative to the amount of the marker before administration. Typically, after administration, the metric is measured at the time when the effect has been achieved, for example, at least one week, one month, 3 months or 6 months after the start of the treatment regimen.

[0055] As used herein, "topical" or "local administration" refers to administration at a specific site of the body intended to have a local rather than systemic effect. Examples of local administration are epidermal, inhalation, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional administration, lymph node administration, intratumoral administration, direct administration to the inner ear or middle ear (e.g., injection through the oval window or round window membrane or transtympanic or intratympanic injection), and administration to the mucous membranes of a subject, where the administration is intended to have a local rather than systemic effect.

[0056] As used herein, the term "operably connected" refers to a first molecule being linked to a second molecule, wherein the arrangement of the molecule causes the first molecule to affect the function of the second molecule. Two molecules may or may not be a part of a single adjacent molecule and may or may not be adjacent. For example, if a promoter regulates the transcription of a transcribable polynucleotide molecule of interest in a cell, the promoter is operably connected to the transcribable polynucleotide molecule. In addition, if two parts of a transcriptional regulatory element are interconnected so that the transcriptional activation function of a part is not adversely affected by the presence of another part, the two parts are operably connected to each other. Two transcriptional regulatory elements may be operably connected to each other by means of a joint polynucleotide (e.g., an intervening non-coding polynucleotide) or may be operably connected to each other in the absence of an intervening nucleotide.

[0057] As used herein, the term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule to which another DNA segment can be connected. A plasmid is a vector, a nucleic acid molecule capable of transporting another nucleic acid connected thereto. Some plasmids can replicate autonomously in the host cell into which they are introduced (e.g., bacterial plasmids and additional mammalian plasmids with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thus replicating with the host genome. Some plasmids can direct the expression of the genes to which they are operably connected.

[0058] As used herein, the term "polynucleotide" refers to a polymer of nucleotides. Generally, polynucleotides are composed of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) naturally present in DNA or RNA connected by phosphodiester bonds. The term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether present in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When the application relates to polynucleotides, it should be understood that DNA, RNA are provided, and single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided in each case. As used herein, "polynucleotide sequence" may refer to polynucleotide material itself and / or biochemical characterization of sequence information of specific nucleic acids (i.e., a series of letters used as base abbreviations). Unless otherwise indicated, the polynucleotide sequence given herein is given in 5' to 3' directions.

[0059] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives transcription of a transgene.

[0060] " Percentage of sequence identity (%) " with respect to reference polynucleotide or peptide sequence is defined as after sequence alignment and introducing room when necessary to realize maximum sequence identity percentage, the percentage of nucleic acid or amino acid identical with the nucleic acid in reference polynucleotide or peptide sequence in candidate sequence.Comparison for determining nucleic acid or amino acid sequence identity percentage can be realized in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2 or Megalign software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for realizing maximum alignment on the total length of the compared sequences.For example, sequence comparison computer program BLAST can be used to generate sequence identity percentage value.For example, given nucleic acid or amino acid sequence A to, with or for given nucleic acid or amino acid sequence B sequence identity percentage (can also be alternatively described as given nucleic acid or amino acid sequence A to, with or for given nucleic acid or amino acid sequence B certain sequence identity percentage) is calculated as follows:

[0061] 100×(fraction X / Y)

[0062] wherein X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and wherein Y is the total number of nucleic acids in B. It will be appreciated that when 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 percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0063] As used herein, the terms "Pendrin" and "SLC26A4" refer to the protein encoded by the SLC26A4 gene and the gene encoding that protein, respectively. SLC26A4 is a member of the solute carrier family 26. Mutations in SLC26A4 cause syndromic or non-syndromic hearing loss. The terms "Pendrin" and "SLC26A4" also refer to variants of wild-type Pendrin and the nucleic acids encoding them, such as variant proteins having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity or higher sequence identity) to the amino acid sequence of wild-type Pendrin (e.g., SEQ ID NO:4 or SEQ ID NO:5) or polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity or higher sequence identity) to the nucleic acid sequence of the wild-type SLC26A4 gene (e.g., SEQ ID NO:6 or SEQ ID NO:7) or a codon-optimized sequence thereof, provided that the encoded Pendrin analog retains the therapeutic function of wild-type (WT) Pendrin (e.g., the ability to transport negatively charged ions (such as chloride, iodide, and bicarbonate) across cell membranes).

[0064] As used herein, the term "SLC26A4 enhancer" refers to a polynucleotide that can be operably linked to a promoter (e.g., the SLC26A4 promoter, minimal promoter, core promoter, or constitutive promoter) to regulate gene expression in cells that express SLC26A4. The SLC26A4 enhancer for use in the compositions and methods described herein has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity or higher sequence identity) to SEQ ID NO:2 or SEQ ID NO:3. The SLC26A4 enhancer described herein can be operably linked to a promoter that is operably linked to a polynucleotide encoding an expression product to increase the expression level of the expression product in cells that express SLC26A4 and / or increase the number of cells that express SLC26A4 in which the expression product is expressed.

[0065] As used herein, the term "SLC26A4 promoter" refers to a polynucleotide or variant thereof that is capable of specifically expressing a transgene in cells expressing SLC26A4, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a SLC26A4 promoter described herein (e.g., a SLC26A4 promoter or enhancer-promoter provided in Table 3). The SLC26A4 promoter for use in the compositions and methods described herein can have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1, at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:17, or at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:18. Any of NOs: 12-16 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0066] As used herein, the term "cell expressing SLC26A4" refers to a cell type in vivo known to endogenously express SLC26A4. Cells expressing SLC26A4 include mammary gland cells (adipocytes, vascular cells, luminal epithelial cells, fibroblasts, dendritic cells, macrophages, basal myoepithelial cells, pericytes and smooth muscle cells); esophageal cells (adipocytes, lymphocytes, vascular cells, epithelial cells of the esophageal mucosa (basal, suprabasal, squamous), fibroblasts (mucosal and muscle), mucous cells, myofibroblasts of the esophageal mucosa, neuronal cells of the esophageal muscle layer, Schwann cells, myocytes (smooth muscle), immune cells (dendritic cells, macrophages, T cells, mast cells), pericytes and smooth muscle cells); cardiac cells (adipocytes, lymphocytes, vascular cells, fibroblasts, Schwann cells, myocytes (cardiac, cytoplasmic), immune cells (dendritic cells, macrophages, T cells, mast cells), pericytes and smooth muscle cells); lung cells (lymphocytes, vascular cells, epithelial cells (alveolar type I, type II, basal cells, ciliated cells and club cells), fibroblasts, immune cells (dendritic cells, macrophages, T cells, alveolar macrophages), pericytes and smooth muscle cells); cells of skeletal muscle (lymphocytes, vascular cells, fibroblasts, dendritic cells, macrophages, satellite cells, myocytes (skeletal muscle, cytoplasmic), pericytes and smooth muscle cells); cells of the prostate (lymphocytes, vascular cells, epithelial cells (hillock cells, luminal cells), fibroblasts, dendritic cells, macrophages, myocytes (smooth muscle)); cells of the skin (vascular cells, epithelial cells (basal keratinocytes, mature keratinocytes, suprabasal keratinocytes), fibroblasts, sebaceous gland cells and sweat gland cells); cells of the kidney (cortical and medullary cells (intercalated cells)); and cells of the thyroid (follicular and parafollicular cells) and inner ear cells expressing SLC26A4.

[0067] As used herein, the term "inner ear cells expressing SLC26A4" refers to cells within the inner ear that endogenously express SLC26A4. Cells within the ear that express SLC26A4 are present in both the cochlea and the vestibule. Cochlear cells expressing SLC26A4 include root cells, fusiform cells, inner sulcus cells, outer sulcus cells, spiral protuberance cells, interdental cells, macrophages, Reissner's membrane, Deiters' cells, vascular cells, marginal cells, and intermediate cells. Vestibular cells expressing SLC26A4 include non-sensory epithelial cells. Additional inner ear cells expressing SLC26A4 include the endolymphatic sac and the endolymphatic ducts of the endolymphatic sac mitochondria-rich cells.

[0068] As used herein, the term "Pan-associated hearing loss" refers to a disease or condition characterized by hearing loss associated with a mutation in SLC26A4, such as DFNB4 (characterized by pre- or post-lingual hearing loss that may be accompanied by an enlarged vestibular aqueduct) and Pendred syndrome (characterized by an enlarged thyroid gland (called a goiter), severe to profound hearing loss (usually beginning at birth), and other abnormalities of the inner ear including an enlarged vestibular aqueduct).

[0069] As used herein, the term "Panin-associated vestibular dysfunction" refers to a disease or condition characterized by vestibular dysfunction (eg, vertigo, dizziness, or imbalance or loss of balance) associated with a mutation in SLC26A4, such as DFNB4 and Pendred syndrome.

[0070] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and / or carriers, to be administered to a subject (such as a mammal, e.g., a human) in order to prevent, treat or control a particular disease or condition that affects or may affect the subject.

[0071] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of subjects (e.g., mammals, such as humans) without excessive toxicity, irritation, allergic response, and other problematic complications and are commensurate with a reasonable benefit / risk ratio.

[0072] As used herein, the term "sample" refers to a specimen isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermal tissue), pancreatic juice, chorionic villus samples, and cells).

[0073] As used herein, the terms "subject" and "patient" refer to an animal (e.g., a mammal, such as a human). A subject to be treated according to the methods described herein can be a subject who has been diagnosed with hearing loss (e.g., panin-associated hearing loss), vestibular dysfunction (e.g., panin-associated vestibular dysfunction or vestibular dysfunction associated with vestibular hair cell loss), or Meniere's disease, or a subject at risk of developing these disorders (e.g., due to a mutation in SLC26A4 or exposure to trauma that may cause vestibular hair cell damage or death, such as exposure to ototoxic drugs, head trauma, or aging). Diagnosis can be made by any method or technique known in the art. One skilled in the art will appreciate that a subject to be treated according to the present disclosure may have already undergone standard testing, or may have been identified as a subject at risk without examination due to the presence of one or more risk factors associated with the disease or disorder.

[0074] As used herein, the terms "transcriptional regulatory element" and "regulatory sequence" refer to polynucleotides that at least partially control the transcription of a gene of interest. Transcriptional regulatory elements can include promoters, enhancers, and other polynucleotides (e.g., polyadenylation signals) that control or help control gene transcription. Examples of transcriptional regulatory elements are described in, for example, Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0075] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, impalefection, and the like.

[0076] As used herein, the term "transduction" and "transduce" refer to a method of introducing a vector construct or a portion thereof into a cell. Where the vector construct is contained in a viral vector such as an AAV vector, transduction refers to viral infection of the cell and subsequent transfer and integration of the vector construct or a portion thereof into the cell genome.

[0077] As used herein, "treatment" and "treating" with respect to a disease or disorder refer to methods for obtaining a beneficial or desired result (e.g., a clinical result). Beneficial or desired results may include, but are not limited to, alleviating or ameliorating one or more symptoms or disorders; reducing the extent of a disease or disorder; stabilizing (i.e., not worsening) the state of a disease, disorder, or illness; preventing the spread of a disease or disorder; delaying or slowing the progression of a disease or disorder; improving or alleviating a disease or disorder; and detectable or undetectable relief (partial or complete). "Improving" or "alleviating" a disease or disorder means that the extent and / or undesirable clinical manifestations of a disease, disorder, or illness are reduced and / or the time course of progression is slowed or prolonged compared to the extent or time course in the absence of treatment. "Treatment" may also mean prolonging survival compared to the expected survival if not receiving treatment. Those in need of treatment include those already suffering from a disease or disorder, as well as those susceptible to a disease or disorder or those in which a disease or disorder will be prevented.

[0078] As used herein, the term "vector" refers to a nucleic acid vector, for example, a DNA vector, such as a plasmid, a cosmid or an artificial chromosome, an RNA vector, a virus or any other suitable replicon (e.g., a viral vector). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described in, for example, Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the compositions and methods described herein include polynucleotide sequences, and, for example, additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express transgenics as described herein include vectors that include regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other available vectors for expressing transgenics include polynucleotide sequences that increase the translation rate of the transgenic or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of the genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain 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 nourseothricin.

[0079] As used herein, the term "wild type" refers to the genotype with the highest frequency for a particular gene in a given organism. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is the plasmid map of plasmid P1236.

[0081] Figure 2 This is the plasmid map of plasmid P1240.

[0082] Figures 3A-3C A series of images showing the inner ear tropism of AAV1 containing a green fluorescent protein (GFP) transgene driven by a ubiquitous promoter (CMV). Sections of the cochlear and vestibular organs of the mouse inner ear demonstrate expression of the enhanced green fluorescent protein (EGFP) transgene ( Figure 3AAs determined by EGFP expression, AAV1 tropism in the cochlea includes, but is not limited to, spiral ganglion neurons (SGNs), Reissner's membrane (RM), fiber cells (F), spiral protuberance cells (SP), and root cells (RC) ( Figure 3B AAV1 tropism in the vestibular system includes, but is not limited to, hair cells (HC), supporting cells (SC), mesenchymal cells (MC), and cells containing the roof of the otolith organ ( Figure 3C ).

[0083] Figures 4A-4D is a series of images showing the lack of EGFP transgene expression in inner ear organs when driven by the murine SLC26A4 promoter of SEQ ID NO: 1 without any enhancer sequence. Sections of the cochlear and vestibular organs of the mouse inner ear show no EGFP expression ( Figure 4A and 4C In the mouse inner ear cochlea ( Figure 4A The square in Figure 4B higher resolution in the ) or the vestibular organs ( Figure 4C The rectangle in Figure 4D No EGFP expression was found in the slices with higher resolution (Fig. 3A). Scale bar, 100 μm.

[0084] Figures 5A-5H is a series of images demonstrating that EGFP transgene expression is restricted to specific cell types of the inner ear when driven by the murine E2 enhancer element of SEQ ID NO: 2 directly fused to the murine SLC26A4 promoter of SEQ ID NO: 1. Sections of the cochlear and vestibular organs of the mouse inner ear from two different animals (top and bottom rows) demonstrate EGFP expression. A lower magnification view of a section from animal #1 is shown in Figure 5A Higher magnification view below: The leftmost rectangle is shown in Figure 5B in; the top rectangle is shown in Figure 5C ; and the rightmost rectangle is shown in Figure 5D A smaller magnified view of the section from animal #2 is shown in Figure 5E Higher magnification view below: The upper left rectangle shows the Fig. 5F Middle; upper right rectangle shows Figure 5G ; and the bottom rectangle appears on Figure 5H EGFP expression was restricted to interdental cells (ID) in two animals ( Figure 5B ), spiral protuberance cells (SP) ( Figure 5D , 5F and 5G) and cochlear root cells (RC) ( Figure 5G ). EGFP expression is restricted to supporting cells (SC) in the vestibular otolith organ ( Figure 5H ). Scale bar, 100 μm.

[0085] Figures 6A-6C is a series of images demonstrating that EGFP transgene expression is restricted to specific cell types of the inner ear when driven by the murine E6 enhancer element of SEQ ID NO:3 directly fused to the murine SLC26A4 promoter of SEQ ID NO:1. Sections of the mouse inner ear and vestibular organ demonstrate EGFP expression ( Fig. 6A ) and its higher magnification (upper rectangle, Figure 6B ; The lower rectangle, Figure 6C ). EGFP expression was shown in the spiral protuberance (SP) and the root cells (RC) of the cochlea ( Figure 6B Weak expression of EGFP was observed in the supporting cells (SC) of the vestibular organ ( Figure 6C ). Scale bar, 100 μm.

[0086] Figure 7 This is the plasmid map of plasmid P1669.

[0087] Figure 8 This is the plasmid map of plasmid P1670.

[0088] Figures 9A-9B is a series of images evaluating Pan protein and EGFP expression. Fig. 9A Images of whole mount views of mouse lateral wall explants stained with an antibody specific for pancreatin. The different layers of the lateral wall are indicated in the images. Fig. 9B A series of images of whole mount views of the lateral wall of a mouse transduced with an AAV vector expressing EGFP under the control of the ubiquitous CMV promoter and stained with an antibody specific for Pan. Images showing staining for Pan alone, GFP alone, and a combination of Pan and GFP are shown.

[0089] Figures 10A-10C Figure 2 depicts whole mount images of mouse lateral wall explants transduced with an AAV1 viral vector expressing EGFP under the control of Figure 1 Series of images: murine minimal SLC26A4 promoter and both murine E2 and E6 enhancers ( Fig. 10A ), the mouse core SLC26A4 promoter and both the mouse E2 and E6 enhancers ( Fig. 10B ) or the mouse core SLC26A4 promoter and only the mouse E2 enhancer ( Fig. 10C ). The top row of each figure depicts both Pan and GFP. The second row depicts only Pan staining in this micrograph. The third row depicts only GFP. The fourth row depicts only GFP with adjusted gain for better visualization Fig. 10CGFP in.

[0090] Fig.11 is a series of images at different magnifications of the same field depicting EGFP expression in the cochlea following administration of an AAV1 vector containing an EGFP expression cassette encoding the E2 enhancer (SEQ ID NO:2) fused directly to the 5' end of the E6 enhancer (SEQ ID NO:3) fused directly to the 5' end of the murine SLC26A4 minimal promoter (SEQ ID NO:17) to Panin knockout mice (see plasmid P1669; Figure 7 ). Panel A is the photomicrograph at the lowest magnification. The area indicated by the box marked "B" in Panel A is shown at a higher magnification in Panel B. The boxes marked "C" and "D" in Panel B are shown at higher magnifications in Panels C and D, respectively.

[0091] Fig.12 are micrographs depicting EGFP expression in the cochlea following in vivo administration of an AAV1 vector containing an EGFP expression cassette encoding the E2 enhancer (SEQ ID NO:2) fused directly to the 5' end of the E6 enhancer (SEQ ID NO:3) fused directly to the 5' end of the murine SLC26A4 minimal promoter (SEQ ID NO:17) to wild-type nonhuman primates (plasmid P1669; Figure 7 ). Arrows point to cells expressing nuclear EGFP. DETAILED DESCRIPTION

[0092] Described herein are compositions and methods for specifically inducing gene expression in cells expressing SLC26A4 or a subpopulation thereof (e.g., inner ear cells expressing SLC26A4, such as interdental cells, spiral eminence cells, root cells, and vestibular supporting cells). The present invention features a SLC26A4 enhancer that can be operably linked to a promoter to induce transgene expression in cells expressing SLC26A4 (e.g., inner ear cells expressing SLC26A4). The SLC26A4 enhancer can also increase the level of gene expression and the number of SLC26A4-expressing cells in which gene expression can be detected. In some embodiments, the SLC26A4 enhancer can reduce or minimize off-target expression in cells that do not express SLC26A4 (e.g., when operably linked to a constitutive promoter). Thus, a SLC26A4 enhancer can be operably linked to a promoter, which in turn can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein or a polynucleotide that can be transcribed to produce an RNA molecule such as an inhibitory RNA molecule) to induce expression of the expression product in cells expressing SLC26A4 with minimal off-target expression in cells that do not endogenously express SLC26A4 (e.g., cochlear hair cells). The invention also features a SLC26A4 promoter that can be used to specifically induce expression of an expression product operably linked thereto in cells expressing SLC26A4. One or more of the SLC26A4 enhancers described herein can be operably linked to a SLC26A4 promoter described herein. The invention also features nucleic acid vectors containing one or more SLC26A4 enhancers operably linked to a promoter (e.g., a SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 3, or an enhancer-promoter, a minimal promoter, a core promoter, or a constitutive promoter) operably linked to a polynucleotide encoding an expression product, and nucleic acid vectors containing a SLC26A4 promoter (e.g., a SLC26A4 promoter having at least 85% sequence identity to SEQ ID NO: 1) operably linked to a polynucleotide encoding an expression product, and methods of using these vectors to treat hearing loss (e.g., panin-related hearing loss), vestibular dysfunction (e.g., imbalance or loss of balance associated with panin-related vestibular dysfunction or vestibular hair cell loss), or Meniere's disease.

[0093] Pantothenic acid

[0094] Pan protein is an anion exchange protein encoded by the SLC26A4 gene and is a member of the solute carrier family 26. Mutations in SLC26A4 are associated with both non-syndromic and syndromic forms of hearing loss. Dozens of SLC26A4 mutations have been identified in subjects with non-syndromic hearing loss (hearing loss unrelated to signs and symptoms affecting other parts of the body) called DFNB4. This form of hearing loss can be pre-linguistic or post-linguistic, and subjects with DFNB4 often have an enlarged vestibular aqueduct. More than 150 mutations in SLC26A4 are associated with Pendred syndrome, which is characterized by an enlarged thyroid gland (goiter), hearing loss, and other abnormalities of the inner ear (including enlarged vestibular aqueduct). Pendred syndrome is the most common form of syndromic deafness, and subjects with Pendred syndrome usually begin to lose their hearing at birth or by the age of three, and show hearing loss that worsens over time, progressing to profound deafness in some subjects. There is no curative treatment for pancreatic-related hearing loss, and supportive care is usually aimed at improving hearing, for example, through the use of hearing aids.

[0095] Gene therapy has recently emerged as an attractive therapeutic approach for treating hearing loss, particularly hearing loss caused by mutations in genes expressed in the inner ear, as delivering a wild-type version of the mutant gene can potentially improve or restore hearing. However, there are many genes associated with hearing loss, and they are expressed in a variety of different cell types. To avoid off-target effects, it is best to induce gene expression only in those cells that express the gene endogenously. This can prove challenging for genes such as SLC26A4 that are expressed in a diverse assortment of cell types in the cochlea and vestibule.

[0096] The present invention is based in part on the discovery of SLC26A4 enhancers and promoters, which can be used to induce gene expression in cells expressing SLC26A4 while minimizing off-target expression in cells that do not express SLC26A4. The SLC26A4 enhancer can be operably linked to a promoter (such as the SLC26A4 promoter), which in turn can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding pan protein). The SLC26A4 enhancers and promoters described herein can be used to induce expression of an expression product in cells expressing SLC26A4 while reducing or eliminating off-target expression in cells that do not express SLC26A4 (e.g., cochlear hair cells). The SLC26A4 enhancer can also be used to increase the expression level of an expression product in cells expressing SLC26A4, and to increase the number of cells expressing SLC26A4 in which the expression product is expressed. Thus, the compositions and methods described herein can be used to express an expression product (e.g., a protein, such as Pan or another protein endogenously expressed in a cell expressing SLC26A4, or an RNA molecule, such as an inhibitory RNA molecule) in a cell expressing SLC26A4 (e.g., an interdental cell, a spiral eminence cell, a root cell, or a vestibular supporting cell) to treat a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss) or deafness (e.g., Pan-associated hearing loss), a subject having or at risk of developing vestibular dysfunction (e.g., Pan-associated vestibular dysfunction or vestibular dysfunction associated with vestibular hair cell loss), or a subject having Meniere's disease. The discovery of SLC26A4 enhancers and promoters that can improve cell type-specific expression can improve the safety and efficacy of gene therapy by reducing toxicity caused by off-target expression.

[0097] The compositions and methods described herein may include a SLC26A4 enhancer listed in Table 2 (e.g., SEQ ID NO: 2 or SEQ ID NO: 3) or a variant thereof, such as a polynucleotide sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the compositions described herein contain two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) SLC26A4 enhancers, which may have the same sequence (e.g., multiple copies of the same SLC26A4 enhancer) or different sequences (e.g., one or more copies of each SLC26A4 enhancer listed in Table 2). In some embodiments, the compositions described herein contain one of each SLC26A4 enhancer listed in Table 2 (e.g., a single copy of SEQ ID NO:2 and a single copy of SEQ ID NO:3). In some embodiments, the compositions described herein contain multiple copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies) of SEQ ID NO:2 or multiple copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies) of SEQ ID NO:3. In some embodiments, the compositions described herein contain a single copy of SEQ NO: 2 and multiple copies of SEQ ID NO: 3 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies), or a single copy of SEQ ID NO: 3 and multiple copies of SEQ ID NO: 2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies). In some embodiments, the compositions described herein contain multiple copies of both SEQ ID NO: 2 and SEQ ID NO: 3 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of each enhancer). In embodiments where the composition contains two or more enhancers (e.g., one or more copies of each of SEQ ID NO:2 and SEQ ID NO:3), the enhancers can be included in any order and can be positioned directly adjacent to each other (e.g., linked without any intervening sequences between the enhancer sequences, e.g., the 3' end of the first enhancer is positioned directly before the 5' end of the second enhancer) or can be linked by a nucleic acid linker (e.g., a nucleic acid linker can be located between each enhancer sequence included in the composition or between at least two enhancer sequences in the composition).

[0098] Exemplary SLC26A4 enhancer sequences are provided in Table 2.

[0099] Table 2. SLC26A4 enhancer sequences

[0100]

[0101] The SLC26A4 enhancer sequence described herein can be contained in a nucleic acid vector and operably linked to a promoter (e.g., a SLC26A4 promoter, such as the promoter of SEQ ID NO: 1 or SEQ ID NO: 17), which itself is operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein of interest (such as pan protein) or an RNA molecule (such as an inhibitory RNA)) to specifically express the expression product in cells expressing SLC26A4 (e.g., in inner ear cells expressing SLC26A4 (such as interdental cells, root cells, spiral eminence cells, or vestibular supporting cells)). According to the methods described herein, a composition containing one or more of the aforementioned polynucleotides (e.g., a SLC26A4 enhancer, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2 or SEQ ID NO: 3) can be administered to a subject, wherein the one or more of the aforementioned polynucleotides is operably linked to a promoter, which is operably linked to a polynucleotide encoding an expression product. The one or more SLC26A4 enhancers can be located 5' to the promoter or 3' to the promoter (e.g., 5' to the promoter or 3' to the coding sequence of the expression product).

[0102] The compositions and methods described herein may also include a SLC26A4 promoter. In some embodiments, the SLC26A4 promoter is operably linked to one or more of the aforementioned enhancer sequences (e.g., SEQ ID NO: 2, SEQ ID NO: 3, or both). The SLC26A4 promoter can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a pan protein, a polynucleotide encoding a protein or RNA molecule endogenously expressed in a cell expressing SLC26A4, or a polynucleotide encoding an inhibitory RNA molecule). Nucleic acid vectors containing a SLC26A4 promoter operably linked to a polynucleotide encoding a pan protein can be used to treat pan protein-related hearing loss (e.g., hearing loss associated with DFNB4 or Pendlade syndrome), pan protein-related vestibular dysfunction (e.g., vestibular dysfunction associated with DFNB4 or Pendlade syndrome), or for treating Meniere's disease. Exemplary SLC26A4 promoters are provided in Table 3 below. In some embodiments, the SLC26A4 promoter for use in the compositions and methods described herein has at least 85% sequence identity to SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some embodiments, the SLC26A4 promoter for use in the compositions and methods described herein has the sequence of SEQ ID NO: 1. In some embodiments, the SLC26A4 promoter for use in the compositions and methods described herein has at least 85% sequence identity to SEQ ID NO: 17 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some embodiments, the SLC26A4 promoter for use in the compositions and methods described herein has the sequence of SEQ ID NO:17.

[0103] Table 3. SLC26A4 promoter and enhancer-promoter sequences

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] In some embodiments, a polynucleotide encoding a wild-type pan protein or a variant thereof (such as a polynucleotide sequence encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) pan protein (e.g., SEQ ID NO:4 or SEQ ID NO:5)) is operably linked to a SLC26A4 promoter described herein (e.g., a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; or a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: NO: 17 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity)) or is operably linked to one or more SLC26A4 enhancers described herein (e.g., to SEQ ID NO: 2 and / or SEQ ID NO: 3). In some embodiments, the polynucleotide encoding the wild-type pan protein or a variant thereof is operably linked to a promoter (e.g., a SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 3 or an enhancer-promoter, a minimal promoter, a core promoter, or a constitutive promoter) operably linked to an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a promoter having a sequence of SEQ ID NO: 17 in the order of 5' to 3'. In some more specific embodiments, the polynucleotide encoding the wild-type pan protein or a variant thereof is operably linked to a sequence of SEQ ID NO: 18.In some embodiments, the polynucleotide sequence encoding pan protein encodes an amino acid sequence containing one or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more conservative amino acid substitutions) relative to SEQ ID NO: 4 or SEQ ID NO: 5, provided that the encoded pan protein analog retains the therapeutic function of wild-type pan protein (e.g., the ability to transport negatively charged ions (such as chloride, iodide and bicarbonate) across cell membranes). No more than 10% of the amino acids in pan protein may be replaced by conservative amino acid substitutions. In some embodiments, the polynucleotide sequence encoding pan protein is any polynucleotide sequence that encodes SEQ ID NO: 4 or SEQ ID NO: 5 by redundancy of the genetic code. The polynucleotide sequence encoding pan protein may be partially or completely codon optimized for expression (e.g., in inner ear cells expressing human SLC26A4). Pan protein can also be encoded by a polynucleotide having a single nucleotide polymorphism (SNP) that has been found to be non-pathogenic in human subjects (e.g., a SNP that does not cause hearing loss). Human Pan protein can be encoded by a polynucleotide having a sequence of SEQ ID NO: 6. Mouse Pan protein can be encoded by a polynucleotide having a sequence of SEQ ID NO: 7. Pan protein can be human Pan protein or can be a homolog of human Pan protein from another mammalian species (e.g., mouse, rat, cattle, horse, goat, sheep, donkey, cat, dog, rabbit, guinea pig or other mammal). Exemplary Pan protein amino acid and polynucleotide sequences are provided in Table 4 below. A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer described herein operably linked to a promoter operably linked to a polynucleotide encoding Pan protein (e.g., an SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 3 or an enhancer-promoter, a minimal promoter, a core promoter, or a constitutive promoter), or a nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 promoter described herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 17) operably linked to a polynucleotide encoding Pan protein can be administered to a subject to treat, reduce, or prevent Pan protein-associated hearing loss (such as hearing loss in a subject with DFNB4 or Pendlade syndrome) or Pan protein-associated vestibular dysfunction (such as vestibular dysfunction associated with DFNB4 or Pendlade syndrome (e.g., imbalance or loss of balance, dizziness or vertigo)), or can be administered to a subject to treat Meniere's disease (e.g., hearing loss, tinnitus, or vestibular dysfunction associated with Meniere's disease).Such nucleic acid vectors can also be administered to a subject to treat vestibular dysfunction (e.g., vertigo, dizziness, or imbalance or loss of balance) associated with damage to or loss of vestibular hair cells (damage to or loss of vestibular hair cells associated with head trauma, disease or infection, ototoxic drugs, or aging, such as age-related vestibular dysfunction, ototoxic drug-induced vestibular dysfunction, disease or infection-related vestibular dysfunction, or head trauma-related vestibular dysfunction).

[0111] Table 4. Pan protein sequence

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] Expression of foreign polynucleotides in mammalian cells

[0118] By administering a nucleic acid vector containing at least one SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) operably linked to a promoter (e.g., an SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 3 or an enhancer-promoter, minimal promoter, core promoter, or constitutive promoter) operably linked to a polynucleotide encoding an expression product (e.g., a protein or RNA molecule of interest, such as an inhibitory RNA), or by administering a nucleic acid vector containing an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) operably linked to a polynucleotide encoding an expression product (e.g., a protein or RNA molecule of interest, such as an inhibitory RNA). NO: 1 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) polynucleotide; or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ In some embodiments, the nucleic acid vector contains an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a promoter having a sequence of SEQ ID NO: 17, in the order of 5' to 3'. In some embodiments, the nucleic acid vector contains a sequence of SEQ ID NO: 18, a sequence of SEQ ID NO: 21, a sequence of SEQ ID NO: 32, and a sequence of SEQ ID NO: 19. In some embodiments, the nucleic acid vector contains a sequence of SEQ ID NO: 23, a sequence of SEQ ID NO: 34, and a sequence of SEQ ID NO: 19. In some embodiments, the nucleic acid vector contains a sequence of SEQ ID NO: 11, a sequence of SEQ ID NO: 12, and a sequence of SEQ ID NO: 13. A variety of methods have been established for delivering proteins to mammalian cells and for stably expressing polynucleotides encoding proteins in mammalian cells.

[0119] A nucleic acid vector (e.g., an AAV vector) described herein (e.g., a nucleic acid vector containing at least one SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) operably linked to a promoter (e.g., a SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 3 or an enhancer-promoter, a minimal promoter, a core promoter or a constitutive promoter) or containing a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity)). A nucleic acid vector comprising a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1 or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:17 can be used to express the polynucleotide in one or more cells expressing SLC26A4 (e.g., inner ear cells expressing SLC26A4). In some embodiments, the nucleic acid vector contains, in 5' to 3' order, an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a promoter having a sequence of SEQ ID NO: 17. In some embodiments, the nucleic acid vector contains a sequence of SEQ ID NO: 18. Exemplary polynucleotides that can be expressed using the nucleic acid vectors described herein include polynucleotides encoding proteins expressed in healthy SLC26A4-expressing cells (such as pan and Atoh1), polynucleotides corresponding to wild-type forms of genes that are endogenously expressed in inner ear cells expressing SLC26A4 and mutated in subjects with hearing loss, deafness, tinnitus, or vestibular dysfunction, and other polynucleotides that can be expressed in inner ear cells expressing SLC26A4 to treat hearing loss, deafness, tinnitus, or vestibular dysfunction.The nucleic acid vectors described herein can also be used to express short hairpin RNA (shRNA), antisense oligonucleotides (ASO), components of a gene editing system (e.g., nucleases such as CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) or guide RNAs (gRNAs)), or microRNAs (e.g., miR-183, miR-96, or miR-182) in cells expressing SLC26A4 (e.g., inner ear cells expressing SLC26A4, such as interdental cells, root cells, spiral eminence cells, and vestibular supporting cells).

[0120] In some embodiments, a polynucleotide encoding a wild-type Atoh1 or a variant thereof (such as a polynucleotide sequence encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) Atoh1 (e.g., SEQ ID NO:8 or SEQ ID NO:10)) is operably linked to a SLC26A4 promoter described herein (e.g., a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; or a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1. NO: 17 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity)) or is operably linked to one or more SLC26A4 enhancers described herein (e.g., to SEQ ID NO: 2 and / or SEQ ID NO: 3). In some embodiments, the polynucleotide encoding the wild-type Atoh1 or a variant thereof is operably linked to a promoter (e.g., a SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 3, or an enhancer-promoter, a minimal promoter, a core promoter, or a constitutive promoter) operably linked to an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a promoter having a sequence of SEQ ID NO: 17 in the order of 5' to 3'. In some embodiments, the polynucleotide encoding the wild-type Atoh1 or a variant thereof is operably linked to a polynucleotide having a sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide sequence encoding the Atoh1 protein encodes an amino acid sequence containing one or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more conservative amino acid substitutions) relative to SEQ ID NO:4, provided that the encoded Atoh1 analog retains the therapeutic function of wild-type Atoh1 (e.g., the ability to promote hair cell development).No more than 10% of the amino acids in the Atoh1 protein may be replaced by conservative amino acid substitutions. In some embodiments, the polynucleotide sequence encoding Atoh1 is any polynucleotide sequence that encodes SEQ ID NO:8 or SEQ ID NO:10 by redundancy of the genetic code. The polynucleotide sequence encoding Atoh1 may be partially or completely codon-optimized for expression (e.g., in inner ear cells expressing human SLC26A4 (such as vestibular supporting cells)). The Atoh1 protein may also be encoded by a polynucleotide having a single nucleotide polymorphism (SNP) that has been found to be non-pathogenic in human subjects (e.g., a SNP that does not cause hearing loss). Human Atoh1 may be encoded by a polynucleotide having a sequence of SEQ ID NO:9. Mouse Atoh1 may be encoded by a polynucleotide having a sequence of SEQ ID NO:11. The Atoh1 protein may be a human Atoh1 protein or may be a homolog of a human Atoh1 protein from another mammalian species (e.g., mouse, rat, cattle, horse, goat, sheep, donkey, cat, dog, rabbit, guinea pig or other mammal). Exemplary Atohl amino acid and polynucleotide sequences are listed in Table 5 below. A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer described herein operably linked to a promoter (e.g., an SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 3 or an enhancer-promoter, a minimal promoter, a core promoter, or a constitutive promoter) operably linked to a polynucleotide encoding Atoh1, or a nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 promoter described herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 17) operably linked to a polynucleotide encoding Atoh1 can be administered to a subject to treat, reduce, or prevent vestibular dysfunction (e.g., imbalance or loss of balance, dizziness, or vertigo) associated with damage or loss of vestibular hair cells (e.g., damage or loss of vestibular hair cells associated with head trauma, disease or infection, ototoxic drugs, or aging, such as age-related vestibular dysfunction, ototoxic drug-induced vestibular dysfunction, disease or infection-related vestibular dysfunction, or head trauma-related vestibular dysfunction).

[0121] Table 5. Atoh1 sequences

[0122]

[0123]

[0124]

[0125] Polynucleotide encoding a protein of interest

[0126] One platform that can be used to achieve therapeutically effective intracellular concentrations of the protein of interest in mammalian cells is by stable expression of a gene encoding the protein of interest (e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell, or by forming an additional concatemer in the nucleus of a mammalian cell). The gene is a polynucleotide encoding the primary amino acid sequence of the corresponding protein. In order to introduce an exogenous gene into a mammalian cell, the gene can be incorporated into a vector. The vector can be introduced into the cell by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and by encapsulating the vector in a liposome. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail in, 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 each of which are incorporated herein by reference.

[0127] Proteins of interest can also be introduced into mammalian cells by targeting a vector containing a gene encoding the protein of interest to cell membrane phospholipids. For example, the vector can be targeted to phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to the VSV-G protein, a viral protein that has an affinity for all cell membrane phospholipids. Such constructs can be generated using methods well known to those skilled in the art.

[0128] Recognition and binding of a polynucleotide encoding a protein of interest by a mammalian RNA polymerase is important for gene expression. Thus, sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and promote assembly of transcription complexes at transcription start sites may be included in the polynucleotide. Such sequence elements include, for example, mammalian promoters, sequences of which can be recognized and bound by specific transcription initiation factors and final RNA polymerases. Examples of mammalian promoters have been described in Smith et al., Mol. Sys. Biol., 3:73, published online, the disclosure of which is incorporated herein by reference. The promoter used in the methods and compositions described herein may be a SLC26A4 promoter (e.g., a SLC26A4 promoter or enhancer-promoter provided in Table 3), a constitutive promoter (e.g., a promoter active in vivo in all cases), a core promoter, or a minimal promoter. Constitutive promoters include CAG promoter, cytomegalovirus (CMV) promoter (e.g., CMV immediate early enhancer and promoter, CMVmini promoter, minCMV promoter, CMV-TATA+INR promoter or min CMV-T6 promoter), smCBA promoter (described in Haire et al., Invest. Opthalmol. Vis. Sci. 47: 3745-3753, 2006), CBA promoter, CASI promoter, dihydrofolate reductase (DHFR) promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, β-globin promoter (e.g., minimal β-globin promoter), HSV promoter (e.g., minimal HSV ICP0 promoter or truncated HSV ICP0 promoter), SV40 promoter (e.g., SV40 minimal promoter) and EF1α promoter. Constitutive promoters may also be referred to as ubiquitous promoters because they are able to induce expression of polynucleotides in a wide range of cell and tissue types. Minimal promoters include CMV minimal promoters (e.g., minCMV promoters), minimal β-globin promoters, minimal HSV promoters (e.g., minimal HSV ICP0 promoters), and SV40 minimal promoters. Alternatively, promoters derived from viral genomes can also be used for stable expression of polynucleotides in mammalian (e.g., human) cells. Examples of functional viral promoters that can be used to express polynucleotides in primate (e.g., human) cells include adenovirus late promoters, vaccinia virus 7.5K promoters, tk promoters of HSV, mouse mammary tumor virus (MMTV) promoters, LTR promoters of HIV, Moloney virus promoters, Epstein Barr virus (EBV) promoters, and Rous sarcoma virus (RSV) promoters.

[0129] Once the polynucleotide encoding the protein of interest is incorporated into a mammalian cell, the transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the mammalian cell to an external chemical reagent (such as an agent that regulates the combination of transcription factors and / or RNA polymerase with mammalian promoters and thus regulates gene expression). Chemical reagents can be used to promote the combination of RNA polymerase and / or transcription factors with mammalian promoters, for example, by removing the repressor protein that has been combined with the promoter. Alternatively, chemical reagents can be used to enhance the affinity of mammalian promoters to RNA polymerase and / or transcription factors, so that in the presence of chemical reagents, the transcription rate of the gene located downstream of the promoter increases. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanism include tetracycline and doxycycline. These reagents are commercially available, and can be applied to mammalian cells according to the scheme established to promote gene expression.

[0130] The nucleic acid vectors described herein may include a woodchuck post-transcriptional regulatory element (WPRE). The WPRE acts at the mRNA level by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of nascent transcripts, thereby increasing the amount of total mRNA in cells. Adding the WPRE to the vector can result in significant improvements in the levels of transgene expression caused by several different promoters in vitro and in vivo.

[0131] In some embodiments, the nucleic acid vectors described herein include a reporter sequence that can be used to verify expression of a gene operably linked to the SLC26A4 promoter and / or SLC26A4 enhancer, for example, in cells and tissues (e.g., in cells expressing SLC26A4, such as interdental cells, root cells, spiral carina cells, and vestibular supporting cells). Reporter gene sequences that can be provided in the transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. When associated with a regulatory element that drives its expression, such as a promoter, the reporter gene sequence provides a signal detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescent or other spectroscopic assays, fluorescence activated cell sorting assays, and immunoassays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the signal-carrying vector is detected by assaying for β-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visualized by color or light production in a luminometer.

[0132] Methods for delivering exogenous polynucleotides to target cells

[0133] Techniques that can be used to introduce polynucleotides (such as polynucleotides operably linked to the SLC26A4 promoter and / or SLC26A4 enhancer described herein) into target cells (e.g., mammalian cells) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the cells of interest. Mammalian cells (such as human cells) subjected to an external electric field in this manner are subsequently susceptible to uptake of exogenous polynucleotides. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. Similar Techniques Nucleofection TM The use of applied electric fields to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. TM Protocols that can be used to perform this technique are described in detail in, for example, Distler et al., Experimental Dermatology 14:315 (2005) and US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0134] Other techniques that can be used to transfect target cells include extrusion perforation methods. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to the applied stress. The advantage of this technique is that a vector is not necessary for delivering polynucleotides to cells (such as human target cells). Extrusion-perforation is described in detail, for example, in Sharei et al., Journal of Visualized Experiments 81: e50980 (2013), the disclosure of which is incorporated herein by reference.

[0135] Lipofection represents another technology that can be used for transfecting target cells.This method involves loading polynucleotides into liposomes, which generally have cationic functional groups (such as quaternary amines or protonated amines) toward the outside of the liposomes.This promotes electrostatic interactions between liposomes and cells due to the anionic nature of the cell membrane, ultimately causing the uptake of exogenous polynucleotides, such as by direct fusion of liposomes with cell membranes or by endocytosis of complexes.Lipofection is described in detail in, 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 cell membranes to cause the uptake of exogenous polynucleotides include contacting cells with cationic polymer-polynucleotide complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that facilitates interaction with cell membranes include activated dendrimers (described, e.g., Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described in detail, e.g., Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to direct the uptake of polynucleotides. This technology is described in detail, e.g., in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0136] Another available tool for inducing uptake of exogenous polynucleotides by target cells is laser transfection, also known as optical transfection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to and in some cases has been found to be superior to electroporation.

[0137] Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-shaped nanostructures are synthesized perpendicular to the substrate surface. DNA containing genes intended for intracellular delivery is connected to the nanostructure surface. A chip with an array of these needles is then pressed against a cell or tissue. Cells pierced by the nanostructure can express one or more delivered genes. Examples of this technology are described in Shalek et al., PNAS 107: 1870 (2010), the disclosure of which is incorporated herein by reference.

[0138] Magnetorfection can also be used to deliver polynucleotides to target cells. The principle of magnetorfection is to associate polynucleotides with cationic magnetic nanoparticles. Magnetic nanoparticles are made of completely biodegradable iron oxide and coated with specific cationic proprietary molecules that vary according to the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells by the influence of the external magnetic field generated by the magnet. This technology is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0139] Another useful tool for inducing uptake of exogenous polynucleotides by target cells is sonoporation, a technique that involves using sound (usually ultrasonic frequencies) to change the permeability of the cell plasma membrane to permeabilize the cell and allow the polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0140] Microvesicles represent another potential medium that can be used to modify the target cell genome according to the methods described herein. For example, microvesicles caused by co-overexpression of glycoprotein VSV-G and, for example, genome modification proteins (such as nucleases) can be used to effectively deliver proteins into cells, which then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of cells for covalent incorporation of polynucleotides of interest (such as genes or regulatory sequences). The use of such vesicles (also referred to as nanovesicles (Gesicles)) in the genetic modification of eukaryotic cells is described in detail, for example, 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; May 13, 2015, in Abstract No. 122.

[0141] Vectors for delivering exogenous polynucleotides to target cells

[0142] In addition to achieving high transcription and translation rates, stable expression of exogenous polynucleotides in mammalian cells can also be achieved by integrating the polynucleotides into the nuclear genome of mammalian cells. A variety of vectors have been developed for delivering and integrating polynucleotides encoding expression products into the nuclear DNA of mammalian cells. Examples of expression vectors are described in, for example, Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). The expression vectors for use in the compositions and methods described herein contain at least one SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) and / or a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1 or a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3 NO:17 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) promoter), operably linked polynucleotides encoding expression products (e.g., encoding a protein of interest or a polynucleotide that can be transcribed to produce an RNA molecule (such as an inhibitory RNA)), and additional sequence elements, for example, for expressing these agents and / or integrating these polynucleotide sequences into the genome of mammalian cells. Vectors that may contain one or more SLC26A4 enhancers and / or SLC26A4 promoters operably linked to a polynucleotide encoding an expression product (e.g., a transgene encoding a protein of interest) include plasmids (e.g., circular DNA molecules that can replicate autonomously in cells), cosmids (e.g., pWE or sCos vectors), artificial chromosomes (e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), and viral vectors. Certain vectors that can be used to express expression products (e.g., proteins of interest) include plasmids containing regulatory sequences that direct gene transcription.Other useful vectors for expressing expression products (e.g., proteins of interest) contain polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide efficient transcription of genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain 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 nourseothricin.

[0143] Viral vectors for polynucleotide delivery

[0144] Viral genomes provide a rich vector source that can be used to effectively deliver genes of interest to the genome of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., Retroviridae vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses (such as picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara, MVA, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, envelope virus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, avian C virus, mammalian C, B virus, D virus, oncorretrovirus, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, foamy virus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, 3rd edition (Lippincott-Raven, Philadelphia, 1996)). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape 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 US Pat. No. 5,801,030, which is incorporated herein by reference for its disclosure relating to viral vectors for use in gene therapy.

[0145] AAV vectors for polynucleotide delivery

[0146] In some embodiments, the polynucleotides of the compositions and methods described herein are incorporated into rAAV vectors and / or virions to facilitate their introduction into cells. The rAAV vectors that can be used in the compositions and methods described herein are recombinant polynucleotide constructs that include (1) a promoter (e.g., an SLC26A4 promoter, such as a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1; or a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1; NO: 17 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) promoter), (2) the sequence to be expressed (e.g., a polynucleotide encoding a protein such as pan protein or Atoh1, or a polynucleotide that can be transcribed to produce an RNA molecule (such as an inhibitory RNA)), and (3) a viral sequence that promotes integration and expression of the sequence to be expressed. The viral sequence can include those sequences of AAV required for DNA cis replication and packaging (e.g., functional ITRs) into virions. In some embodiments, the rAAV vector further comprises at least one SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2 or SEQ ID NO: 3). In some embodiments, the rAAV vectors useful in the compositions and methods described herein contain, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and a promoter having the sequence of SEQ ID NO: 17. In some embodiments, the rAAV vector contains the sequence of SEQ ID NO: 18. In a typical application, the sequence to be expressed encodes a wild-type form of a protein expressed in inner ear cells expressing SLC26A4 that is mutated in subjects with a hereditary form of hearing loss (such as a wild-type form of pan protein), or a protein or RNA molecule that can promote the differentiation of vestibular supporting cells into vestibular hair cells (such as Atoh1). Such rAAV vectors may also contain a marker or reporter gene. Available rAAV vectors have one or more AAV WT genes that are deleted in whole or in part but still retain functional flanking ITR sequences. The AAV ITRs may be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs may be AAV2 ITRs.Methods for using rAAV vectors are described in, for example, Tal et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000), each of which is incorporated herein by reference for its disclosure relating to AAV vectors for gene delivery.

[0147] The polynucleotides and vectors described herein (e.g., SLC26A4 enhancers and / or SLC26A4 promoters operably linked to polynucleotides encoding expression products) can be incorporated into rAAV virions to facilitate the introduction of polynucleotides or vectors into cells. The capsid protein of AAV constitutes the external non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins VP1, VP2, and VP3 required for virion assembly. The construction of rAAV virions has been described in, for example, US 5,173,414; US 5,139,941; US ​​5,863,541; US ​​5,869,305; US 6,057,152; and US 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

[0148] The rAAV virions used in conjunction with the compositions and methods described herein include virions derived from a variety of AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S. For targeting cells expressing SLC26A4, AAV1, AAV2, AAV2quad (YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, AAV-DJ, AAV-DJ / 9, 7m8, and PHP.B may be particularly useful. Serotypes evolved for transduction of the retina can also be used in the methods and compositions described herein. The construction and use of AAV vectors of different serotypes and AAV proteins are described in, for example, Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0149] Also used in combination with the compositions and methods described herein is a pseudotyped rAAV vector. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) that are derived from capsid genes of serotypes other than a given serotype (e.g., AAV1, AAV2, AAV2quad (YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.) pseudotyped. The construction and use of pseudotyped rAAV virions are known in the art and are described in, for example, 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).

[0150] AAV virions with mutations in the virion capsid can be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for promoting AAV targeting to specific cell types. The construction and characterization of AAV capsid mutants (including insertion mutants, alanine screening mutants, and epitope tag mutants) are described in Wu et al., J. Virol. 74: 8635 (2000). Other rAAV virions that can be used in the methods described herein include those capsid hybrids 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).

[0151] Pharmaceutical composition

[0152] The SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) and / or the SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3 NO: 17 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) can be operably linked to a polynucleotide encoding an expression product (e.g., a transgene encoding a protein of interest or an RNA molecule (such as an inhibitory RNA)) and incorporated into a vector for administration to a patient, such as a human patient with sensorineural hearing loss (e.g., pan protein-associated hearing loss) or vestibular dysfunction (e.g., vestibular dysfunction associated with vestibular hair cell damage or loss or pan protein-associated vestibular dysfunction). Pharmaceutical compositions containing vectors (such as viral vectors) containing the SLC26A4 enhancer and / or SLC26A4 promoter described herein operably linked to a polynucleotide encoding an expression product can be prepared using methods known in the art. For example, such compositions can be prepared using, for example, physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacology 22nd Edition, Allen, L. Ed. (2013); incorporated herein by reference) and in a desired form (e.g., in the form of a lyophilized formulation or an aqueous solution).

[0153] The invention also provides a method for preparing an SLC26A4 enhancer as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) and / or a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3; NO:17 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) of nucleic acid vectors (e.g., viral vectors) mixtures can be prepared in water appropriately mixed with one or more excipients, carriers or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and their mixtures and in oils. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders (described in US 5,466,468, the disclosure of which is incorporated herein by reference) for the extemporaneous preparation of sterile injectable solutions or dispersions. In either case, the preparation may be sterile and may be fluid to the extent that there is easy injectability. The preparation can be stable under the conditions of manufacture and storage and can be preserved under the conditions of preventing the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and / or vegetable oils. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. The effects of microorganisms can be prevented by a variety of antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugars or sodium chloride, will preferably be included. Prolonging the absorption of the injectable composition can be achieved by using an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0154] For example, if desired, a solution comprising a pharmaceutical composition as described herein can be appropriately buffered, and the liquid diluent can first be made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous medium that can be used will be known to those skilled in the art according to the present disclosure. For example, a dose can be dissolved in 1ml isotonic NaCl solution and added to 1000ml subcutaneous infusion fluid or injected at the infusion site of the suggestion. Depending on the disease of the treated subject, a certain dosage variation will necessarily occur. In order to be applied topically to the inner ear, the composition can be formulated to include synthetic perilymph. Exemplary synthetic perilymph includes 20-200mM NaCl, 1-5mM KCl, 0.1-10mM CaCl 2 , 1-10 mM glucose and 2-50 mM HEPE, with a pH between about 6 and 9 and an osmotic pressure of about 300 mOsm / kg. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject. In addition, for human administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0155] Treatment

[0156] The compositions described herein can be administered to a subject suffering from or at risk of developing sensorineural hearing loss, vestibular dysfunction, or Meniere's disease by a variety of routes, such as topical administration to the middle ear or inner ear (e.g., administration to the perilymph or endolymph, such as administration to or through the oval window, round window, or semicircular canal (e.g., the transverse canal), or by transtympanic or intratympanic injection, e.g., administration to inner ear cells expressing SLC26A4), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, infusion, lavage, and oral administration. In any given case, the most appropriate route of administration will depend on the specific composition being administered, the patient, the method of drug formulation, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. The composition can be administered once or more than once (eg, once a year, twice a year, three times a year, once every two months, once a month, or once every two weeks).

[0157] A subject that can be treated as described herein is a subject having or at risk for developing sensorineural hearing loss. In some embodiments, the compositions described herein are used to treat pan protein-related hearing loss (e.g., DFNB4 or Pendelaide syndrome). DFNB4 and Pendelaide syndrome can be treated by administering a nucleic acid vector containing an SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) operably linked to a polynucleotide encoding pan protein (e.g., a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5) and / or a SLC26A4 promoter (e.g., a polynucleotide encoding SEQ ID NO:5) operably linked to a polynucleotide encoding pan protein (e.g., a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5) NO:1; or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:17. In some embodiments, the nucleic acid vector contains, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO:2, an enhancer having the sequence of SEQ ID NO:3, and a promoter having the sequence of SEQ ID NO:17, the promoter being operably linked to a polynucleotide encoding a pan protein. In some embodiments, the nucleic acid vector contains a polynucleotide having a sequence of SEQ ID NO: 18 operably linked to a polynucleotide encoding a pan protein. The subject can have or be identified as having a mutation in SLC26A4 and can have severe, moderate, or mild hearing loss at the time of initiation of treatment, or can be treated prior to the onset of symptoms (e.g., prophylactic treatment). In some embodiments, the composition is administered as a prophylactic treatment to a subject at risk of developing hearing loss, such as a subject who carries a SLC26A4 mutation associated with hearing loss but has not yet exhibited hearing impairment.

[0158] In some embodiments, the compositions described herein are used to treat a subject with Meniere's disease. Both a subject with a mutation in SLC26A4 and a subject with Meniere's disease have endolymphatic edema, and thus, a composition (e.g., containing a SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) operably linked to a polynucleotide encoding pan protein (e.g., a polynucleotide encoding SEQ ID NO: 4 or SEQ ID NO: 5) and / or a SLC26A4 promoter (e.g., a polynucleotide encoding SEQ ID NO: 5) operably linked to a polynucleotide encoding pan protein (e.g., a polynucleotide encoding SEQ ID NO: 4 or SEQ ID NO: 5) NO: 1 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) polynucleotide; or with SEQ ID NO: 17 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) polynucleotide) nucleic acid vector) can also be effective in reducing or improving endolymphatic edema in subjects with Meniere's disease. Such treatment can be used to treat hearing loss, tinnitus or vestibular dysfunction (e.g., vertigo) in subjects with Meniere's disease, and can reduce the feeling of fullness or congestion in the ear.

[0159] In some embodiments, the compositions described herein are used to treat subjects suffering from or at risk of developing vestibular dysfunction (e.g., vertigo, dizziness, imbalance, oscillopsia, balance disorders, or bilateral vestibular lesions). In some embodiments, the vestibular dysfunction is a Pan protein-related vestibular dysfunction associated with DFNB4 or Pendred syndrome (e.g., imbalance or loss of balance associated with DFNB4 or Pendred syndrome). Panin-related vestibular dysfunction can be treated by administering a nucleic acid vector comprising a SLC26A4 enhancer as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) and / or a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3; NO:17 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity)), the SLC26A4 enhancer and / or SLC26A4 promoter operably linked to a polynucleotide encoding Pan protein (e.g., a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the subject may have or may have been identified as having a mutation in SLC26A4 and may have severe, moderate or mild vestibular dysfunction at the time of initiation of treatment, or may be treated prior to the onset of symptoms (e.g., prophylactic treatment).

[0160] Vestibular dysfunction may also be caused by damage or loss of vestibular hair cells. Therefore, the compositions and methods described herein can be used to treat subjects suffering from or at risk of developing vestibular hair cell damage or loss (e.g., vestibular hair cell damage or loss associated with disease or infection, head trauma, ototoxic drugs (e.g., vestibular toxic drugs) or aging), subjects suffering from or at risk of developing vestibular dysfunction (e.g., dizziness, vertigo, imbalance, bilateral vestibular lesions, oscillopsia or balance disorders), subjects carrying genetic mutations associated with vestibular dysfunction, or subjects with a family history of hereditary vestibular dysfunction. In some embodiments, the disease associated with damage or loss of hair cells (e.g., vestibular hair cells) is an autoimmune disease or disorder, in which the autoimmune response leads to hair cell damage or death. Autoimmune diseases associated with vestibular dysfunction include autoimmune inner ear disease (AIED), polyarteritis nodosa (PAN), Cogan's syndrome, relapsing polychondritis, systemic lupus erythematosus (SLE), Wegener's granulomatosis, Sjögren's syndrome, syndrome and Behcet's disease disease). Some infectious diseases (such as Lyme disease and syphilis) can also cause vestibular dysfunction (e.g., by triggering autoantibody production). Viral infections (such as rubella, cytomegalovirus (CMV), lymphocytic choriomeningitis virus (LCMV), HSV type 1 and type 2, West Nile virus (WNV), human immunodeficiency virus (HIV), varicella zoster virus (VZV), measles and mumps) can also cause vestibular dysfunction. In some embodiments, the subject suffers from vestibular dysfunction associated with or caused by loss of hair cells (e.g., vestibular hair cells). In some embodiments, the compositions and methods described herein can be used to treat subjects suffering from or at risk of developing oscillopsia. In some embodiments, the compositions and methods described herein can be used to treat subjects suffering from or at risk of developing bilateral vestibular lesions. In some embodiments, the compositions and methods described herein can be used to treat subjects suffering from or at risk of developing balance disorders (e.g., imbalance). The compositions and methods described herein can also be applied as a preventive treatment to subjects at risk of developing vestibular dysfunction, e.g., subjects with a family history of vestibular dysfunction (e.g., hereditary vestibular dysfunction), subjects carrying a gene mutation associated with vestibular dysfunction but not yet showing symptoms of vestibular dysfunction, or subjects exposed to acquired vestibular dysfunction risk factors (e.g., disease or infection, head trauma, ototoxic drugs, or aging). The compositions and methods described herein can also be used to treat subjects with idiopathic vestibular dysfunction.

[0161] The compositions and methods described herein can be used to induce or increase vestibular hair cell regeneration in a subject. Subjects who may benefit from compositions that promote or induce vestibular hair cell regeneration include subjects who suffer from or are at risk of developing vestibular dysfunction due to hair cell loss (e.g., vestibular hair cell loss associated with trauma (e.g., head trauma), disease or infection, ototoxic drugs, or aging), and subjects with abnormal vestibular hair cells (e.g., vestibular hair cells that function abnormally compared to normal vestibular hair cells), vestibular hair cells with damage (e.g., vestibular hair cell damage associated with trauma (e.g., head trauma), disease or infection, ototoxic drugs, or aging), or subjects with reduced vestibular hair cell numbers due to genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase vestibular hair cell maturation, which can result in improved vestibular function. In some embodiments, the compositions and methods described herein promote or increase the maturation of regenerated vestibular hair cells.

[0162] Compositions and methods described herein can also be used to prevent or reduce vestibular dysfunction caused by vestibular hair cell damage or death (e.g., vestibular hair loss induced by vestibular toxic drugs) induced by ototoxic drugs in subjects who have received ototoxic drug treatment or are receiving or are about to receive ototoxic drug treatment. Ototoxic drugs are toxic to the cells of the inner ear and can cause vestibular dysfunction (e.g., vertigo, dizziness, imbalance, bilateral vestibular lesions or oscillopsia). It has been found that drugs with ototoxicity include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin and netilmicin), puromycin, antitumor drugs (e.g., platinum-containing chemotherapeutics, such as cisplatin, carboplatin and oxaliplatin, or other chemotherapeutics, such as nitrogen mustard and vincristine), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, particularly at high doses) and quinine. Some of these drugs, such as nitrogen mustard, vincristine, gentamicin, streptomycin, and tobramycin, have been specifically identified as vestibular toxic drugs. In some embodiments, the methods and compositions described herein can be used to treat bilateral vestibular lesions or oscillopsia due to aminoglycoside ototoxicity (e.g., the methods and compositions described herein can be used to promote or increase vestibular hair cell regeneration in a subject with aminoglycoside-induced bilateral vestibular lesions or oscillopsia).

[0163] Vestibular dysfunction associated with damage or loss of vestibular hair cells (e.g., damage or loss of vestibular hair cells associated with disease or infection, head trauma, ototoxic drugs (e.g., vestibulotoxic drugs), or aging) can be treated by administering a nucleic acid vector comprising a SLC26A4 enhancer as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) and / or a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3). NO:1 or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:17, wherein the SLC26A4 enhancer and / or SLC26A4 promoter is operably linked to a polynucleotide encoding Atoh1 (e.g., a polynucleotide encoding SEQ ID NO:8 or SEQ ID NO:10) or a polynucleotide encoding Pan protein (e.g., a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5). In some embodiments, the nucleic acid vector contains, in 5' to 3' order, an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a promoter having a sequence of SEQ ID NO: 17, which is operably linked to a polynucleotide encoding Atoh1 or Pan protein. In some embodiments, the nucleic acid vector contains a polynucleotide having a sequence of SEQ ID NO: 18, which is operably linked to a polynucleotide encoding Atoh1 or Pan protein. Such a nucleic acid vector can also be used to induce or increase vestibular hair cell regeneration or vestibular hair cell maturation in a subject in need thereof.

[0164] The methods described herein may include a step of screening a subject for one or more mutations in a gene (e.g., SLC26A4) known to be associated with hearing loss or vestibular dysfunction prior to treatment or administration with a composition as described herein. Subjects may 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 assessing the hearing of a subject prior to treatment or administration of a composition as described herein. Hearing may be assessed using standard tests such as audiometry, auditory brainstem response (ABR), electrocochlear osmography (ECOG), and otoacoustic emissions. These tests may also be used to assess the hearing of a subject after treatment or administration with a composition as described herein. In some embodiments, the methods described herein include a step of assessing the vestibular function of a subject prior to treatment or administration of a composition as described herein. Vestibular function can be assessed using standard tests such as eye movement tests (e.g., electronystagmogram (ENG) or videonystagmogram (VNG)), vestibular-ocular reflex (VOR) tests (e.g., head impact tests (Halmagyi–Curthoys test), or caloric reflex tests, which can be performed at the bedside or using video impact testing (VHIT), posturography, swivel chair testing, ECOG, vestibular evoked myogenic potentials (VEMPs), and specialized clinical balance tests such as those described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010). These tests can also be used to assess vestibular function in a subject after treatment with or administration of a composition described herein.

[0165] 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:17) and / or a SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2. The polynucleotide encoding the expression product operably linked to the polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO:3 can be a polynucleotide encoding Pan protein (e.g., a polynucleotide encoding the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5), a polynucleotide encoding Atoh1 (e.g., a polynucleotide encoding SEQ ID NO:8 or SEQ ID NO:9), a polynucleotide encoding Atoh2 (e.g., a polynucleotide encoding SEQ ID NO:10 or SEQ ID NO:11), a polynucleotide encoding Atoh3 (e.g., a polynucleotide encoding SEQ ID NO:12 or SEQ ID NO:13), a polynucleotide encoding Atoh4 (e.g., a polynucleotide encoding SEQ ID NO:14 or SEQ ID NO:15), a polynucleotide encoding Atoh5 (e.g., a polynucleotide encoding SEQ ID NO:15), a polynucleotide encoding Atoh6 (e.g., a polynucleotide encoding SEQ ID NO:16 or SEQ ID NO:17), a polynucleotide encoding Atoh7 (e.g., a polynucleotide encoding SEQ ID NO:17), a polynucleotide encoding Atoh8 (e.g., a polynucleotide encoding SEQ ID NO:18), a polynucleotide encoding Atoh9 (e.g., a polynucleotide encoding SEQ ID NO:19), a polynucleotide encoding Atoh10 (e.g., a polynucleotide encoding SEQ ID NO:11 NO:10), a polynucleotide encoding a wild-type form of a protein expressed in inner ear cells expressing SLC26A4 (which is mutated in subjects with sensorineural hearing loss or vestibular dysfunction), a polynucleotide encoding another protein of interest (e.g., a reporter protein such as a fluorescent protein, lacZ, or luciferase), or a polynucleotide that can be transcribed to produce an RNA molecule (such as shRNA), an ASO, a component of a gene editing system (e.g., a nuclease, such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN) or a zinc finger nuclease (ZFN) or a guide RNA (gRNA)), or a microRNA. In some embodiments, the expression product is operably linked to a polynucleotide sequence that comprises, in 5' to 3' order, an enhancer having a sequence of SEQ ID NO:2, an enhancer having a sequence of SEQ ID NO:3, and a promoter having a sequence of SEQ ID NO:17. In some embodiments, the expression product is operably linked to a polynucleotide having a sequence of SEQ ID NO:18.The polynucleotide can be selected based on the cause of the subject's hearing loss or vestibular dysfunction (e.g., if the subject's hearing loss is associated with a mutation in SLC26A4, the polynucleotide can encode wild-type Pan protein, or if the subject's vestibular dysfunction is age-related or ototoxic drug-induced vestibular dysfunction associated with hair cell loss, the polynucleotide can encode Atoh1), the severity of the subject's hearing loss, the health of the subject's inner ear cells, the subject's age, the subject's family history of hearing loss, or other factors.

[0166] Treatment may include administering a composition containing a nucleic acid vector (e.g., an AAV vector) containing the SLC26A4 enhancer and / or the SLC26A4 promoter described herein in various unit doses. Each unit dose will generally contain a predetermined amount of the therapeutic composition. The amount to be administered, as well as the specific route of administration and formulation, are within the skill of those skilled in the clinical field. The unit dose need not be administered as a single injection, but may comprise continuous infusion over a set period of time. Administration may be performed using a syringe pump to control the infusion rate so as to minimize damage to the inner ear (e.g., cochlea and / or vestibular system). In the case where the nucleic acid vector is an AAV vector (e.g., AAV1, AAV2, AAV2quad (YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, or PHP.S vector), the viral vector may be, for example, about 1 x 10 9 vector genomes (VG) / mL to about 1x10 16 VG / mL (e.g., 1x 10 9 VG / mL, 2x 10 9 VG / mL, 3x 10 9 VG / mL, 4x 10 9 VG / mL, 5x 10 9 VG / mL, 6x 10 9 VG / mL, 7x 10 9 VG / mL, 8x10 9 VG / mL, 9x 10 9 VG / mL, 1x 10 10 VG / mL, 2x 10 10 VG / mL, 3x 10 10 VG / mL, 4x10 10 VG / mL, 5x 10 10 VG / mL, 6x 10 10VG / mL、7x 10 10 VG / mL、8x 10 10 VG / mL、9x 10 10 VG / mL、1x10 11 VG / mL、2x 10 11 VG / mL、3x 10 11 VG / mL、4x 10 11 VG / mL、5x 10 11 VG / mL、6x 10 11 VG / mL、7x10 11 VG / mL、8x 10 11 VG / mL、9x 10 11 VG / mL、1x 10 12 VG / mL、2x 10 12 VG / mL、3x 10 12 VG / mL、4x10 12 VG / mL、5x 10 12 VG / mL、6x 10 12 VG / mL、7x 10 12 VG / mL、8x 10 12 VG / mL、9x 10 12 VG / mL、1x10 13 VG / mL、2x 10 13 VG / mL、3x 10 13 VG / mL、4x 10 13 VG / mL、5x 10 13 VG / mL、6x 10 13 VG / mL、7x10 13 VG / mL、8x 10 13 VG / mL、9x 10 13 VG / mL、1x 10 14 VG / mL、2x 10 14 VG / mL、3x 10 14 VG / mL、4x10 14 VG / mL、5x 10 14 VG / mL、6x 10 14 VG / mL、7x 10 14 VG / mL、8x 10 14 VG / mL、9x 10 14 VG / mL、1x10 15 VG / mL、2x 10 15 VG / mL、3x 10 15VG / mL, 4x 10 15 VG / mL, 5x 10 15 VG / mL, 6x 10 15 VG / mL, 7x10 15 VG / mL, 8x 10 15 VG / mL, 9x 10 15 VG / mL, or 1x 10 16 VG / mL) is administered to a patient in a volume of 1 μL to 200 μL (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μL). AAV vectors can be administered in a volume of about 1×10 7 VG / ear to approx. 2x 10 15 VG / ear (e.g., 1x 10 7 VG / ear, 2x 10 7 VG / ear, 3x 10 7 VG / ear, 4x10 7 VG / ear, 5x 10 7 VG / ear, 6x 10 7 VG / ear, 7x 10 7 VG / ear, 8x 10 7 VG / ear, 9x 10 7 VG / ear, 1x 10 8 VG / ear, 2x 10 8 VG / ear, 3x 10 8 VG / ear, 4x 10 8 VG / ear, 5x 10 8 VG / ear, 6x10 8 VG / ear, 7x 10 8 VG / ear, 8x 10 8 VG / ear, 9x 10 8 VG / ear, 1x 10 9 VG / ear, 2x 10 9 VG / ear, 3x 10 9 VG / ear, 4x 10 9 VG / ear, 5x 10 9 VG / ear, 6x 10 9 VG / ear, 7x 10 9 VG / ear, 8x10 9 VG / ear, 9x 109 VG / ear, 1x 10 10 VG / ear, 2x 10 10 VG / ear, 3x 10 10 VG / ear, 4x 10 10 VG / ear, 5x 10 10 VG / ear, 6x 10 10 VG / ear, 7x 10 10 VG / ear, 8x10 10 VG / ear, 9x 10 10 VG / ear, 1x 10 11 VG / ear, 2x 10 11 VG / ear, 3x 10 11 VG / ear, 4x 10 11 VG / ear, 5x 10 11 VG / ear, 6x10 11 VG / ear, 7x 10 11 VG / ear, 8x 10 11 VG / ear, 9x 10 11 VG / ear, 1x 10 12 VG / ear, 2x10 12 VG / ear, 3x 10 12 VG / ear, 4x 10 12 VG / ear, 5x10 12 VG / ear, 6x 10 12 VG / ear, 7x 10 12 VG / ear, 8x 10 12 VG / ear, 9x 10 12 VG / ear, 1x 10 13 VG / ear, 2x 10 13 VG / ear, 3x 10 13 VG / ear, 4x 10 13 VG / ear, 5x 10 13 VG / ear, 6x 10 13 VG / ear, 7x 10 13 VG / ear, 8x 10 13 VG / ear, 9x10 13 VG / ear, 1x 10 14 VG / ear, 2x10 14 VG / ear, 3x 10 14 VG / ear, 4x 10 14 VG / ear, 5x 10 14 VG / ear, 6x 1014 VG / ear, 7x 10 14 VG / ear, 8x 10 14 VG / ear, 9x 10 14 VG / ear, 1x 10 15 VG / ear or 2x 10 15 VG / ear) was administered to the subjects.

[0167] The compositions described herein are administered in an amount sufficient to improve or restore (e.g., rescue) hearing, inhibit or slow the progression of hearing loss (e.g., sensorineural hearing loss), reduce tinnitus (e.g., in subjects with Meniere's disease), reduce vestibular dysfunction, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), treat bilateral vestibular lesions, treat oscillopsia, inhibit or slow the progression of vestibular dysfunction, reduce the feeling of fullness in the ear (e.g., in subjects with Meniere's disease), increase or promote vestibular hair cell regeneration, increase or induce hair cell maturation (e.g., maturation of regenerated vestibular hair cells), or increase or induce expression of an expression product in cells expressing SLC26A4 (e.g., interdental cells, spiral ridge cells, root cells, or vestibular supporting cells). Hearing can be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochlear oscopy (ECOG), and otoacoustic emissions) and can be improved 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 audiometric results obtained before treatment. In some embodiments, the composition is administered in an amount sufficient to improve the subject's ability to understand conversation. The compositions described herein can also be administered in an amount sufficient to delay or prevent the development of sensorineural hearing loss or deafness (e.g., in subjects who carry a SLC26A4 mutation but do not exhibit hearing loss at the time of treatment, or in subjects who exhibit mild to moderate hearing loss at the time of treatment). Vestibular function can be evaluated using standard tests of balance and vertigo (e.g., eye movement tests (e.g., ENG or VNG), VOR tests (e.g., head impact test (Ha-Ke test, such as VHIT) or caloric reflex test), posturography, swivel chair test, ECOG, VEMP, and specialist clinical balance tests) and can improve by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to measurements obtained before treatment. The compositions described herein can also be administered in an amount sufficient to slow or prevent the development or progression of vestibular dysfunction (e.g., in subjects carrying an SLC26A4 mutation associated with vestibular dysfunction, or in subjects who have been exposed to risk factors associated with vestibular dysfunction (e.g., ototoxic medications, head trauma or disease or infection) but do not exhibit vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), or in subjects exhibiting mild to moderate vestibular dysfunction).Expression of a protein encoded by a transgene operably linked to the SLC26A4 promoter and / or enhancer in a nucleic acid vector administered to a subject or administered to a cell can be evaluated using immunohistochemistry, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detecting protein or mRNA, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to expression prior to administration of a composition described herein. Vestibular hair cell regeneration can be assessed indirectly based on vestibular function tests and can be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to vestibular hair cell regeneration before administration of a composition described herein or compared to an untreated subject. The compositions and methods described herein can also reduce toxicity associated with administration of a nucleic acid vector compared to toxicity observed after administration of a nucleic acid vector that does not contain a SLC26A4 promoter and / or enhancer described herein (e.g., administration of a nucleic acid vector in which the same transgene is expressed using a ubiquitous promoter and / or without an SLC26A4 enhancer described herein). These effects can occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks or more after administration of a composition described herein. Depending on the dosage and route of administration for treatment, the patient may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer after administration of the composition. Depending on the results of the evaluation, the patient may receive additional treatment.

[0168] Reagent test kit

[0169] The compositions described herein can be provided in a kit for treating sensorineural hearing loss or vestibular dysfunction. The compositions can include one or more SLC26A4 enhancers described herein (e.g., polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) and / or SLC26A4 promoters (e.g., polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; or polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3; NO:17 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), a nucleic acid vector containing such a polynucleotide, or a nucleic acid vector containing an SLC26A4 enhancer and / or promoter described herein operably linked to a polynucleotide encoding an expression product (e.g., a transgene encoding a protein of interest, such as a protein that can be expressed in inner ear cells expressing SLC26A4 to treat hearing loss (e.g., Pan protein) or vestibular dysfunction (e.g., Pan protein or Atoh1)), or a transgene encoding an RNA molecule, such as an inhibitory RNA molecule). The nucleic acid vector can be packaged in an AAV viral capsid (e.g., AAV1, AAV2, AAV2quad (YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, AAV-DJ, AAV-DJ / 9, 7m8, or PHP.B). The kit may also include a package insert that instructs the user of the kit (such as a physician) to perform the methods described herein. The kit may optionally include a syringe or other device for administering the composition.

[0170] Example

[0171] The following examples are put forth so as to provide one of ordinary skill in the art with a description of how to use, prepare, and evaluate the compositions and methods described herein and are intended solely as exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0172] Example 1. Demonstration of AAV1-CMV.H2B.EGFP tropism across mouse inner ear tissues

[0173] To test the hypothesis that AAV1 transduces multiple cell types across the inner ear, including those that do not express Pan protein, AAV1 virus was injected into the posterior semicircular canal (intralabyrinthine (IL)) at a rate of 7.19 × 10 10 vg / ear was delivered to adult C57BL / 6 mice. The AAV1 virus was delivered to adult C57BL / 6 mice at a dose of 3.59 x 10 13 vg / mL of titer packaged with a plasmid containing an expression cassette encoding the cytomegalovirus (CMV) promoter driving expression of nuclear-targeted enhanced green fluorescent protein (EGFP fused to the H2B fragment of the histone 2b gene). Two weeks after virus injection, whole ears were fixed, decalcified, paraffin-embedded, and sections were imaged for EGFP using fluorescence microscopy. EGFP fluorescence was detected in the nuclei of many cell types across the inner ear, including but not limited to spiral ganglion neurons, Reissner's membrane, fiber cells of the lateral wall, spiral eminence cells, and root cells of the cochlea ( Figure 3A and 3B In the vestibule, EGFP fluorescence was detected in the nuclei of hair cells, supporting cells, mesenchymal cells, and roof cells of the otolith organ ( Figure 3A and 3C ).

[0174] Example 2. EGFP transgene expression is severely reduced in mouse inner ear tissue when under the control of the murine SLC26A4 core promoter

[0175] In vivo experiments were performed to evaluate whether expression of the AAV1 EGFP transgene could be restricted to SLC26A4-expressing cells of the cochlea and vestibule by replacing the ubiquitous CMV promoter with the murine SLC26A4 core promoter. To test this, a plasmid containing an expression cassette encoding the murine SLC26A4 core promoter (SEQ ID NO: 1) driving expression of nuclear-targeted EGFP without any enhancer sequence was expressed at 6.28 x 10 13 vg / mL was packaged into AAV1. The resulting AAV virus was transfected via the posterior semicircular canal (IL) at a titer of 6.28 x 10 10 vg / ear was topically administered to adult C57BL / 6 mice. Two weeks after virus injection, whole ears were fixed, decalcified, paraffin-embedded, and sections were imaged for EGFP using fluorescence microscopy. Figures 4A-4D EGFP fluorescence was not detected in any cell type in the cerebral canal (data not shown). Figure 4A and 4C The fluorescence observed in the wider view of FIG. 1 is not related to EGFP expression and is a result of naturally fluorescent structures in the mouse ear that can be observed when the display intensity is significantly increased, as here, in order to detect any EGFP-specific staining.

[0176] Example 3. Addition of an enhancer to the murine SLC26A4 core promoter results in increased EGFP transgene expression in target cell types within the cochlea and vestibule

[0177] To test whether the combination of the murine SLC26A4 core promoter and enhancer elements would increase EGFP expression in target cell types within both the cochlea and vestibule, a plasmid containing the protein encoding the E2 enhancer (SEQ ID NO: 2) fused directly to the 5′ end of the murine SLC26A4 core promoter (SEQ ID NO: 1) driving expression of nuclear-targeted EGFP was constructed (plasmid P1240; Figure 2 ) or the E6 enhancer (SEQ ID NO: 3) fused directly to the 5′ end of the murine SLC26A4 core promoter (SEQ ID NO: 1) (plasmid P1236; Figure 1 ) will be expressed as 7.12x10 13 and 6.68x 10 13 Each of these AAV1 vectors was separately packaged into AAV1 at a titer of 7.12×10 10 and 6.68x 10 10 vg / ear were topically administered to different adult C57BL / 6 mice. Two weeks after virus injection, whole ears were fixed, decalcified, paraffin-embedded, and sections were imaged for EGFP using a fluorescence microscope. In ears treated with AAV1 containing the E2 enhancer, EGFP was expressed in interdental cells (ID), spiral protuberance cells (SP), and root cells (RC) of the cochlea ( Figure 5A-5G ) and supporting cells of the vestibular otolith organs ( Figure 5E and 5H ). In ears treated with AAV1 containing the E6 enhancer, EGFP fluorescence was detected in the nuclei of the spiral protuberance cells (SP) and the root cells (RC) of the cochlea ( Figure 6A-6B Weak EGFP fluorescence signals were detected in the supporting cells (SC) of the vestibular otolith organ ( Fig. 6A and 6C ).

[0178] Example 4. The presence of multiple enhancers in the AAV1 vector increases SLC26A4 promoter-driven GFP expression in mouse cochlear lateral wall explants without affecting cell specificity

[0179] The lateral wall of the cochlea was excised from 6-8 week old male C57BL / 6J mice (000664, The Jackson Laboratory) and used for incubation with AAV1 viral vectors derived from transgenic plasmids containing a nuclear-targeted H2B-EGFP fusion transgene driven by various promoters (CMV and various SLC26A4 promoters described herein, without enhancer), and AAV1 vectors containing a murine core SLC26A4 promoter (SEQ ID NO: 1) and both the murine E2 enhancer (SEQ ID NO: 2) and the murine E6 enhancer (SEQ ID NO: 3) (P1670; Figure 8 ); or by the murine minimal SLC26A4 promoter (SEQ ID NO: 17) and both the murine E2 enhancer and the murine E6 enhancer (P1669; Figure 7 )-driven H2B-EGFP fusion transgene AAV1 vector. 2 After euthanasia, the temporal bones were harvested and the cochlear lateral walls were dissected in ice-cold DMEM / F-12 solution (11039021, Gibco) and cultured in DMEM / F-12, GlutaMax medium (10565018, Gibco) supplemented with 10% FBS (F4135, Sigma) and 10 μg / ml ciprofloxacin (AC456880050, Fisher Scientific) in glass bottom culture dishes (10810-054, Matsunami Glass). AAV was added to the culture medium in 250 μl of culture medium and placed in the culture medium for 3 days, and then washed with 2 ml of fresh culture medium prepared as described above. The cultured lateral walls were then kept in culture for another 2 days (a total of 5 days of culture after adding AAV to the culture medium).

[0180] At the end of the incubation, the samples were fixed with fresh 4% formaldehyde in 1X PBS for 1 hour at room temperature (RT) and rinsed 3 times with 1X PBS for 5 minutes each. The tissue was blocked with 10% standard donkey serum, 0.5% TritonX-100 in PBS at pH 7.4 for 1 hour at room temperature, and then incubated overnight at 4°C with a primary antibody against pan protein (BiCell Scientific 20501) diluted 1:100 with 0.5% TritonX-100 in 1X PBS. The next day, after washing with PBS (3 times, 5 minutes), the tissue was incubated with a secondary antibody (1:500; Invitrogen A10042: donkey (host), rabbit IgG (target species) coupled to Alexa Fluor568) at room temperature for 2 hours. After secondary antibody incubation, tissues were washed with PBS (3x, 5 min) and then mounted in Slowfade Diamond anti-fading mounting medium (DAKO) (ThermoFisher Molecular probes, s36963).

[0181] After fixation, the sidewalls were imaged using a Zeiss LSM 880 confocal microscope on the 488 (EGFP) and 568 (pan protein) channels. The laser power and gain were set to achieve the highest EGFP signal without saturating the detector. After establishing the imaging settings, all groups were imaged in one study with the same laser power and gain to allow comparison between groups.

[0182] from Fig. 9A As can be seen in Figure 3, most of the cells expressing SLC26A4 were present in the spiral ridges and tight junctions of the stria vascularis. AAV expressing EGFP under the control of the ubiquitous CMV promoter produced EGFP expression throughout the lateral wall that was not limited to the SLC26A4 expressing region, demonstrating the ubiquitous presence of AAV1 tropism in the lateral wall with this promoter ( Fig. 9B Replacing the CMV promoter with the murine core or minimal promoter, in combination with the E2 and / or E6 enhancers disclosed herein, results in EGFP expression being restricted primarily to cells that also express Pan protein, with little or no EGFP expression in other cells of the lateral wall ( Figures 10A-10C ).

[0183] Inclusion of the murine E6 and E2SLC26A4 enhancer sequences fused to the 5' end of the minimal or core murine promoter in an AAV vector expressing EGFP results in expression of EGFP without loss of specificity for cells expressing SLC26A4 ( Fig. 10A and 10B Interestingly, when both the mouse E2 and mouse E6 enhancers are combined with the mouse minimal promoter ( Fig. 10A ) or mouse core promoter ( Fig. 10B ) were coupled, both resulted in specific expression of EGFP, which was stronger than the case where only the mouse E2 enhancer was coupled to the mouse core promoter ( Fig. 10C ).

[0184] Example 5. In vivo administration of AAV1 vectors containing multiple enhancers increases SLC26A4 promoter-driven GFP expression in mouse cochlea

[0185] As described in Example 3, to test whether the combination of E2 and E6 enhancers would further increase EGFP expression in vivo, a plasmid containing the gene encoding the 5′-HRP-1 promoter fused directly to the murine SLC26A4 core promoter (SEQ ID NO: 1) (plasmid P1670; Figure 8 ) or the murine SLC26A4 minimal promoter (SEQ ID NO: 17) (plasmid P1669; Figure 7 ) directly fused to the 5' end of the E6 enhancer (SEQ ID NO: 3) and the 5' end of the E2 enhancer (SEQ ID NO: 2) at 4.41 x 10 13 (Batch 1), 2.70x 10 13 (Batch 2) and 4.60x 10 13 vg / mL were packaged into AAV1.

[0186] Batch 2 of the AAV1 vector with the minimal SLC26A4 promoter containing expression cassettes encoding the E2 enhancer (SEQ ID NO: 2) and the E6 enhancer (SEQ ID NO: 3) was injected via the posterior semicircular canal (IL) at 1.5 x 10 10 Doses of gc / ear were topically applied bilaterally to newborn Pan protein knockout mice at the age of P1-P3. These Pan protein KO mice were generated by CRISPR / Cas9-mediated deletion of exons 3-5 of the SLC26A4 gene on mouse chromosome 12 in mice with a C57BL / 6 background. At the age of P21, the whole ears were fixed, decalcified and paraffin-embedded, and the sections were imaged for EGFP using fluorescence microscopy. The sections were additionally stained for Kcnj10 (Abnova H00003766-M01) to visualize the cochlear structures. In ears treated with AAV1 containing the E2 and E6 enhancers coupled to a minimal promoter, EGFP fluorescence was detected in the nuclei of interdental cells (ID), spiral protuberance cells (SP), and root cells (RC) of the cochlea ( Fig.11 ).

[0187] Example 6. In vivo administration of AAV1 vectors containing multiple SLC26A4 enhancers and the SLC26A4 promoter induces GFP expression in SLC26A4-expressing cells in the cochlea of ​​non-human primates

[0188] A plasmid (plasmid P1669) containing an EGFP expression cassette encoding the E2 enhancer (SEQ ID NO:2) fused directly to the 5' end of the E6 enhancer (SEQ ID NO:3) fused directly to the 5' end of the murine SLC26A4 minimal promoter (SEQ ID NO:17) was packaged into AAV. The virus was topically administered to 2-4 year old cynomolgus monkeys (Macaca fascicularis). Administration was performed bilaterally by first creating fenestrations of the PSCC to allow fluid outflow, followed by delivery of 60 μL / ear of virus through the round window membrane. After 2 weeks of life, the animals were perfused, the entire ear was collected, decalcified, paraffin embedded, and sections were imaged for EGFP using fluorescence microscopy. In ears treated with AAV1 containing the E2 and E6 enhancers coupled to a minimal promoter, EGFP fluorescence was detected in the nuclei of the spiral protuberance cells (SP), the root cells (RC) of the cochlea, and the outer sulcus cells ( Fig.12 ).

[0189] Example 7. Administering a composition comprising a nucleic acid vector containing a SLC26A4 enhancer and a SLC26A4 promoter to a subject with sensorineural hearing loss

[0190] According to the methods disclosed herein, a physician skilled in the art can treat a patient, such as a human patient, with hearing loss (e.g., pan protein-related hearing loss, such as DFNB4 or Pendrede syndrome), thereby improving or restoring hearing. To this end, a physician skilled in the art can administer to a human patient a composition containing an AAV vector (e.g., AAV1, AAV2, AAV2quad (YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB or PHP.S vector) containing at least one SLC26A4 enhancer described herein (e.g., with SEQ ID NO: 2 and / or SEQ ID NO: 3). NO:3), the SLC26A4 enhancer operably linked to a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) operably linked to a polynucleotide encoding an expression product (e.g., a wild-type version of pan protein, such as a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5); or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; NO:17 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) polynucleotides. Compositions containing AAV vectors can be administered to a patient, for example, by topical administration to the inner ear (e.g., injection into the perilymph or endolymph or through the round window membrane) to treat sensorineural hearing loss.

[0191] After the composition is applied to the patient, a person skilled in the art can monitor the improvement of the patient's response to the therapy by a variety of methods. For example, a physician can monitor the patient's hearing by performing standard tests such as audiometry, ABR, electrocochlear osmography (ECOG), and otoacoustic emissions after the composition is applied. Compared with the hearing test results before the application of the composition, it is found that the patient shows an improvement in hearing in one or more tests after the application of the composition, which indicates that the patient has a good response to the treatment. Subsequent doses can be determined and applied as needed.

[0192] Example 8. Administering a composition comprising a nucleic acid vector containing a SLC26A4 enhancer and a SLC26A4 promoter to a subject with vestibular dysfunction

[0193] According to the methods disclosed herein, a physician skilled in the art can treat a patient (such as a human patient) suffering from vestibular dysfunction (e.g., vestibular dysfunction associated with hair cell loss, such as age-related vestibular dysfunction or ototoxic drug-induced vestibular dysfunction) to improve or restore vestibular function. To this end, a physician skilled in the art can administer to a human patient a composition containing an AAV vector (e.g., AAV1, AAV2, AAV2quad (YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB or PHP.S vector) containing at least one SLC26A4 enhancer described herein (e.g., with SEQ ID NO: 2 and / or SEQ ID NO: 3). NO:3), the SLC26A4 enhancer operably linked to a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a wild-type form of Atoh1, such as a polynucleotide encoding SEQ ID NO:8 or SEQ ID NO:10); or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; NO:17 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity)). A composition containing an AAV vector can be administered to a patient, for example, by topical administration to the inner ear (e.g., injection into the perilymph or endolymph, through the round window membrane, or injection into the semicircular canal) to treat vestibular dysfunction.

[0194] After the composition is administered to the patient, a skilled practitioner in the art can monitor the expression of the therapeutic protein encoded by the transgene, as well as the improvement of the patient in response to the therapy, by a variety of methods. For example, a doctor can monitor the vestibular function of the patient by performing standard tests such as electronystagmography, video nystagmography, VOR test (e.g., head impulse test (Halmagyi–Curthoys test, such as VHIT) or caloric reflex test), rotation test, vestibular evoked myogenic potential, or computerized dynamic posturography. Compared to the test results obtained before the administration of the composition, it is found that the patient exhibits an improvement in vestibular function in one or more tests after the administration of the composition, which indicates that the patient has a good response to the treatment. Subsequent doses can be determined and administered as needed.

[0195] Example 9. Administering a composition comprising a nucleic acid vector containing a SLC26A4 enhancer and a SLC26A4 promoter to a subject suffering from pre-Meniere's disease

[0196] According to the methods disclosed herein, a physician skilled in the art can treat a patient (e.g., a human patient) with Meniere's disease to reduce vertigo, improve hearing, reduce tinnitus, or reduce ear fullness. To this end, a physician skilled in the art can administer to a human patient a composition containing an AAV vector (e.g., AAV1, AAV2, AAV2quad (YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vector) containing at least one SLC26A4 enhancer described herein (e.g., with SEQ ID NO: 2 and / or SEQ ID NO: 3). NO:3), the SLC26A4 enhancer operably linked to a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) operably linked to a polynucleotide encoding an expression product (e.g., a wild-type version of pan protein, such as a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5); or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1; NO:17 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity)). A composition containing an AAV vector can be administered to a patient, for example, by topical administration to the inner ear (e.g., injection into the perilymph or endolymph, through the round window membrane, or injection into the semicircular canal) to treat Meniere's disease.

[0197] After the composition is administered to the patient, a skilled practitioner in the art can monitor the expression of the therapeutic protein encoded by the transgene, and the improvement of the patient in response to the therapy by a variety of methods. For example, the physician can monitor the vestibular function of the patient by performing standard tests after administering the composition, such as electronystagmography, video nystagmography, VOR test (e.g., head impulse test (Halmagyi–Curthoys test, e.g., VHIT) or caloric reflex test), rotation test, vestibular evoked myogenic potential, or computerized dynamic posturography; and can monitor the patient's hearing by performing standard tests such as audiometry, ABR, electrocochlear mapping (ECOG), and otoacoustic emissions. The physician can also rely on the patient's report on vertigo, tinnitus, and ear fullness. It is found that after administering the composition, the patient shows improved vestibular function or hearing in one or more tests or reports reduced vertigo, tinnitus, or reduced ear fullness compared to the test results obtained before administering the composition, indicating that the patient has a good response to treatment. Subsequent doses can be determined and administered as needed.

[0198] Exemplary embodiments of the invention are described in the following enumerated paragraphs.

[0199] E1. A polynucleotide comprising an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3, wherein the enhancer is operably linked to a promoter, wherein the enhancer in the polynucleotide is less than 3 kilobases (kb) from the promoter. 3kb) (e.g., about 3.0kb, 2.75kb, 2.5kb, 2.25kb, 2.0kb, 1.75kb, 1.5kb, 1.25kb, 1.0kb, 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases, 100 bases, 50 bases or less).

[0200] E2. The polynucleotide of E1, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 2 kb.

[0201] E3. The polynucleotide of E2, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 1 kb.

[0202] E4. The polynucleotide of E3, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 0.5 kb.

[0203] E5. The polynucleotide of E4, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 100 bases.

[0204] E6. The polynucleotide of any one of E1-E5, wherein the enhancer is located 5' to the promoter in the polynucleotide.

[0205] E7. The polynucleotide of any one of E1-E5, wherein the enhancer is located 3' to the promoter in the polynucleotide.

[0206] E8. A polynucleotide as described in any of E1-E7, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0207] E9. The polynucleotide of E8, wherein the enhancer has the sequence of SEQ ID NO: 2.

[0208] E10. A polynucleotide as described in any of E1-E7, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0209] E11. The polynucleotide of E10, wherein the enhancer has the sequence of SEQ ID NO: 3.

[0210] E12. The polynucleotide of any one of E1-E11, wherein the enhancer is fused directly to the promoter.

[0211] E13. A polynucleotide as described in any of E1-E11, wherein the enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleic acids).

[0212] E14. The polynucleotide of any one of E1-E13, wherein the promoter is a minimal promoter, a core promoter or a constitutive promoter.

[0213] E15. A polynucleotide as described in E14, wherein the promoter is a CAG promoter, a CBA promoter, a smCBA promoter, a CASI promoter, a dihydrofolate reductase (DHFR) promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, a HSV promoter or a SV40 promoter.

[0214] E16. A polynucleotide as described in E15, wherein the promoter is a minimal β-globin promoter, a CMVmini promoter, a minCMV promoter, a CMV-TATA+INR promoter, a min CMV-T6 promoter, a minimal HSV ICP0 promoter, a truncated HSV ICP0 promoter or a SV40 minimal promoter.

[0215] E17. The polynucleotide of any one of E1-E13, wherein the promoter is a minimal promoter.

[0216] E18. The polynucleotide of any one of E1-E13, wherein the promoter is a mammalian SLC26A4 promoter.

[0217] E19. The polynucleotide of E18, wherein the SLC26A4 promoter is a human or mouse SLC26A4 promoter.

[0218] E20. The polynucleotide of E19, wherein the SLC26A4 promoter has at least 85% sequence identity to SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0219] E21. The polynucleotide of E20, wherein the SLC26A4 promoter has a sequence of SEQ ID NO: 1.

[0220] E22. The polynucleotide of E19, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 12-16.

[0221] E23. The polynucleotide of E22, wherein the SLC26A4 promoter has the sequence of any one of SEQ ID NOs: 12-16.

[0222] E24. A polynucleotide as described in E19, wherein the SLC26A4 promoter has at least 85% sequence identity to SEQ ID NO:17 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0223] E25. The polynucleotide of E24, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:17.

[0224] E26. A polynucleotide as described in any one of E1-E25, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0225] E27. The polynucleotide of E26, wherein the expression product is a heterologous expression product.

[0226] E28. The polynucleotide of E26, wherein the expression product is an expression product endogenously expressed in a cell expressing SLC26A4.

[0227] E29. The polynucleotide of E28, wherein the expression product is an expression product endogenously expressed in inner ear cells expressing SLC26A4.

[0228] E30. The polynucleotide of E29, wherein the expression product is an expression product that is endogenously expressed in interdental cells, spiral eminence cells, cochlear root cells and / or vestibular supporting cells (eg, expressed in at least one of these cell types).

[0229] E31. The polynucleotide of any one of E28-E30, wherein the expression product is pan protein (eg, mammalian pan protein).

[0230] E32. The polynucleotide of E31, wherein the pan protein (mammalian pan protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0231] E33. A polynucleotide as described in any of E31 or E32, wherein the pan protein (mammalian pan protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:4 or SEQ ID NO:5.

[0232] E34. The polynucleotide of E33, wherein the pan protein (mammalian pan protein) has the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0233] E35. The polynucleotide of any one of E27-E30, wherein the expression product is Atoh1 (eg, mammalian Atoh1).

[0234] E36. The polynucleotide of E35, wherein the Atoh1 (mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0235] E37. A polynucleotide as described in any of E35 or E36, wherein the Atoh1 (mammalian Atoh1 protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:8 or SEQ ID NO:10.

[0236] E38. The polynucleotide of E37, wherein the Atoh1 (mammalian Atoh1 protein) has the sequence of SEQ ID NO: 8 or SEQ ID NO: 10.

[0237] E39. A polynucleotide as described in E26 or E27, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease, such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN) or a guide RNA (gRNA)), or a microRNA.

[0238] E40. A polynucleotide as described in any of E1-E39, wherein the polynucleotide comprises an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0239] E41. The polynucleotide of E40, wherein the polynucleotide comprises an enhancer having the sequence of SEQ ID NO: 2 and an enhancer having the sequence of SEQ ID NO: 3.

[0240] E42. A polynucleotide as described in E41, wherein the polynucleotide comprises, in 5' to 3' order, an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a SLC26A4 promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 17 (e.g., an enhancer having a sequence of SEQ ID NO: 2 is directly fused to an enhancer having a sequence of SEQ ID NO: 3 that is directly fused to a SLC26A4 promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 17).

[0241] E43. The polynucleotide of E42, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:17.

[0242] E44. The polynucleotide of E42 or E43, wherein the polynucleotide comprises the sequence of SEQ ID NO:18.

[0243] E45. A polynucleotide as described in any of E1-E41, wherein the polynucleotide comprises two or more copies of an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or two or more copies of an enhancer (e.g., two or more copies of one or two enhancers) having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0244] E46. The polynucleotide of E45, wherein the polynucleotide comprises two or more copies of an enhancer having the sequence of SEQ ID NO: 2 and / or SEQ ID NO: 3 (eg, two or more copies of one or two enhancers).

[0245] E47. A nucleic acid vector comprising the polynucleotide described in any one of E1-E46.

[0246] E48. A nucleic acid vector comprising a polynucleotide comprising an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0247] E49. A nucleic acid vector as described in E48, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0248] E50. The nucleic acid vector as described in E49, wherein the enhancer has the sequence of SEQ ID NO:2.

[0249] E51. A nucleic acid vector as described in E48, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0250] E52. The nucleic acid vector of E51, wherein the enhancer has a sequence of SEQ ID NO: 3.

[0251] E53. A nucleic acid vector as described in any one of E48-E52, wherein the enhancer is operably linked to a promoter.

[0252] E54. The nucleic acid vector of E53, wherein the enhancer is located 5' to the promoter.

[0253] E55. The nucleic acid vector of E53, wherein the enhancer is located 3' to the promoter.

[0254] E56. A nucleic acid vector as described in any one of E53-E55, wherein the enhancer is directly fused to the promoter.

[0255] E57. A nucleic acid vector as described in any one of E53-E55, wherein the enhancer is linked to the promoter via a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleic acids).

[0256] E58. A nucleic acid vector as described in any one of E53-E57, wherein the promoter is a minimal promoter, a core promoter or a constitutive promoter.

[0257] E59. A nucleic acid vector as described in E58, wherein the promoter is a CAG promoter, a CBA promoter, a smCBA promoter, a CASI promoter, a dihydrofolate reductase (DHFR) promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, a HSV promoter or a SV40 promoter.

[0258] E60. The nucleic acid vector as described in E59, wherein the promoter is a minimal β-globin promoter, a CMVmini promoter, a minCMV promoter, a CMV-TATA+INR promoter, a min CMV-T6 promoter, a minimal HSV ICP0 promoter, a truncated HSV ICP0 promoter or a SV40 minimal promoter.

[0259] E61. The nucleic acid vector of E58, wherein the promoter is a mammalian promoter.

[0260] E62. The nucleic acid vector of any one of E53-E57, wherein the promoter is a mammalian SLC26A4 promoter.

[0261] E63. The nucleic acid vector of E62, wherein the SLC26A4 promoter is a human or mouse SLC26A4 promoter.

[0262] E64. The nucleic acid vector of E63, wherein the SLC26A4 promoter has at least 85% sequence identity to SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0263] E65. The nucleic acid vector of E64, wherein the SLC26A4 promoter has a sequence of SEQ ID NO: 1.

[0264] E66. The nucleic acid vector of E63, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 12-16.

[0265] E67. The nucleic acid vector of E66, wherein the SLC26A4 promoter has the sequence of any one of SEQ ID NOs: 12-16.

[0266] E68. A nucleic acid vector as described in E63, wherein the SLC26A4 promoter has at least 85% sequence identity to SEQ ID NO:17 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0267] E69. The nucleic acid vector of E68, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:17.

[0268] E70. A nucleic acid vector as described in any one of E53-E69, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0269] E71. The nucleic acid vector of E70, wherein the expression product is a heterologous expression product.

[0270] E72. The nucleic acid vector of E70, wherein the expression product is an expression product endogenously expressed in a cell expressing SLC26A4.

[0271] E73. The nucleic acid vector of E72, wherein the expression product is an expression product endogenously expressed in inner ear cells expressing SLC26A4.

[0272] E74. A nucleic acid vector as described in E73, wherein the expression product is an expression product that is endogenously expressed in interdental cells, spiral ridge cells, cochlear root cells and / or vestibular supporting cells (e.g., expressed in at least one of these cell types).

[0273] E75. The nucleic acid vector of any one of E72-E74, wherein the expression product is pan protein (eg, mammalian pan protein).

[0274] E76. The nucleic acid vector of E75, wherein the pan protein (mammalian pan protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0275] E77. A nucleic acid vector as described in E75 or E76, wherein the pan protein (mammalian pan protein) has at least 85% sequence identity with SEQ ID NO:4 or SEQ ID NO:5 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0276] E78. The nucleic acid vector of E77, wherein the pan protein (mammalian pan protein) has the sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0277] E79. The nucleic acid vector of any one of E71-E74, wherein the expression product is Atoh1 (eg, mammalian Atoh1 protein).

[0278] E80. The nucleic acid vector of E79, wherein the Atoh1 (mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0279] E81. A nucleic acid vector as described in any of E79 or E80, wherein the Atoh1 (mammalian Atoh1 protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:8 or SEQ ID NO:10.

[0280] E82. The nucleic acid vector of E81, wherein the Atoh1 (mammalian Atoh1 protein) has the sequence of SEQ ID NO: 8 or SEQ ID NO: 10.

[0281] E83. A nucleic acid vector as described in E70 or E71, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease, such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN) or a guide RNA (gRNA)), or a microRNA.

[0282] E84. A nucleic acid vector as described in any of E48 to E83, wherein the polynucleotide comprises an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0283] E85. The nucleic acid vector of E84, wherein the polynucleotide comprises an enhancer having the sequence of SEQ ID NO: 2 and an enhancer having the sequence of SEQ ID NO: 3.

[0284] E86. A nucleic acid vector as described in E85, wherein the polynucleotide comprises, in 5' to 3' order, an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a SLC26A4 promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 17 (for example, the enhancer having a sequence of SEQ ID NO: 2 is directly fused to an enhancer having a sequence of SEQ ID NO: 3 that is directly fused to a SLC26A4 promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 17).

[0285] E87. The nucleic acid vector of E86, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:17.

[0286] E88. The nucleic acid vector of E86 or E87, wherein the polynucleotide comprises the sequence of SEQ ID NO:18.

[0287] E89. A nucleic acid vector as described in any of E48-E85, wherein the polynucleotide comprises two or more copies of an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or two or more copies of an enhancer (e.g., two or more copies of one or two enhancers) having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0288] E90. The nucleic acid vector of E89, wherein the polynucleotide comprises two or more copies of an enhancer having the sequence of SEQ ID NO: 2 and / or SEQ ID NO: 3 (eg, two or more copies of one or two enhancers).

[0289] E91. A nucleic acid vector comprising a polynucleotide comprising a SLC26A4 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0290] E92. The nucleic acid vector of E91, wherein the SLC26A4 promoter has a sequence of SEQ ID NO: 1.

[0291] E93. A nucleic acid vector comprising a polynucleotide comprising a SLC26A4 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:17.

[0292] E94. The nucleic acid vector of E93, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:17.

[0293] E95. A nucleic acid vector as described in any one of E91-E94, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0294] E96. The nucleic acid vector of E95, wherein the expression product is a heterologous expression product.

[0295] E97. The nucleic acid vector of E95, wherein the expression product is an expression product endogenously expressed in a cell expressing SLC26A4.

[0296] E98. The nucleic acid vector of E97, wherein the expression product is an expression product endogenously expressed in inner ear cells expressing SLC26A4.

[0297] E99. A nucleic acid vector as described in E98, wherein the expression product is an expression product that is endogenously expressed in interdental cells, spiral ridge cells, cochlear root cells and / or vestibular supporting cells (e.g., expressed in at least one of these cell types).

[0298] E100. The nucleic acid vector of any one of E97-E99, wherein the expression product is pan protein (eg, mammalian pan protein).

[0299] E101. The nucleic acid vector of E100, wherein the pan protein (mammalian pan protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0300] E102. A nucleic acid vector as described in E100 or E101, wherein the pan protein (mammalian pan protein) has at least 85% sequence identity with SEQ ID NO:4 or SEQ ID NO:5 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0301] E103. The nucleic acid vector of E102, wherein the pan protein (mammalian pan protein) has the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0302] E104. The nucleic acid vector of any one of E95-E99, wherein the expression product is Atoh1 (eg, mammalian Atoh1 protein).

[0303] E105. The nucleic acid vector of E104, wherein the Atoh1 (mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0304] E106. A nucleic acid vector as described in E104 or E105, wherein the Atoh1 (mammalian Atoh1 protein) has at least 85% sequence identity with SEQ ID NO:8 or SEQ ID NO:10 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0305] E107. The nucleic acid vector of E106, wherein the Atoh1 (encoded mammalian Atoh1) has the sequence of SEQ ID NO:8 or SEQ ID NO:10.

[0306] E108. A nucleic acid vector as described in E95 or E96, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease, such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN) or a guide RNA (gRNA)), or a microRNA.

[0307] E109. The nucleic acid vector of any one of E91-E108, wherein the promoter is operably linked to an enhancer.

[0308] E110. A nucleic acid vector as described in E109, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) or has at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0309] E111. A nucleic acid vector as described in E110, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0310] E112. The nucleic acid vector as described in E111, wherein the enhancer has the sequence of SEQ IDNO:2.

[0311] E113. A nucleic acid vector as described in E110, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0312] E114. The nucleic acid vector as described in E113, wherein the enhancer has the sequence of SEQ ID NO:3.

[0313] E115. The nucleic acid vector of any one of E109-E114, wherein the enhancer is located 5' of the promoter.

[0314] E116. The nucleic acid vector of any one of E109-E114, wherein the enhancer is located 3' to the promoter.

[0315] E117. The nucleic acid vector of any one of E109-E116, wherein the enhancer is directly fused to the promoter.

[0316] E118. A nucleic acid vector as described in any one of E109-E116, wherein the enhancer is linked to the promoter through a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleic acids).

[0317] E119. A nucleic acid vector as described in any one of E109-E118, wherein the polynucleotide comprises an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0318] E120. The nucleic acid vector of E119, wherein the polynucleotide comprises an enhancer having the sequence of SEQ ID NO: 2 and an enhancer having the sequence of SEQ ID NO: 3.

[0319] E121. A nucleic acid vector as described in E120, wherein the polynucleotide comprises, in 5' to 3' order, an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a SLC26A4 promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 17 (e.g., an enhancer having a sequence of SEQ ID NO: 2 is directly fused to an enhancer having a sequence of SEQ ID NO: 3 that is directly fused to a SLC26A4 promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO: 17).

[0320] E122. The nucleic acid vector of E121, wherein the SLC26A4 promoter has a sequence of SEQ ID NO:17.

[0321] E123. The nucleic acid vector of E121 or E122, wherein the polynucleotide comprises the sequence of SEQ ID NO:18.

[0322] E124. A nucleic acid vector as described in any of E109-E120, wherein the polynucleotide comprises two or more copies of an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or two or more copies of an enhancer (e.g., two or more copies of one or two enhancers) having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0323] E125. The nucleic acid vector of E1124, wherein the polynucleotide comprises two or more copies of an enhancer having the sequence of SEQ ID NO: 2 and / or SEQ ID NO: 3 (eg, two or more copies of one or two enhancers).

[0324] E126. A polynucleotide comprising a SLC26A4 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1, wherein the SLC26A4 promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0325] E127. The polynucleotide of E126, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 1.

[0326] E128. A polynucleotide comprising a SLC26A4 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17, wherein the SLC26A4 promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0327] E129. The polynucleotide of E128, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:17.

[0328] E130. The polynucleotide of any one of E126-E129, wherein the expression product is a heterologous expression product.

[0329] E131. The polynucleotide of any one of E126-E129, wherein the expression product is an expression product endogenously expressed in a cell expressing SLC26A4.

[0330] E132. The polynucleotide of E131, wherein the expression product is an expression product endogenously expressed in inner ear cells expressing SLC26A4.

[0331] E133. The polynucleotide of E132, wherein the expression product is an expression product that is endogenously expressed in interdental cells, spiral eminence cells, cochlear root cells and / or vestibular supporting cells (eg, expressed in at least one of these cell types).

[0332] E134. The polynucleotide of any one of E126-E129 and E131-E133, wherein the expression product is pan protein (eg, mammalian pan protein).

[0333] E135. The polynucleotide of E134, wherein the pan protein (mammalian pan protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0334] E136. A polynucleotide as described in E134 or E135, wherein the pan protein (mammalian pan protein) has at least 85% sequence identity with SEQ ID NO:4 or SEQ ID NO:5 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0335] E137. The polynucleotide of E136, wherein the pan protein (mammalian pan protein) has the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0336] E138. The polynucleotide of any one of E126-E133, wherein the expression product is Atoh1 (eg, a mammalian Atoh1 protein).

[0337] E139. The polynucleotide of E138, wherein the Atoh1 (mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0338] E140. A polynucleotide as described in E138 or E139, wherein the Atoh1 (mammalian Atoh1 protein) has at least 85% sequence identity with SEQ ID NO:8 or SEQ ID NO:10 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0339] E141. The polynucleotide of E140, wherein the Atoh1 (encoded mammalian Atoh1) has the sequence of SEQ ID NO:8 or SEQ ID NO:10.

[0340] E142. A polynucleotide as described in any one of E126-E133, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease, such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN) or a guide RNA (gRNA)), or a microRNA.

[0341] E143. The polynucleotide of any one of E126-E142, wherein the promoter is operably linked to an enhancer.

[0342] E144. The polynucleotide of E143, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) or has at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0343] E145. A polynucleotide as described in E144, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0344] E146. The polynucleotide of E145, wherein the enhancer has the sequence of SEQ ID NO:2.

[0345] E147. A polynucleotide as described in E144, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0346] E148. The polynucleotide of E147, wherein the enhancer has the sequence of SEQ ID NO:3.

[0347] E149. The polynucleotide of any one of E143-E148, wherein the enhancer is located 5' to the promoter.

[0348] E150. The polynucleotide of any one of E143-E148, wherein the enhancer is located 3' to the promoter.

[0349] E151. The polynucleotide of any one of E143-E150, wherein the enhancer is fused directly to the promoter.

[0350] E152. A polynucleotide as described in any of E143-E150, wherein the enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleic acids).

[0351] E153. A polynucleotide as described in any one of E143 to E152, wherein the polynucleotide comprises an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0352] E154. The polynucleotide of E153, wherein the polynucleotide comprises an enhancer having the sequence of SEQ ID NO: 2 and an enhancer having the sequence of SEQ ID NO: 3.

[0353] E155. A polynucleotide as described in E154, wherein the polynucleotide comprises, in 5' to 3' order, an enhancer having a sequence of SEQ ID NO:2, an enhancer having a sequence of SEQ ID NO:3, and a SLC26A4 promoter having a sequence of SEQ ID NO:1 or SEQ ID NO:17 (e.g., an enhancer having a sequence of SEQ ID NO:2 is directly fused to an enhancer having a sequence of SEQ ID NO:3 that is directly fused to a SLC26A4 promoter having a sequence of SEQ ID NO:1 or SEQ ID NO:17).

[0354] E156. The polynucleotide of E155, wherein the SLC26A4 promoter has a sequence of SEQ ID NO:17.

[0355] E157. The polynucleotide of E155 or E156, wherein the polynucleotide comprises the sequence of SEQ ID NO:18.

[0356] E158. A polynucleotide as described in any one of E143 to E154, wherein the polynucleotide comprises two or more copies of an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or two or more copies of an enhancer (e.g., two or more copies of one or two enhancers) having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0357] E159. The polynucleotide of E158, wherein the polynucleotide comprises two or more copies of an enhancer having the sequence of SEQ ID NO: 2 and / or SEQ ID NO: 3 (eg, two or more copies of one or two enhancers).

[0358] E160. A nucleic acid vector comprising the polynucleotide described in any one of E126-E159.

[0359] E161. The nucleic acid vector of any one of E47-E125 and E160, wherein the nucleic acid vector is a viral vector, a plasmid, a cosmid or an artificial chromosome.

[0360] E162. The nucleic acid vector of any one of E47-E125, E160 and E161, wherein the nucleic acid vector is a viral vector.

[0361] E163. The nucleic acid vector as described in E162, wherein the viral vector is an adeno-associated virus (AAV) viral vector, an adenoviral viral vector or a lentiviral viral vector.

[0362] E164. The nucleic acid vector as described in E163, wherein the viral vector is an AAV vector.

[0363] E165. A nucleic acid vector as described in E164, wherein the AAV vector has an AAV1, AAV2, AAV2quad (YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB or PHP.S capsid.

[0364] E166. A composition comprising the nucleic acid vector of any one of E47-E125 and E160-E165 and a pharmaceutically acceptable carrier, diluent or excipient.

[0365] E167. A cell comprising the polynucleotide of any one of E1-E46 and E126-E159 or the nucleic acid vector of any one of E47-E125 and E160-E165.

[0366] E168. The cell as described in E167, wherein the cell is a cell expressing SLC26A4.

[0367] E169. The cell of E168, wherein the cell is an inner ear cell expressing SLC26A4.

[0368] E170. The cell of any one of E167-E169, wherein the cell is a mammalian cell.

[0369] E171. The cell as described in E170, wherein the mammalian cell is a human cell.

[0370] E172. The cell of any one of E167-E171, wherein the cell is an interdental cell, a spiral eminence cell, a cochlear root cell, or a vestibular supporting cell.

[0371] E173. A method for expressing an expression product in a cell, the method comprising the step of contacting the cell with the nucleic acid vector described in any one of E47-E125 and E160-E165 or the composition described in E166.

[0372] E174. The method of E173, wherein said cells are inner ear cells.

[0373] E175. The method of E173 or E174, wherein the cell is a cell expressing SLC26A4.

[0374] E176. The cell of any one of E173-E175, wherein the cell is an inner ear cell expressing SLC26A4.

[0375] E177. The method of E176, wherein the inner ear cells expressing SLC26A4 are interdental cells, spiral eminence cells, cochlear root cells, or vestibular supporting cells.

[0376] E178. The method of any one of E173-E177, wherein the cell is a mammalian cell.

[0377] E179. The method of E178, wherein the mammalian cell is a human cell.

[0378] E180. The method of any one of E173-E179, wherein said contacting is performed in a subject (eg, in vivo).

[0379] E181. A method of treating a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss or deafness), the method comprising administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector described in any one of E47-E125 and E160-E165 or a composition described in E166.

[0380] E182. The method of E181, wherein the hearing loss is panin-related hearing loss.

[0381] E183. The method of E182, wherein the expression product is pan protein.

[0382] E184. The method of E182 or E183, wherein the pan protein-related hearing loss is hearing loss associated with Pendred syndrome or DFNB4.

[0383] E185. A method for treating hearing loss associated with Meniere's disease in a subject in need thereof, the method comprising the step of administering to the inner ear of the subject an effective amount of the nucleic acid vector of any one of E47-E125 and E160-E165 or the composition of E166.

[0384] E186. A method for treating tinnitus associated with Meniere's disease in a subject in need thereof, the method comprising the step of administering to the inner ear of the subject an effective amount of the nucleic acid vector of any one of E47-E125 and E160-E165 or the composition of E166.

[0385] E187. The method of E185 or E186, wherein the expression product is pan protein.

[0386] E188. A method for treating vestibular dysfunction associated with Meniere's disease in a subject in need thereof, the method comprising the step of administering to the inner ear of the subject an effective amount of the nucleic acid vector of any one of E47-E125 and E160-E165 or the composition of E166.

[0387] E189. The method of E188, wherein the expression product is pan protein or Atoh1.

[0388] E190. The method of E188 or E189, wherein the vestibular dysfunction is vertigo.

[0389] E191. A method of treating a subject having or at risk of developing vestibular dysfunction, the method comprising administering to the inner ear of the subject a therapeutically effective amount of the nucleic acid vector of any one of E47-E125 and E160-E165 or the composition of E166.

[0390] E192. The method of E191, wherein the vestibular dysfunction is panin-related vestibular dysfunction.

[0391] E193. The method of E192, wherein the expression product is pan protein.

[0392] E194. The method of E192 or E193, wherein the Pan protein-related vestibular dysfunction is vestibular dysfunction associated with Pendred syndrome or DFNB4.

[0393] E195. The method of E191, wherein the expression product is pan protein or Atoh1.

[0394] E196. A method for inducing or increasing the differentiation of vestibular supporting cells into vestibular hair cells, the method comprising the step of contacting the vestibular supporting cells with the nucleic acid vector described in any one of E47-E125 and E160-E165 or the composition described in E166, wherein the expression product is Atoh1.

[0395] E197. The method of E196, wherein the contacting is in vivo (eg, in a subject).

[0396] E198. The method of E197, wherein the subject suffers from or is at risk of developing vestibular dysfunction.

[0397] E199. A method for inducing or increasing vestibular hair cell regeneration in a subject in need thereof, the method comprising the step of administering to the inner ear of the subject an effective amount of a nucleic acid vector described in any one of E47-E125 and E160-E165 or a composition described in E166, wherein the expression product is Atoh1.

[0398] E200. The method of E199, wherein the subject suffers from or is at risk of developing vestibular dysfunction.

[0399] E201. A method for improving the function of a cell expressing SLC26A4, the method comprising the step of contacting the cell expressing SLC26A4 with the nucleic acid vector described in any one of E47-E125 and E160-E165 or the composition described in E166.

[0400] E202. The method of E201, wherein the contacting is performed in vivo (eg, in a subject).

[0401] E203. The method of E202, wherein the subject has or is at risk of developing hearing loss (eg, sensorineural hearing loss) or vestibular dysfunction.

[0402] E204. A method as described in any one of E191-E203, wherein the vestibular dysfunction is vertigo, dizziness, imbalance (e.g., loss of balance or balance disorder), oscillopsia, or bilateral vestibular lesions.

[0403] E205. The method of any one of E201-E204, wherein the vestibular dysfunction is associated with damage or loss of vestibular hair cells.

[0404] E206. A method as described in E205, wherein the damage or loss of the vestibular hair cells is related to: age (the vestibular dysfunction is age-related vestibular dysfunction), exposure to ototoxic (e.g., vestibular toxic) drugs (the vestibular dysfunction is ototoxic drug-induced vestibular dysfunction), disease or infection (the vestibular dysfunction is disease or infection-related vestibular dysfunction) or head trauma (the vestibular dysfunction is head trauma-related vestibular dysfunction).

[0405] E207. A method as described in E206, wherein the ototoxic drug is an aminoglycoside (aminoglycoside antibiotics, such as gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin and netilmicin), puromycin, an anti-tumor drug (e.g., a platinum-containing chemotherapeutic agent, such as cisplatin, carboplatin or oxaliplatin, or another chemotherapeutic agent, such as nitrogen mustard or vincristine), a loop diuretic (e.g., ethacrynic acid or furosemide), a salicylate or quinine.

[0406] E208. The method of any one of E181-E187, wherein the method further comprises evaluating the hearing of the subject before administering the nucleic acid vector or composition.

[0407] E209. The method of any one of E181-E187 and E208, wherein the method further comprises evaluating the hearing of the subject after administering the nucleic acid vector or composition.

[0408] E210. The method of any one of E188-E207, wherein the method further comprises evaluating the vestibular function of the subject before administering the nucleic acid vector or composition.

[0409] E211. The method of any one of E188-E207 and E210, wherein the method further comprises evaluating the vestibular function of the subject after administering the nucleic acid vector or composition.

[0410] E212. The method of any one of E173-E211, wherein the nucleic acid vector or composition is administered topically.

[0411] E213. The method of E212, wherein the nucleic acid vector or composition is administered to the inner ear.

[0412] E214. The method of E212, wherein the nucleic acid vector or composition is administered to the middle ear.

[0413] E215. The method of E212, wherein the nucleic acid vector or composition is administered transtympanically or intratympanically.

[0414] E216. The method of E212, wherein the nucleic acid vector or composition is administered into the perilymph.

[0415] E217. The method of E212, wherein the nucleic acid vector or composition is administered into the endolymph.

[0416] E218. The method of E212, wherein the nucleic acid vector or composition is administered to or through the oval window.

[0417] E219. The method of E212, wherein the nucleic acid vector or composition is administered to or through the round window.

[0418] E220. The method of E212, wherein the nucleic acid vector or composition is administered to the semicircular canal.

[0419] E221. A method as described in any of E173-E220, wherein the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, delay the development of hearing loss, slow the progression of hearing loss, improve hearing, increase or induce the expression of an expression product in a cell expressing SLC26A4, reduce tinnitus, improve vestibular function, reduce vertigo, improve balance, increase the number of vestibular hair cells, inhibit or slow the progression of vestibular dysfunction, reduce ear fullness, increase vestibular hair cell regeneration, induce or increase vestibular supporting cells to differentiate into vestibular hair cells, increase or induce hair cell maturation (e.g., maturation of regenerated hair cells), or improve vestibular supporting cell function.

[0420] E222. The method of any one of E173-E221, wherein the subject is a human subject.

[0421] E223. A kit comprising the polynucleotide of any one of E1-E46 or E126-E159, the nucleic acid vector of any one of E47-E125 or E160-E165, or the composition of E166.

[0422] Other Implementations

[0423] Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described manner for carrying out the invention will be apparent to those skilled in the art and are intended to be included within the scope of the invention. Other embodiments are within the scope of the claims.

Claims

1. A polynucleotide comprising an enhancer, wherein the enhancer has at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and the enhancer is operably linked to a promoter, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 3 kilobases (3kb).

2. The polynucleotide of claim 1, wherein the distance between the enhancer and the promoter is less than 500 bases.

3. The polynucleotide of claim 1, wherein the distance between the enhancer and the promoter is less than 100 bases.

4. The polynucleotide of claim 1, wherein the enhancer is fused directly to the promoter.

5. The polynucleotide of any one of claims 1 to 4, wherein the enhancer is located 5' to the promoter.

6. The polynucleotide of any one of claims 1 to 5, wherein the enhancer has a sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

7. The polynucleotide of any one of claims 1-6, wherein the promoter is a constitutive promoter, a core promoter or a minimal promoter.

8. The polynucleotide of any one of claims 1-6, wherein the promoter is a mammalian SLC26A4 promoter.

9. The polynucleotide of claim 8, wherein the promoter is the mouse or human SLC26A4 promoter.

10. The polynucleotide of claim 9, wherein the murine SLC26A4 promoter has at least 85% sequence identity to the sequence of SEQ ID NO:

1.

11. The polynucleotide of claim 10, wherein the murine SLC26A4 promoter has the sequence of SEQ ID NO:

1.

12. The polynucleotide of claim 9, wherein the murine SLC26A4 promoter has at least 85% sequence identity to the sequence of SEQ ID NO:

17.

13. The polynucleotide of claim 12, wherein the murine SLC26A4 promoter has the sequence of SEQ ID NO:

17.

14. The polynucleotide of any one of claims 1-13, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

15. The polynucleotide of claim 14, wherein the expression product is a heterologous expression product.

16. The polynucleotide of claim 14, wherein the expression product is an expression product endogenously expressed in inner ear cells expressing SLC26A4.

17. The polynucleotide of claim 16, wherein the inner ear cell expressing SLC26A4 is an interdental cell, a spiral eminence cell, a cochlear root cell, or a vestibular supporting cell.

18. The polynucleotide of claim 14, wherein the expression product is mammalian pan protein.

19. The polynucleotide of claim 18, wherein the mammalian Pan protein is a wild-type isoform that is endogenously expressed in the ear of a mammal.

20. The polynucleotide of claim 19, wherein the mammalian pan protein has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:

5.

21. The polynucleotide of any one of claims 1-20, wherein the polynucleotide comprises an enhancer having at least 85% sequence identity to SEQ ID NO:2 and an enhancer having at least 85% sequence identity to SEQ ID NO:

3. 22 . The polynucleotide of claim 21 , wherein the polynucleotide comprises, in 5′ to 3′ order, an enhancer having a sequence of SEQ ID NO: 2, an enhancer having a sequence of SEQ ID NO: 3, and a SLC26A4 promoter having a sequence of SEQ ID NO: 1 or SEQ ID NO:

17.

23. The polynucleotide of claim 22, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:

17.

24. A nucleic acid vector comprising the polynucleotide according to any one of claims 1 to 23.

25. The nucleic acid vector of claim 24, wherein the nucleic acid vector is a viral vector.

26. The nucleic acid vector of claim 25, wherein the viral vector is an adeno-associated viral vector.

27. The nucleic acid vector of any one of claims 24-26, wherein the expression product is wild-type mammalian pan protein.

28. The nucleic acid vector of any one of claims 24-26, wherein the expression product is a wild-type mammalian Atoh1 protein.

29. A composition comprising the nucleic acid vector of any one of claims 24-28 and a pharmaceutically acceptable carrier, diluent or excipient.

30. A method for expressing an expression product in an inner ear cell, the method comprising contacting the inner ear cell with the nucleic acid vector of any one of claims 24 to 28 or the composition of claim 29.

31. The method of claim 30, wherein the contacting is performed in a subject.

32. A method of treating a subject having or at risk of developing Panin-associated hearing loss, the method comprising the step of administering to the subject a therapeutically effective amount of the nucleic acid vector of claim 27.

33. The method of claim 32, wherein the Panin-related hearing loss is Pendred syndrome or DFNB4.

34. A method of treating hearing loss associated with Meniere's disease in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of the nucleic acid vector of claim 27.

35. A method of treating a subject having or at risk for developing Panin-associated vestibular dysfunction, the method comprising the step of administering to the subject a therapeutically effective amount of the nucleic acid vector of claim 27.

36. A method of treating vestibular dysfunction associated with Meniere's disease in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of the nucleic acid vector of claim 27.

37. A method of treating a subject suffering from or at risk of developing vestibular dysfunction associated with damage to or loss of vestibular hair cells, the method comprising the step of administering to the subject a therapeutically effective amount of the nucleic acid vector of claim 27 or 28.

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