SLC26A4 regulatory element and application thereof

Abstract

The present disclosure provides SL26A4 promoters and SLC26A4 enhancers, as well as vectors containing the same, that can be used to limit gene expression to cells expressing SLC26A4 and to increase gene expression in these cells, 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 format and is hereby incorporated by reference in its entirety. The XML copy was created on September 25, 2023, named 51124-101WO3_Sequence_Listing_9_25_23.xml, and is 73,879 bytes in size. Background of the Invention

[0003] Hearing loss is the most common human sensory deficit, affecting nearly 15% of school-age children and one-third of people aged 65 and older. In the United States, congenital hearing loss occurs in approximately one out of every 500 newborns 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-lingual or post-lingual non-syndromic hearing loss, or may be born with hearing impairment that progresses to profound deafness over time. Pendrin is expressed in the apical membranes of specialized epithelial cells in the inner ear (cochlea, vestibular labyrinth, and endolymphatic sac, as well as non-sensory epithelium of the saccule, utricle, and ampulla), thyroid (thyroid cells), kidney (type B intercalated cells of the renal collecting duct), airway, breast, salivary gland ducts, and liver. Within the inner ear, Pendrin regulates pH and fluid absorption by exchanging chloride and bicarbonate anions between the epithelial and endolymphatic fluid compartments. There is currently no curative therapy for this population. Therefore, there is a need for a therapeutic agent to restore and / or salvage the progression of hearing loss in patients with these mutations. Summary of the Invention

[0004] The present invention provides compositions and methods for promoting the expression of a gene of interest (such as a gene endogenously expressed in cells expressing SLC26A4) in a specific cell type. The present invention features an 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 the expression of the expression product in cells 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 pendrin-related hearing loss), Meniere's disease (e.g., hearing loss, tinnitus, or vestibular dysfunction associated with Meniere's disease) and / or vestibular dysfunction (e.g., pendrin-related vestibular dysfunction or vestibular dysfunction associated with vestibular hair cell damage or loss).

[0005] In a first aspect, the present invention provides a nucleic acid vector comprising an SLC26A4 promoter of the formula 5'-B-A-C-3', wherein: A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); B is absent or has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and C is absent or has at least 85% sequence identity with SEQ ID NO:3 or a portion thereof comprising at least the first 159 consecutive nucleotides of SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), wherein the SLC26A4 promoter is no longer than 1481 bases.

[0006] In another aspect, the present invention provides a polynucleotide comprising an SLC26A4 promoter of the formula 5'-B-A-C-3' operably linked to a polynucleotide that can be transcribed to produce an expression product, wherein: A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); B is absent or has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides from the 3'-end of SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and C is absent or has at least 85% sequence identity with SEQ ID NO:3 or a portion thereof comprising at least the first 159 consecutive nucleotides of SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), wherein the SLC26A4 promoter is not longer than 1481 bases.

[0007] In another aspect, the present invention provides a polynucleotide comprising an SLC26A4 enhancer having at least 85% sequence identity with any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), the enhancer being operably linked to a promoter, wherein the enhancer is linked to the promoter directly or through a nucleic acid linker of 1 to 500 nucleotides (e.g., 1-50, 1-100, 1-150, 1-200, 1-250, 1-300, 1-350, 1-400, 1-450, 1-500, 50-500, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, or 450-500 nucleotides, e.g., about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides).

[0008] In another aspect, the present invention provides a nucleic acid vector comprising a polynucleotide that comprises an SLC26A4 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 higher sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35. In some embodiments, the SLC26A4 enhancer is operably linked to a promoter.

[0009] In another aspect, the present invention provides a polynucleotide, the polynucleotide comprising: (a) an SLC26A4 promoter of the formula 5'-B-A-C-3', wherein: A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); B is absent or has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides from the 3'-end of SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and C is absent or has at least 85% sequence identity with SEQ ID NO:3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and (b) an SLC26A4 enhancer having at least 85% sequence identity with any one of SEQ ID NO:4-9, 34 and 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), wherein the enhancer is linked to the promoter directly or via a nucleic acid linker of 1 to 500 nucleotides (e.g., 1-50, 1-100, 1-150, 1-200, 1-250, 1-300, 1-350, 1-400, 1-450, 1-500, 50-500, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500 or 450-500 nucleotides, e.g., about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 nucleotides). In some embodiments, the SLC26A4 promoter is not longer than 1481 bases.

[0010] In another aspect, the present invention provides a nucleic acid vector containing the polynucleotide of any one of the foregoing aspects.

[0011] In some embodiments of any of the foregoing aspects, 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 phosphoglycerate kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, an HSV promoter, or an 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 an SV40 minimal promoter. In some embodiments, the promoter is a minimal promoter.

[0012] In some embodiments of any of the foregoing aspects, the promoter is a mammalian SLC26A4 promoter. In some embodiments, the SLC26A4 promoter is a human or murine SLC26A4 promoter. In some embodiments, the SLC26A4 promoter has the formula 5'-B-A-C-3', wherein: A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); B is absent or has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides from the 3'-end of SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and C is absent or has at least 85% sequence identity with SEQ ID NO:3 or a portion thereof comprising at least the first 159 consecutive nucleotides of SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), wherein the SLC26A4 promoter is no longer than 1481 bases.

[0013] In some embodiments of any of the foregoing aspects, A has the sequence of SEQ ID NO:1.

[0014] In some embodiments of any of the foregoing aspects, B is absent.

[0015] In some embodiments of any of the foregoing aspects, B has the sequence of SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2. In some embodiments, B has a sequence of a portion of SEQ ID NO:2, the portion comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2. In some embodiments, the portion of SEQ ID NO:2 is the first 307 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2 (as shown in SEQ ID NO:14). In some embodiments, B has the sequence of SEQ ID NO:2.

[0016] In some embodiments of any of the foregoing aspects, C is absent.

[0017] In some embodiments of any of the foregoing aspects, both B and C are absent.

[0018] In some embodiments of any of the foregoing aspects, C has the sequence of SEQ ID NO:3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:3. In some embodiments, C has a sequence of a portion of SEQ ID NO:3, the portion comprising at least the first 159 nucleotides of SEQ ID NO:3. In some embodiments, the portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:15, 16, 17, 18, and 19, respectively). In some embodiments, the indicated portion of SEQ ID NO:3 has the sequence of SEQ ID NO:15. In some embodiments, the indicated portion of SEQ ID NO:3 has the sequence of SEQ ID NO:16. In some embodiments, the indicated portion of SEQ ID NO:3 has the sequence of SEQ ID NO:17. In some embodiments, the indicated portion of SEQ ID NO:3 has the sequence of SEQ ID NO:18. In some embodiments, the indicated portion of SEQ ID NO:3 has the sequence of SEQ ID NO:19. In some embodiments, C has the sequence of SEQ ID NO:3.

[0019] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:1.

[0020] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:20.

[0021] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:21 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:21.

[0022] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:22 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:22.

[0023] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:23 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:23.

[0024] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:24 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:24.

[0025] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:25 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:25.

[0026] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:26 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:26.

[0027] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:27 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:27.

[0028] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:28 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:28.

[0029] In some embodiments of any of the foregoing aspects, 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 higher sequence identity) with any one of SEQ ID NOs: 29 - 33. In some embodiments, the SLC26A4 promoter has the sequence of any one of SEQ ID NOs: 29 - 33.

[0030] In some embodiments of any one of the foregoing aspects, 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 higher sequence identity) with SEQ ID NO: 41. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 41.

[0031] In some embodiments of any of the foregoing aspects, the SLC26A4 promoter is operably linked to the SLC26A4 enhancer.

[0032] In some embodiments of any of the foregoing aspects, the SLC26A4 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 higher sequence identity) with any one of SEQ ID NOs: 4 - 9, 34 and 35.

[0033] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer is directly linked (e.g., fused) to the promoter.

[0034] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer is linked to the promoter by a nucleic acid linker of 1 to 500 nucleotides (e.g., 1 - 50, 1 - 100, 1 - 150, 1 - 200, 1 - 250, 1 - 300, 1 - 350, 1 - 400, 1 - 450, 1 - 500, 50 - 500, 100 - 500, 150 - 500, 200 - 500, 250 - 500, 300 - 500, 350 - 500, 400 - 500 or 450 - 500 nucleotides, e.g., about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 nucleotides).

[0035] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleotides (e.g., 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, or 90-100 nucleotides, such as about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0036] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer is located 5' of the promoter.

[0037] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer is located 3' of the promoter.

[0038] 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.

[0039] In some embodiments of any of the foregoing aspects, the expression product is a heterologous expression product.

[0040] In some embodiments of any of the foregoing aspects, the expression product is an expression product that is endogenously expressed in cells that express SLC26A4. In some embodiments, the expression product is an expression product that is endogenously expressed in inner ear cells that express SLC26A4. In some embodiments, the expression product is an expression product that is endogenously expressed (e.g., expressed in at least one of these cell types) in interdental cells, spiral prominence cells, cochlear root cells, and / or vestibular supporting cells.

[0041] In some embodiments of any of the foregoing aspects, the expression product is pannexin (e.g., mammalian pannexin). In some embodiments, pannexin (mammalian pannexin) is the wild-type isoform that is endogenously expressed in the inner ear of a mammal. In some embodiments, pannexin (mammalian pannexin) has at least 85% sequence identity with SEQ ID NO:10 or SEQ ID NO:11 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). In some embodiments, pannexin (mammalian pannexin) has the sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0042] In some embodiments of any of the foregoing aspects, the expression product is Atoh1 (e.g., mammalian Atoh1). In some embodiments, Atoh1 (mammalian Atoh1 protein) is the wild-type isoform endogenously expressed in the inner ear of a mammal. In some embodiments, Atoh1 (mammalian Atoh1 protein) has at least 85% sequence identity with SEQ ID NO: 36 or SEQ ID NO: 38 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, Atoh1 (mammalian Atoh1 protein) has the sequence of SEQ ID NO: 36 or SEQ ID NO: 38.

[0043] In some embodiments of any of the foregoing aspects, the expression product is a protein, short hairpin RNA (shRNA), antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), transcription activator-like effector nuclease (TALEN), or zinc finger nuclease (ZFN) or a guide RNA (gRNA)), or a microRNA.

[0044] In some embodiments of any of the foregoing aspects, the nucleic acid vector or polynucleotide contains two or more different SLC26A4 enhancers, each of which is independently selected from 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 higher sequence identity) with any one of SEQ ID NOs: 4-9, 34, and 35. In some embodiments, each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NOs: 4-9, 34, and 35. In some embodiments in which the nucleic acid vector or polynucleotide contains two or more different SLC26A4 enhancers, the first 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 higher sequence identity) with SEQ ID NO: 6, and the second 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 higher sequence identity) with SEQ ID NO: 7. In some embodiments, the nucleic acid vector or polynucleotide contains a first enhancer having the sequence of SEQ ID NO: 6 and a second enhancer having the sequence of SEQ ID NO: 7. In some embodiments, both the enhancer of SEQ ID NO: 6 and the enhancer of SEQ ID NO: 7 are located 5' of the SLC26A4 promoter (e.g., the SLC26A4 promoter of SEQ ID NO: 1). In some embodiments, both the enhancer of SEQ ID NO: 6 and the enhancer of SEQ ID NO: 7 are located 5' of the SLC26A4 promoter, and one enhancer is directly linked to the other enhancer, which is directly linked to the SLC26A4 promoter, e.g., in the following 5' to 3' order: SEQ ID NO: 6-SEQ ID NO: 7-SLC26A4 promoter. In a particular embodiment, the nucleic acid vector or polynucleotide contains SEQ ID NO: 6-SEQ ID NO: 7-SEQ ID NO: 1 linked directly in 5' to 3' order.

[0045] In some embodiments of any of the foregoing aspects, the nucleic acid vector or polynucleotide contains two or more copies of the SLC26A4 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 higher sequence identity) with any one of SEQ ID NOs: 4-9, 34, and 35. In some embodiments, each copy of the two or more copies of the SLC26A4 enhancer has the sequence of one of SEQ ID NOs: 4-9, 34, and 35.

[0046] In some embodiments of any of the foregoing aspects, the SLC26A4 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 higher sequence identity) with SEQ ID NO: 4. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 4.

[0047] In some embodiments of any of the foregoing aspects, the SLC26A4 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 higher sequence identity) with SEQ ID NO: 5. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 5.

[0048] In some embodiments of any of the foregoing aspects, the SLC26A4 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 higher sequence identity) with SEQ ID NO: 6. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 6.

[0049] In some embodiments of any of the foregoing aspects, the SLC26A4 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 higher sequence identity) with SEQ ID NO: 7. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 7.

[0050] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:8 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO:8.

[0051] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:9 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO:9.

[0052] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:34 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, the SLC26A4 enhancer has the sequence of SEQ IDNO:34.

[0053] In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity). In some embodiments, the SLC26A4 enhancer has the sequence of SEQ IDNO:35.

[0054] In some embodiments of any of the foregoing aspects, the nucleic acid vector is a viral vector, plasmid, cosmid or 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) vector, an adenovirus vector or a lentiviral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector has an AAV1, AAV2, AAV2quad(Y-F), 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(Y-F) 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.

[0055] In another aspect, the present invention provides a composition comprising the nucleic acid vector of any one of the foregoing aspects and embodiments. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0056] In another aspect, the present 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 prominence cell, a cochlear root cell, or a vestibular supporting cell.

[0057] In another aspect, the present invention provides a method for 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 prominence 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 contacting is carried out in a subject (e.g., in vivo).

[0058] In another aspect, the present invention provides a method for treating a subject suffering from or at risk of developing hearing loss (e.g., sensorineural hearing loss), 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 hearing loss is pendrin-related hearing loss. In some embodiments, the expression product is pendrin (e.g., the wild-type isoform of mammalian pendrin). In some embodiments, the pendrin-related hearing loss is hearing loss associated with Pendred syndrome or DFNB4.

[0059] In another aspect, the present invention provides a method for treating a subject in need thereof for hearing loss associated with Meniere's disease, 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 pendrin (e.g., the wild-type isoform of mammalian pendrin).

[0060] In another aspect, the present invention provides 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 a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the expression product is pendrin (e.g., the wild-type isoform of mammalian pendrin).

[0061] 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 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 pannexin (e.g., a wild-type isoform of mammalian pannexin) or Atoh1 (e.g., a wild-type isoform of mammalian Atoh1). In some embodiments, the vestibular dysfunction is vertigo.

[0062] In another aspect, 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 pannexin-related vestibular dysfunction. In some embodiments, the expression product is pannexin (e.g., a wild-type isoform of mammalian pannexin). In some embodiments, the pannexin-related vestibular dysfunction is vestibular dysfunction associated with Pendred syndrome or DFNB4. In some embodiments, the expression product is pannexin (e.g., a wild-type isoform of mammalian pannexin) or Atoh1 (e.g., a wild-type isoform of mammalian Atoh1).

[0063] In another aspect, the present invention provides a method for 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 (e.g., a wild-type isoform of mammalian Atoh1). In some embodiments, the contacting is carried out in vivo (e.g., in a subject). In some embodiments, the subject suffers from vestibular dysfunction or is at risk of developing vestibular dysfunction.

[0064] In another aspect, 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 (e.g., a wild-type isoform of mammalian Atoh1). In some embodiments, the contacting is carried out in vivo (e.g., in a subject). In some embodiments, the subject suffers from vestibular dysfunction or is at risk of developing vestibular dysfunction.

[0065] In another aspect, the present invention provides a method for improving the function of cells expressing SLC26A4, the method comprising the step of contacting the cells expressing SLC26A4 with the nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the contacting is carried out in vivo (e.g., in a subject). In some embodiments, the subject has hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction or is at risk of developing hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction.

[0066] 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 of any of the foregoing aspects, the vestibular dysfunction is associated with damage or loss of vestibular hair cells. In some embodiments, the damage or loss of vestibular hair cells is associated with: age (i.e., the vestibular dysfunction is age-related vestibular dysfunction), exposure to ototoxic (e.g., vestibulotoxic) 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 antibiotic, such as gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin or netilmicin), viomycin, an antitumor 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.

[0067] In some embodiments of any of the foregoing aspects, the method further comprises evaluating the hearing of the subject before administering the nucleic acid vector or composition.

[0068] 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.

[0069] In some embodiments of any of the foregoing aspects, the method further comprises evaluating the vestibular function of the subject before administering the nucleic acid vector or composition.

[0070] In some embodiments of any of the foregoing aspects, the method further comprises evaluating the vestibular function of the subject after administering the nucleic acid vector or composition.

[0071] In some embodiments of any of the foregoing aspects, the nucleic acid vector or composition is administered locally. In some embodiments, the nucleic acid vector or composition is administered to the inner ear. In some embodiments, the nucleic acid vector or composition is administered to the middle ear. In some embodiments, the nucleic acid vector or composition is administered via the tympanic cavity or intratympanically. In some embodiments, the nucleic acid vector or composition is administered into the perilymph. In some embodiments, the nucleic acid vector or composition is administered into the endolymph. In some embodiments, the nucleic acid vector or composition is administered to or via the oval window. In some embodiments, the nucleic acid vector or composition is administered to or via the round window. In some embodiments, the nucleic acid vector or composition is administered to the semicircular canals.

[0072] 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 the expression of the expression product in cells 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 a feeling of ear fullness, increase vestibular hair cell regeneration, induce or increase the differentiation of vestibular supporting cells into vestibular hair cells, increase or induce the maturation of vestibular hair cells (e.g., the maturation of regenerated vestibular hair cells), or improve the function of cells expressing SLC26A4 (e.g., inner ear cells expressing SLC26A4).

[0073] In some embodiments of any of the foregoing aspects, the subject is a human subject.

[0074] In another aspect, the present invention provides a kit comprising a polynucleotide, nucleic acid vector, or composition of any one of the foregoing aspects and embodiments.

[0075] Definitions

[0076] As used herein, the term "about" refers to a value within 10% of the stated value.

[0077] As used herein, "administering" refers to providing or giving a therapeutic agent (e.g., a nucleic acid vector comprising an SLC26A4 promoter and / or SLC26A4 enhancer) to a subject by any effective route. Exemplary routes of administration are described below.

[0078] As used herein, the phrase "administering to the inner ear" refers to providing or giving 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 the oval window, round window, or semicircular canals (e.g., horizontal canal), or through the oval window, round window, or semicircular canals, or by transtympanic or intratympanic injection, such as administration to inner ear cells that express SLC26A4.

[0079] As used herein, the term "cell type" refers to a group of cells that share a phenotype that is statistically separable based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation characteristic profiles. Cells of a common cell type may include those isolated from a common tissue (e.g., epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those isolated from a common organ, tissue system, blood vessel, or other structure and / or region of an organism.

[0080] As used herein, the terms "conservative mutation", "conservative substitution", and "conservative amino acid substitution" refer to the substitution of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties (such as polarity, net charge, and spatial volume). These properties for each of the twenty naturally occurring amino acids are summarized in Table 1 below.

[0081] Table 1. Representative Physicochemical Properties of Naturally Occurring Amino Acids

[0082]

[0083] It should be appreciated from this table that the conservative amino acid families include (i) G, A, V, L, and I; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Thus, a conservative mutation or substitution is a mutation or substitution in which one amino acid is replaced with a member of the same amino acid family (e.g., Ser replaced with Thr or Lys replaced with Arg).

[0084] 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 result (including a clinical result) when administered to a subject, including a mammal (e.g., a human), and thus, the "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 Pendred syndrome), it is the amount of the composition, vector construct, or viral vector sufficient to achieve a therapeutic response as compared to the response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein corresponding to such an amount will vary depending on various factors such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, subject being treated (e.g., age, sex, weight), or identity of the host, etc., but can still be routinely determined by one of ordinary skill in the art. Similarly, as used herein, a "therapeutically effective amount" of a composition, vector construct, or viral vector of the present disclosure is an amount that produces a beneficial or desired result in a subject as 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 an ordinary skilled person by conventional methods known in the art. The dosage regimen can be adjusted to provide an optimal therapeutic response.

[0085] 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 at 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).

[0086] As used herein, the term "expression" refers to one or more of the following events: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein. The term "expression product" refers to a protein or RNA molecule produced by any of these events.

[0087] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally present in a particular organism (e.g., a human) or at 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 provided to an organism from an external source or provided to a culture material extracted from said organism.

[0088] As used herein, the term "heterologous" refers to a combination of elements that are not naturally occurring. For example, a heterologous transgene refers to a transgene that is not naturally expressed by the promoter to which it is operably linked.

[0089] As used herein, the terms “increase” and “decrease” refer to a modulation that results in a greater or lesser amount of a function, expression, or activity, respectively, relative to a reference. For example, after administration of a composition in the methods described herein, the amount of a marker of a measure (e.g., transgenic expression level or auditory brainstem response) as described herein may increase or decrease 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, the measure is measured after administration, at a time when the administration has achieved the effect, e.g., at least one week, one month, 3 months, or 6 months after the start of a treatment regimen.

[0090] As used herein, “local” or “locally administered” refers to an administration that is intended to act locally rather than systemically on a particular site of the body. Examples of local administrations are epidermal, inhalation, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional administration, lymph node administration, intratumoral administration, direct administration to the inner or middle ear (e.g., by injection through the oval or round window membrane or by transtympanic or intratympanic injection), and administration to the mucosa of a subject, where the administration is intended to have a local rather than systemic effect.

[0091] As used herein, the term “operably linked” refers to a first molecule being linked to a second molecule, where the arrangement of the molecules is such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous 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, then the promoter is operably linked to the transcribable polynucleotide molecule. Additionally, if two portions of a transcriptional regulatory element are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion, then the two portions are operably linked to each other. Two transcriptional regulatory elements may be operably linked to each other by means of a linker polynucleotide (e.g., an intervening non-coding polynucleotide) or may be operably linked to each other in the absence of intervening nucleotides.

[0092] As used herein, the term “plasmid” refers to an extrachromosomal circular double-stranded DNA molecule to which additional DNA segments can be ligated. A plasmid is a vector, a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been ligated. Certain plasmids are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial plasmids with a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell after being introduced into the host cell and thereby replicate along with the host genome. Certain plasmids are capable of directing the expression of genes to which they are operably linked.

[0093] 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) that are naturally occurring in DNA or RNA and are linked by phosphodiester bonds. The term encompasses molecules that contain nucleosides or nucleoside analogs with chemically or biologically modified bases, modified backbones, etc., whether or not they are present in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to polynucleotides, it should be understood that DNA, RNA are provided, and in each case, single-stranded and double-stranded forms (as well as the complement of each single-stranded molecule) are provided. As used herein, a "polynucleotide sequence" may refer to the polynucleotide material itself and / or the sequence information that biochemically characterizes a particular nucleic acid (i.e., a series of letters used as abbreviations for bases). Unless otherwise specified, the polynucleotide sequences given herein are presented in the 5' to 3' direction.

[0094] As used herein, a "polynucleotide that can be transcribed to produce RNA or protein" refers to a polynucleotide that can direct the production of RNA or protein (e.g., via transcription or translation). Such polynucleotides can be incorporated into a nucleic acid vector or expression cassette, for example, operably linked to the SLC26A4 promoter and / or SLC26A4 enhancer described herein. The terms "polynucleotide that can be transcribed to produce..." and "polynucleotide encoding..." are used interchangeably herein and refer to a polynucleotide that can direct the production of a protein (e.g., pannexin or Atoh1) or an RNA molecule (e.g., an inhibitory RNA molecule such as siRNA, shRNA, or miRNA).

[0095] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives the transcription of a transgene.

[0096] "Percent sequence identity (%)" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. The alignment for determining the percent nucleic acid or amino acid sequence identity can be achieved in various ways within the capabilities of one of ordinary skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2 or Megalign software. One of ordinary skill in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve the maximum alignment over the full length of the sequences being compared. For example, the sequence comparison computer program BLAST can be used to generate percent sequence identity values. For example, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which can also alternatively be phrased as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows:

[0097] 100 × (fraction X / Y)

[0098] where X is the number of nucleotides or amino acids scored as identical matches in the alignment of A and B by the sequence alignment program (e.g., BLAST), and where Y is the total number of nucleic acids in B. It should be understood 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.

[0099] As used herein, the terms "Pendrin" and "SLC26A4" refer to the protein encoded by the SLC26A4 gene and the gene encoding the 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 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) with the amino acid sequence of wild-type Pendrin (e.g., SEQ ID NO:10 or SEQ ID NO:11) or variants of proteins or 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) with the nucleic acid sequence of wild-type SLC26A4 gene (e.g., SEQ ID NO:12 or SEQ ID NO:13) or its codon-optimized sequence, 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 ions, iodide ions, and bicarbonate ions) across cell membranes).

[0100] As used herein, the term "SLC26A4 enhancer" refers to a polynucleotide that can be operably linked to a promoter (e.g., SLC26A4 promoter, minimal promoter, core promoter, or constitutive promoter) to regulate gene expression in cells that express SLC26A4. The SLC26A4 enhancer used 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) with any one of SEQ ID NO:4-9, 34, and 35. 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 and express the expression product.

[0101] As used herein, the term "SLC26A4 promoter" refers to a polynucleotide or variant thereof that is capable of specifically expressing a transgene in cells that express 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 higher sequence identity) to the SLC26A4 promoter described herein (e.g., the SLC26A4 promoter of formula B-A-C as described herein or the promoters or enhancer-promoters provided in Table 2). 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 higher sequence identity) to any one of SEQ ID NOs: 1 and 20 - 27 or 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) to any one of SEQ ID NOs: 28 - 33 and 41.

[0102] As used herein, the term "SLC26A4-expressing cells" refers to cell types that are known to endogenously express SLC26A4 in vivo. SLC26A4-expressing cells include cells of the mammary gland (adipocytes, vascular cells, luminal epithelial cells, fibroblasts, dendritic cells, macrophages, basal myoepithelial cells, pericytes, and smooth muscle cells); cells of the esophagus (adipocytes, lymphocytes, vascular cells, epithelial cells of the esophageal mucosa (basal, supra-basal, squamous), fibroblasts (mucosal and muscular), mucous cells, myofibroblasts of the esophageal mucosa, neuronal cells of the esophageal muscle layer, Schwann cells, muscle cells (smooth muscle), immune cells (dendritic cells, macrophages, T cells, mast cells), pericytes, and smooth muscle cells); cells of the heart (adipocytes, lymphocytes, vascular cells, fibroblasts, Schwann cells, muscle cells (cardiac, cytoplasmic), immune cells (dendritic cells, macrophages, T cells, mast cells), pericytes, and smooth muscle cells); cells of the lung (lymphocytes, vascular cells, epithelial cells (type I alveolar, type II alveolar, 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, muscle cells (skeletal muscle, cytoplasmic), pericytes, and smooth muscle cells); cells of the prostate (lymphocytes, vascular cells, epithelial cells (trabecular cells, luminal cells), fibroblasts, dendritic cells, macrophages, muscle cells (smooth muscle)); cells of the skin (vascular cells, epithelial cells (basal keratinocytes, mature keratinocytes, supra-basal 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 that express SLC26A4.

[0103] As used herein, the term "inner ear cells that express SLC26A4" refers to cells that endogenously express SLC26A4 within the inner ear. Inner ear cells that express SLC26A4 are present in both the cochlea and the vestibule. Cochlear cells that express SLC26A4 include root cells, fusiform cells, inner sulcus cells, outer sulcus cells, spiral prominence cells, interdental cells, macrophages, Reissner’s membrane, Deiters’ cells, vascular cells, marginal cells, and intermediate cells. Vestibular cells that express SLC26A4 include non-sensory epithelial cells. Additional inner ear cells that express SLC26A4 include mitochondrion-rich endolymphatic sac cells of the endolymphatic sac and endolymphatic duct.

[0104] As used herein, the term "Pendrin-related hearing loss" refers to a disease or disorder characterized by hearing loss associated with mutations in SLC26A4, such as DFNB4 (characterized by pre-lingual or post-lingual hearing loss that may be associated with an enlarged vestibular aqueduct) and Pendred syndrome (characterized by an enlarged thyroid gland (goiter), severe to profound hearing loss (usually from birth), and other abnormalities of the inner ear (including an enlarged vestibular aqueduct)).

[0105] As used herein, the term "Pendrin-related vestibular dysfunction" refers to a disease or disorder characterized by vestibular dysfunction (e.g., vertigo, dizziness, or imbalance or loss of balance) associated with mutations in SLC26A4, such as DFNB4 and Pendred syndrome.

[0106] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally combined 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 specific disease or disorder that affects or may affect the subject.

[0107] 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 a subject (e.g., a mammal, e.g., a human) without undue toxicity, irritation, allergic response, and other problem complications and that are commensurate with a reasonable benefit / risk ratio.

[0108] 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 sample, and cells).

[0109] 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 diagnosed with hearing loss (e.g., Pendrin-related hearing loss), vestibular dysfunction (e.g., Pendrin-related 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 mutations in SLC26A4 or exposure to trauma that may cause vestibular hair cell injury 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. Those skilled in the art will understand that a subject to be treated according to the present disclosure may have already undergone standard testing or may be identified as at risk without examination due to the presence of one or more risk factors associated with the disease or condition.

[0110] As used herein, the terms "transcription regulatory element" and "regulatory sequence" refer to a polynucleotide that at least partially controls the transcription of a gene of interest. Transcription regulatory elements can include promoters, enhancers, and other polynucleotides that control or assist in controlling gene transcription (e.g., polyadenylation signals). Examples of transcription regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0111] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, impalefection, etc.

[0112] As used herein, the terms "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 within 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.

[0113] 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 can 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, condition, or disorder; 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 remission (partial or complete). "Improving" or "alleviating" a disease or disorder means that the extent and / or the time course of progression of the disease, condition, or disorder and / or the undesired clinical manifestations are reduced and / or the time course of progression is slowed or extended as compared to the extent or time course in the absence of treatment. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment. Those in need of treatment include those who already have a disorder or condition, as well as those who are predisposed to a disorder or condition or who will prevent a disorder or condition.

[0114] As used herein, the term "vector" refers to a nucleic acid vector, e.g., a DNA vector such as a plasmid, cosmid or 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, e.g., Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the compositions and methods described herein contain polynucleotide sequences and, e.g., additional sequence elements for expressing the proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express transgenes as described herein include vectors that contain regulatory sequences (such as promoter and enhancer regions) that direct gene transcription. Other available vectors for expressing transgenes contain polynucleotide sequences that increase the translation rate of the transgene or improve the stability or nuclear export of the mRNA produced by gene transcription. These sequence elements include, e.g., 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 a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0115] As used herein, the term "wild-type" refers to the genotype having the highest frequency for a particular gene in a given organism. Brief Description of the Drawings

[0116] Figure 1A-1C Depicts the general design of various plasmids for generating an AAV-DJ vector for the expression of enhanced green fluorescent protein (EGFP) targeted to the nucleus by fusion with H2B. Figure 1A Depicts the general design of a plasmid in which the expression of the H2B-EGFP fusion protein is driven by a promoter without the addition of any SLC26A4 enhancer. Figure 1A The general design depicted in also contains barcodes for the scRNA-seq experiments described herein. Figure 1B Depicts the general design of a plasmid in which the expression of the H2B-EGFP fusion protein is driven by the SLC26A4 promoter disclosed herein with the SLC26A4 enhancer fused to the 5' end of the promoter. Figure 1BThe general design depicted also contains barcodes for the scRNA-seq experiments described herein. Figure 1C Depicted is the general design of a plasmid in which the expression of the H2B-EGFP fusion protein is driven by the SLC26A4 promoter disclosed herein in the case where two SLC26A4 enhancers (Enhancer #1 and Enhancer #2) are fused to each other and to the 5'-end of the promoter.

[0117] Figure 2A-2B Is a series of micrographs depicting panprotein immunostaining. Figure 2A Is a micrograph depicting the orientation of the cultured lateral wall and cochlear anatomy stained with an antibody specific for panprotein. Figure 2B Depicted is an embedded whole-mount view of the mouse lateral wall transfected with an AAV-DJ viral vector and counterstained with an antibody specific for panprotein, the viral vector expressing an enhanced green fluorescent protein (EGFP) transgene that is targeted to the nucleus by fusion with H2B and is under the control of the ubiquitous CMV promoter. The first (left) image shows the panprotein channel alone; the second (middle) image shows both the panprotein and GFP channels; and the third (right) image shows the GFP channel alone.

[0118] Figure 3A-3I Depicted is an embedded whole-mount view of the mouse lateral wall transfected with an AAV-DJ viral vector and counterstained with an antibody specific for panprotein after five days of culture, the viral vector expressing an enhanced green fluorescent protein (EGFP) transgene that is targeted to the nucleus by fusion with H2B and is under the control of various human SLC26A4 promoters disclosed herein. Figure 3A –Pend3.1 (SEQ ID NO:40); Figure 3B –Pend3.1.1 (SEQ ID NO:27); Figure 3C –Pend3.1.3 (SEQID NO:25); Figure 3D –Pend3.1.4 (SEQ ID NO:24); Figure 3E –Pend3.1.5 (SEQ ID NO:21); Figure 3F –Pend3.1.6 (SEQ ID NO:23); Figure 3G –Pend3.1.7 (SEQ ID NO:22); Figure 3H –Pend3.1.8 (SEQ IDNO:1); Figure 3I –Pend3.1.9 (SEQ ID NO:20) human core promoter. Scale bar, 100 μm. The top images in each panel show the panprotein and GFP channels; the middle images show the panprotein channel alone; and the bottom images show the GFP channel alone.

[0119] Figure 4A-4B Depicts a whole-mount view of the mouse lateral wall transfected with an AAV-DJ viral vector and counterstained with an antibody specific for pendrin after five days of culture. The viral vector expresses an enhanced green fluorescent protein (EGFP) transgene that is targeted to the nucleus by fusion with H2B and is under the control of the Pend3.1.8 promoter (SEQ ID NO:1) and the mouse or human SLC26A4 enhancer indicated below. Figure 4A – Left inset - mouse E2 enhancer (mE2; SEQ ID NO:6); middle inset - mouse E6 enhancer (mE6; SEQ ID NO:7); right inset - human 6.4 enhancer (hE6.4; SEQ ID NO:5); Figure 4B - Left inset - human 6.1 enhancer (hE6.1; SEQ ID NO:4); middle inset - human 6.2 enhancer (hE6.2; SEQ ID NO:8); right inset - human 6.3 enhancer (hE6.3; SEQ ID NO:9). Scale bar, 100 μm. The top row in each panel shows the pendrin and GFP channels; the second row shows the pendrin channel alone; the third row shows the GFP channel alone; and the fourth row shows the GFP channel alone with adjusted gain.

[0120] Figure 5 Depicts a whole-mount view of the mouse lateral wall transfected with an AAV-DJ viral vector and counterstained with an antibody specific for pendrin after five days of culture. The viral vector expresses an enhanced green fluorescent protein (EGFP) transgene that is targeted to the nucleus by fusion with H2B and is under the control of the sole Pend3.1.6 promoter (SEQ ID NO:23) in combination with the mouse or human SLC26A4 enhancer indicated below. Left inset – human 6.4 enhancer (hE6.4; SEQ ID NO:5); middle inset - mouse E6 enhancer (mE6; SEQ ID NO:7); right inset - Pend3.1.6 without any enhancer. Scale bar, 100 μm. The top row in each panel shows the pendrin and GFP channels; the second row shows the pendrin channel alone; the third row shows the GFP channel alone; and the fourth row shows the GFP channel alone with adjusted gain.

[0121] Figure 6 is a series of bar graphs that depict Figure 3H (the sole Pend 3.1.8 promoter), Figure 4ALeft small panel, middle small panel, and right small panel (Pend 3.1.8 promoter + mouse E2 enhancer (left small panel); Pend3.1.8 promoter + mouse E6 enhancer (middle small panel); Pend3.1.8 promoter + human 6.4 enhancer (right small panel)) and Figure 4B Quantification of GFP signal in whole mounts of the mouse lateral wall shown in the left small panel (Pend3.1.8 promoter + human 6.1 enhancer) ((n = 3 - 5 per group; mean ± SEM)). Panel A depicts the percentage of paneth cells that are also GFP positive. Panel B depicts the number of GFP positive cells in the paneth cell positive area of the lateral wall. Panel C depicts the number of GFP positive cells outside the paneth cell positive area of the lateral wall. Panel D depicts the mean intensity of the GFP signal in paneth cells.

[0122] Figure 7 Depicts the sequence overlap of the various SLC26A4 promoters described herein and how those promoters overlap with the genomic sequence of the human SLC26A4 gene. Figure 7 Also depicts the correlation between various regions of these promoters and SEQ ID NOs: 1 - 3.

[0123] Figure 8 Depicts the results of single cell RNA-seq analysis in different cell types present in the mouse lateral wall of the various promoters disclosed herein alone or in combination with the various enhancers disclosed herein. Controls are the ubiquitously expressed Pgk promoter and constructs lacking any promoter (promoterless). These results also include the results of the Pend3.1.8 promoter combined with the human 5.1 enhancer (SEQ ID NO: 34) (“hE5.1+3.1.8”) and combined with the human 5.4 enhancer (SEQ ID NO: 35) (“hE5.4+3.1.8”).

[0124] Figure 9 Depicts the plasmid map of P1708.

[0125] Figure 10A-10B Depicts the results of transfecting the mouse lateral wall with an AAV-DJ viral vector and counterstaining with an antibody specific for paneth after five days of culture, the viral vector expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion to H2B and under the control of the Pend3.1.8 promoter (SEQ ID NO: 1) and one or both of the murine E2 and E6 SLC26A4 enhancers. Figure 10ADepicts whole - mount views of the murine lateral wall for each AAV - DJ vector - left inset - murine E2 enhancer (SEQ ID NO:6) and murine E6 enhancer (SEQ ID NO:7); middle inset - murine E2 enhancer; right inset - murine E6 enhancer. Figure 10B Is a violin plot depicting GFP intensity in cells expressing pannexin after transfection with AAV - DJ vectors containing both murine E2 and E6 and AAV - DJ vectors containing only the murine E6 enhancer.

[0126] Figure 11 Depicts the plasmid map of P1527.

[0127] Figure 12 Are images at a series of different magnifications of the same field of view, which depict the AAV - DJ vector containing the EGFP expression cassette with the E6.4 enhancer (SEQ ID NO:5) directly fused to the 5' end of the human SLC26A4 minimal promoter (SEQ ID NO:1) administered into pannexin - knockout mice (see plasmid P1527; Figure 11 )( Figure 12 , insets A - D); or the AAV1 vector containing the EGFP expression cassette driven by the CMV promoter ( Figure 12 , insets E - F) after cochlear EGFP expression. Figure 12 , insets A and E are micrographs at the lowest magnification. The area indicated by the box labeled "B" in Figure 12 inset A is shown at a higher magnification in Figure 12 inset B. Figure 12 The boxes labeled "C" and "D" in inset B are shown at higher magnifications in Figure 12 insets C and D respectively. The area indicated by the box labeled "F" in Figure 12 inset E is shown at a higher magnification in Figure 12 inset F. The field of view is counterstained with an antibody against Kcnj10 to better show the cochlear outline. RM - Reissner's membrane; ID - interdental cells; SP - spiral prominence. Scale bar = 100 μm. Detailed Description

[0128] The present invention describes compositions and methods for specifically inducing gene expression in cells or subpopulations thereof that express SLC26A4 (e.g., inner ear cells that express SLC26A4, such as interdental cells, spiral prominence cells, root cells, and vestibular supporting cells). The present invention features an SLC26A4 promoter that can induce transgene expression in cells that express SLC26A4 (e.g., inner ear cells that express SLC26A4) and an SLC26A4 enhancer that can be operably linked to the promoter to induce or increase transgene expression in cells that express SLC26A4. The SLC26A4 enhancer can be operably linked to a promoter (e.g., the SLC26A4 promoter) to increase the level of transgene expression and the number of cells that express SLC26A4 in which transgene 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, the SLC26A4 promoter and enhancer described herein 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 that express SLC26A4, with minimal off-target expression in cells that do not endogenously express SLC26A4 (e.g., cochlear hair cells). The present invention also features nucleic acid vectors containing an SLC26A4 promoter and optionally one or more SLC26A4 enhancers operably linked to a polynucleotide encoding an expression product, and nucleic acid vectors containing one or more SLC26A4 enhancers operably linked to a promoter (e.g., the SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or an enhancer-promoter, minimal promoter, core promoter, or constitutive promoter) operably linked to a polynucleotide encoding an expression product, and methods of using these vectors to treat hearing loss (e.g., pendrin-related hearing loss), vestibular dysfunction (e.g., imbalance or loss of balance associated with pendrin-related vestibular dysfunction or damage or loss of vestibular hair cells), or Meniere's disease.

[0129] Pendrin

[0130] Pendrin is an anion exchanger 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 called DFNB4 (hearing loss not associated with signs and symptoms affecting other parts of the body). This form of hearing loss can be pre-lingual or post-lingual, and subjects with DFNB4 typically have an enlarged vestibular aqueduct. More than 150 mutations in SLC26A4 are associated with Pendred syndrome, which is characterized by an enlarged thyroid (goiter), hearing loss, and other abnormalities of the inner ear (including an enlarged vestibular aqueduct). Pendred syndrome is the most common form of syndromic deafness, and subjects with Pendred syndrome typically begin to lose their hearing at birth or by three years of age and exhibit hearing loss that worsens over time, progressing to profound deafness in some subjects. There is no curative treatment for pendrin-related hearing loss, and supportive therapies typically aim to improve hearing, for example, by using hearing aids.

[0131] Gene therapy has recently emerged as an attractive treatment for hearing loss, particularly hearing loss caused by mutations in genes expressed in the inner ear, because delivery of the wild-type version of the mutated 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 endogenously express the gene. This can prove challenging for genes such as SLC26A4 that are expressed in different cell type classifications in the cochlea and vestibule.

[0132] The present invention is based in part on the discovery of the SLC26A4 promoter and enhancer, which can be used to induce gene expression in cells that express SLC26A4 while minimizing off-target expression in cells that do not express SLC26A4. The SLC26A4 promoter can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding prestin), and optionally operably linked to an SLC26A4 enhancer (e.g., the SLC26A4 enhancer described herein). The SLC26A4 enhancer can be operably linked to a promoter (e.g., the SLC26A4 promoter, a constitutive promoter, a core promoter, or a minimal promoter), which is operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding prestin). The SLC26A4 promoter and enhancer described herein can be used to induce the expression of an expression product in cells that express 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 that express SLC26A4 and to increase the number of cells that express 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 prestin, Atoh1, or another protein endogenously expressed in cells that express SLC26A4, or an RNA molecule, such as an inhibitory RNA molecule) in cells that express SLC26A4 (e.g., inner ear cells that express SLC26A4, such as interdental cells, spiral prominence cells, root cells, or vestibular supporting cells) to treat a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss) or deafness (e.g., prestin-related hearing loss), a subject having or at risk of developing vestibular dysfunction (e.g., prestin-related vestibular dysfunction or vestibular dysfunction associated with damage or loss of vestibular hair cells), 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.

[0133] The compositions and methods described herein may include an SLC26A4 promoter. In some embodiments, the SLC26A4 promoter has the formula B-A-C, where: A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), B is absent or has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof that includes 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), and C is absent or has at least 85% sequence identity with SEQ ID NO:3 or a portion thereof that includes at least the first 159 nucleotides of SEQ ID NO:3 (i.e., the first 159 consecutive nucleotides starting from the 5'-end of SEQ ID NO:3), wherein the SLC26A4 promoter is no longer than 1481 bases. In some embodiments, C is absent and the SLC26A4 promoter has the formula B-A, where A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), and B has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof that includes 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).In some embodiments, B is absent and the SLC26A4 promoter has the formula A-C, where A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity), and C has at least 85% sequence identity with SEQ ID NO:3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:3 (i.e., the first 159 consecutive nucleotides from the 5' end of SEQ ID NO:3) (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, both B and C are absent, and the SLC26A4 promoter has the sequence of SEQ ID NO:1 or a variant thereof having at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity). In some embodiments, A has the sequence of SEQ ID NO:1. In some embodiments, B has the sequence of SEQ ID NO:2. In some embodiments, C has the sequence of SEQ ID NO:3. In some embodiments, the portion of SEQ ID NO:2 comprises 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, or 800-917 consecutive nucleotides from the 3' end of SEQ ID NO:2 (e.g., the portion of SEQ ID NO:2 comprises at least 1, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, or 917 consecutive nucleotides from the 3' end of SEQ ID NO:2 in an SLC26A4 promoter having the formula B-A-C or B-A).In some embodiments, the portion of SEQ ID NO:2 is the first 307 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2 (nucleotides 611 - 917 of SEQ ID NO:3, as shown in SEQ ID NO:14). In some embodiments, the portion of SEQ ID NO:3 comprises at least the first 159, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100 or 1117 consecutive nucleotides starting from the 5'-end of SEQ ID NO:3. In some embodiments, the portion of SEQ ID NO:3 comprises the first 159, 324, 341, 716 or 723 consecutive nucleotides starting from the 5'-end of SEQ ID NO:3 (shown as SEQ ID NO:15, 16, 17, 18 and 19 respectively).

[0134] In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:1 (formula B-A-C, where B and C are absent). In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:20 (formula B-A-C, where C is absent, B has the sequence of SEQ ID NO:2, and A has the sequence of SEQ ID NO:1). In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:21 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:21 (formula B-A-C, where B is absent, A has the sequence of SEQ ID NO:1, and C has the sequence of SEQ ID NO:3). In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:22 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:22 (formula B-A-C, where C is absent, B has the sequence of the first 307 consecutive nucleotides starting from the 3' end of SEQ ID NO:2 (as shown in SEQ ID NO:14), and A has the sequence of SEQ ID NO:1). In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:23 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:23 (formula B-A-C, where B is absent, C has the sequence of the first 341 consecutive nucleotides starting from the 5' end of SEQ ID NO:3 (as shown in SEQ ID NO:17), and A has the sequence of SEQ ID NO:1).In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:24 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:24 (the formula B-A-C, where B is absent, C has the sequence of the first 723 consecutive nucleotides starting from the 5' end of SEQ ID NO:3 (as shown in SEQ ID NO:19), and A has the sequence of SEQ ID NO:1). In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:25 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:25 (the formula B-A-C, where B has the sequence of the first 307 consecutive nucleotides starting from the 3' end of SEQ ID NO:2 (as shown in SEQID NO:14), A has the sequence of SEQ ID NO:1, and C has the sequence of the first 716 consecutive nucleotides starting from the 5' end of SEQ ID NO:3 (as shown in SEQ ID NO:18)). In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ IDNO:26 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:26 (the formula B-A-C, where B has the sequence of the first 307 consecutive nucleotides starting from the 3' end of SEQ ID NO:2 (as shown in SEQ ID NO:14), A has the sequence of SEQ ID NO:1, and C has the sequence of the first 324 consecutive nucleotides starting from the 5' end of SEQ ID NO:3 (as shown in SEQ ID NO:16)).In some embodiments, the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:27 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) or has the sequence of SEQ ID NO:27 (formula B-A-C, where B has the sequence of the first 307 consecutive nucleotides starting from the 3' end of SEQ ID NO:2 (as shown in SEQ ID NO:14), A has the sequence of SEQ ID NO:1, and C has the sequence of the first 159 consecutive nucleotides starting from the 5' end of SEQ ID NO:3 (as shown in SEQ ID NO:15)). The SLC26A4 promoter sequences described above are provided in Table 2 below.

[0135] Table 2. SLC26A4 Promoter Sequences

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] In some embodiments, the SLC26A4 promoter described herein (e.g., the SLC26A4 promoter of formula B-A-C as described above, such as an SLC26A4 promoter having at least 85% sequence identity to any one of SEQ ID NOs: 1 and 20-27) is operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding prestin or Atoh1, a polynucleotide encoding a protein or RNA molecule that is endogenously expressed in a cell expressing SLC26A4, or a polynucleotide encoding an inhibitory RNA molecule). A nucleic acid vector containing an SLC26A4 promoter operably linked to a polynucleotide encoding prestin can be used to treat prestin-related hearing loss (e.g., hearing loss associated with DFNB4 or Pendred syndrome), prestin-related vestibular dysfunction (e.g., vestibular dysfunction associated with DFNB4 or Pendred syndrome), or Meniere's disease. A nucleic acid vector containing an SLC26A4 promoter operably linked to a polynucleotide encoding Atoh1 can be used to treat vestibular dysfunction associated with vestibular hair cell damage or loss (e.g., vestibular hair cell damage or loss due to aging, disease or infection, head trauma, or exposure to ototoxic drugs). In some embodiments, the SLC26A4 promoter is operably linked to an enhancer, such as the SLC26A4 enhancer described below herein.

[0155] The compositions and methods described herein may include the SLC26A4 enhancers listed in Table 3 (e.g., any one of SEQ ID NOs: 4-9, 34, and 35) or variants thereof, such as polynucleotide sequences having 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 any one of SEQ ID NOs: 4-9, 34, and 35. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of any one of SEQ ID NOs: 4-9, 34, and 35. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of SEQ ID NO: 4. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of SEQ ID NO: 5. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of SEQ ID NO: 6. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of SEQ ID NO: 7. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of SEQ ID NO: 8. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of SEQ ID NO: 9. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of SEQ ID NO: 34. In some embodiments, the SLC26A4 enhancer used in the compositions and methods described herein has the sequence of SEQ ID NO: 35. 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 at least two of the SLC26A4 enhancers listed in Table 3, e.g., 2, 3, 4, 5 or 6 different enhancer sequences).In embodiments where the composition contains two or more SLC26A4 enhancers (e.g., multiple copies of a single enhancer listed in Table 3 or one or more copies of at least two enhancers listed in Table 3), the enhancers can be included in any order and can be positioned directly adjacent to each other (e.g., joined without any intervening sequence 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 joined by a nucleic acid linker (e.g., the nucleic acid linker can be positioned between each enhancer sequence included in the composition or between at least two enhancer sequences in the composition) (such as a nucleic acid linker comprising from one to five hundred nucleotides (e.g., 1 - 50, 1 - 100, 1 - 150, 1 - 200, 1 - 250, 1 - 300, 1 - 350, 1 - 400, 1 - 450, 1 - 500, 50 - 500, 100 - 500, 150 - 500, 200 - 500, 250 - 500, 300 - 500, 350 - 500, 400 - 500, or 450 - 500 nucleotides)). In some embodiments where the composition contains two or more SLC26A4 enhancers, the composition contains two different SLC26A4 enhancers. In some embodiments where the composition contains two different SLC26A4 enhancers, the first enhancer is mE2 (SEQ ID NO:6), and the second enhancer is mE6 (SEQ ID NO:7). In some embodiments where the composition contains mE2 (SEQ ID NO:6) and mE6 (SEQ ID NO:7), both mE2 and mE6 are located 5' of the SLC26A4 promoter (e.g., the SLC26A4 promoter of SEQ ID NO:1). In some embodiments where the composition contains mE2 (SEQ ID NO:6) and mE6 (SEQ ID NO:7) (both located 5' of the SLC26A4 promoter), one enhancer is directly joined to the other enhancer, and the other enhancer is directly joined to the SLC26A4 promoter, e.g., in the order 5'-mE2-mE6-SLC26A4 promoter-3'. In a particular embodiment, the 5'-mE2-mE6-SLC26A4 promoter-3' polynucleotide sequence is SEQ ID NO:6-SEQ ID NO:7-SEQ ID NO:1 in the 5' to 3' order.

[0156] Exemplary SLC26A4 enhancer sequences are provided in Table 3.

[0157] Table 3. SLC26A4 Enhancer Sequences

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] The SLC26A4 enhancer sequences described herein can be included in a nucleic acid vector and operably linked to a promoter (e.g., an SLC26A4 promoter, such as an SLC26A4 promoter of the formula B-A-C (e.g., an SLC26A4 promoter having at least 85% sequence identity to any one of SEQ ID NOs: 1 and 20-27), any other SLC26A4 promoter listed in Table 2 (e.g., an SLC26A4 promoter of any one of SEQ ID NOs: 28-33 and 41), a minimal promoter, a core promoter, or a constitutive promoter), which promoter itself can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein of interest (such as prestin or Atoh1) or an RNA molecule (such as an inhibitory RNA)) to specifically express the expression product in cells that express SLC26A4 (e.g., in inner ear cells that express SLC26A4, such as interdental cells, root cells, spiral prominence cells, or vestibular supporting cells). The SLC26A4 enhancer can be directly linked (e.g., fused) to the promoter or can be operably linked to the promoter via a nucleic acid linker, such as a nucleic acid linker comprising from one to five hundred nucleotides (e.g., 1-50, 1-100, 1-150, 1-200, 1-250, 1-300, 1-350, 1-400, 1-450, 1-500, 50-500, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, or 450-500 nucleotides). One or more SLC26A4 enhancers can be located 5' of the promoter or 3' of the promoter (e.g., 5' of the promoter or 3' of the coding sequence of the expression product).

[0165] In some embodiments, a polynucleotide encoding wild-type pendrin 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 higher sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) pendrin (e.g., SEQ ID NO:10 or SEQ ID NO:11)) is operably linked to the SLC26A4 promoter described herein (e.g., a promoter of formula B-A-C, e.g., 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 higher sequence identity) to any one of SEQ ID NO:1 and 20-27), which is optionally operably linked to an SLC26A4 enhancer (e.g., the SLC26A4 enhancer provided in Table 3), or is operably linked to a promoter (e.g., an SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or an enhancer-promoter, minimal promoter, core promoter or constitutive promoter) that is operably linked to one or more SLC26A4 enhancers described herein (e.g., one 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 higher sequence identity) to any one of SEQ ID NO:4, 5, 8, 9, 34 and 35). In some embodiments, the polynucleotide sequence encoding pendrin 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:10 or SEQ ID NO:11, provided that the encoded pendrin analog retains the therapeutic function of wild-type pendrin (e.g., the ability to transport negatively charged ions (such as chloride ions, iodide ions and bicarbonate ions) across cell membranes). No more than 10% of the amino acids in pendrin may be replaced by conservative amino acid substitutions. In some embodiments, the polynucleotide sequence encoding pendrin is any polynucleotide sequence that encodes SEQ ID NO:10 or SEQ ID NO:11 by the redundancy of the genetic code. The polynucleotide sequence encoding pendrin may be partially or fully codon-optimized for expression (e.g., in inner ear cells expressing human SLC26A4).Pendrin 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., an SNP that does not result in hearing loss). Human pendrin can be encoded by a polynucleotide having the sequence of SEQ ID NO:12. Mouse pendrin can be encoded by a polynucleotide having the sequence of SEQ ID NO:13. Pendrin can be human pendrin or can be a homolog of human pendrin from another mammalian species (e.g., mouse, rat, bovine, equine, caprine, ovine, donkey, feline, canine, rabbit, guinea pig, or other mammal). Exemplary pendrin amino acid and polynucleotide sequences are provided in Table 4 below. A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer (e.g., an SLC26A4 enhancer having at least 85% sequence identity to any of SEQ ID NO:4, 5, 8, 9, 34, and 35) operably linked to a promoter (e.g., an SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2, or an enhancer-promoter, minimal promoter, core promoter, or constitutive promoter) operably linked to a polynucleotide encoding pendrin, or a nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 promoter (e.g., a promoter of formula B-A-C, such as an 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 higher sequence identity) to any of SEQ ID NO:1 and 20-27) operably linked to a polynucleotide encoding pendrin and optionally an SLC26A4 enhancer (e.g., the SLC26A4 enhancer of Table 3) described herein can be administered to a subject to treat, reduce, or prevent pendrin-related hearing loss (such as hearing loss in subjects with DFNB4 or Pendred syndrome) or pendrin-related vestibular dysfunction (such as vestibular dysfunction associated with DFNB4 or Pendred 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 or loss of vestibular hair cells (vestibular hair cell damage or loss 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).

[0166] Table 4. Pendrin Sequences

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] Expression of exogenous polynucleotides in mammalian cells

[0175] By administering a nucleic acid vector comprising 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 higher sequence identity) to any one of SEQ ID NO: 4, 5, 8 or 9) operably linked to a promoter (e.g., the SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 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 molecule), or by administering a nucleic acid vector comprising an SLC26A4 promoter (e.g., the SLC26A4 promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NO: 1 and 20-27) optionally operably linked to an SLC26A4 enhancer (e.g., the SLC26A4 enhancer of Table 3) and operably linked to a polynucleotide encoding an expression product (e.g., a protein or RNA molecule (such as an inhibitory RNA molecule) of interest), the compositions and methods described herein can be used to induce or increase the expression of an exogenous polynucleotide (e.g., a gene endogenously expressed in cells expressing SLC26A4, such as a polynucleotide encoding prestin) specifically in cells expressing SLC26A4 (e.g., inner ear cells expressing SLC26A4, such as interdental cells, root cells, spiral prominence cells and vestibular supporting cells). A variety of methods have been established for delivering proteins to mammalian cells and for stably expressing polynucleotides encoding proteins in mammalian cells.

[0176] The nucleic acid vectors described herein (e.g., AAV vectors) (e.g., nucleic acid vectors containing at least one SLC26A4 enhancer operably linked to a promoter (e.g., the SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or an enhancer-promoter, minimal promoter, core promoter, or constitutive 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 higher sequence identity) to any one of SEQ ID NO:4, 5, 8, or 9), or nucleic acid vectors containing an SLC26A4 promoter (e.g., the SLC26A4 promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NO:1 and 20-27) optionally operably linked to an SLC26A4 enhancer (e.g., the SLC26A4 enhancer of Table 3)) can be used to express polynucleotides in one or more cells that express SLC26A4 (e.g., inner ear cells that express SLC26A4). Exemplary polynucleotides that can be expressed using the nucleic acid vectors described herein include polynucleotides encoding proteins (such as prestin and Atoh1) that are expressed in healthy SLC26A4-expressing cells, polynucleotides corresponding to the wild-type form of a gene that is endogenously expressed in inner ear cells that express SLC26A4 and is mutated in a subject with hearing loss, deafness, tinnitus, or vestibular dysfunction, and other polynucleotides that can be expressed in inner ear cells that express SLC26A4 to treat hearing loss, deafness, tinnitus, or vestibular dysfunction. The nucleic acid vectors described herein can also be used to express short hairpin RNAs (shRNAs), antisense oligonucleotides (ASOs), components of gene editing systems (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 that express SLC26A4 (e.g., inner ear cells that express SLC26A4, such as interdental cells, root cells, spiral prominence cells, and vestibular supporting cells).

[0177] In some embodiments, a polynucleotide encoding 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 higher sequence identity) with the amino acid sequence of wild-type mammalian (e.g., human or mouse) Atoh1 (e.g., SEQ ID NO: 36 or SEQ ID NO: 38)) is operably linked to the SLC26A4 promoter described herein (e.g., a promoter of formula B-A-C, such as 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 higher sequence identity) with any one of SEQ ID NOs: 1 and 20-27), which may optionally be operably linked to an SLC26A4 enhancer (e.g., the SLC26A4 enhancer provided in Table 3), or is operably linked to a promoter (e.g., an SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or an enhancer-promoter, minimal promoter, core promoter or constitutive promoter) operably linked to one or more SLC26A4 enhancers described herein (e.g., one 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 higher sequence identity) with any one of SEQ ID NOs: 4, 5, 8, 9, 34 and 35). 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: 36, 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: 36 or SEQ ID NO: 38 by the redundancy of the genetic code. The polynucleotide sequence encoding Atoh1 may be partially or fully codon-optimized for expression (e.g., in inner ear cells (such as vestibular supporting cells) expressing human SLC26A4).The Atoh1 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., an SNP that does not cause hearing loss). Human Atoh1 can be encoded by a polynucleotide having the sequence of SEQ ID NO:37. Mouse Atoh1 can be encoded by a polynucleotide having the sequence of SEQ ID NO:39. The Atoh1 protein can be a human Atoh1 protein or can be a homolog of the human Atoh1 protein from another mammalian species (e.g., mouse, rat, bovine, horse, goat, sheep, donkey, cat, dog, rabbit, guinea pig, or other mammal). Exemplary Atoh1 amino acid and polynucleotide sequences are listed in Table 5 below. A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer (e.g., an SLC2A4 enhancer of any of SEQ ID NO:4, 5, 8, 9, 34, and 35) operably linked to a promoter (e.g., the SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or an enhancer - promoter, minimal promoter, core promoter, or constitutive promoter) operably linked to a polynucleotide encoding Atoh1, or a nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 promoter (e.g., a promoter of formula B - A - C, e.g., an 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 higher sequence identity) with any of SEQ ID NO:1 and 20 - 27) operably linked to a polynucleotide encoding Atoh1 and optionally an SLC26A4 enhancer (e.g., an SLC26A4 enhancer of Table 3) described herein 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).

[0178] Table 5. Atoh1 Sequences

[0179]

[0180]

[0181]

[0182] Polynucleotide encoding the protein of interest

[0183] One platform that can be used to achieve therapeutically effective intracellular concentrations of a protein of interest in mammalian cells is through 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 formation of an episomal concatemer in the nucleus of a mammalian cell). The gene is a polynucleotide encoding the primary amino acid sequence of the corresponding protein. To introduce a foreign 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, for example, in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0184] The protein of interest can also be introduced into mammalian cells by targeting a vector containing the gene encoding the protein of interest to cell membrane phospholipids. For example, by linking a vector molecule to the VSV-G protein (a viral protein that has an affinity for all cell membrane phospholipids), the vector can be targeted to phospholipids on the extracellular surface of the cell membrane. Such constructs can be generated using methods well known to those skilled in the art.

[0185] The recognition and binding of mammalian RNA polymerase to a polynucleotide encoding a protein of interest are important for gene expression. Accordingly, sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and facilitate the assembly of the transcription complex at the transcription start site can be included within the polynucleotide. Such sequence elements include, for example, mammalian promoters, the sequences of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters have been described in the online publication by Smith et al., Mol. Sys. Biol., 3:73, the disclosure of which is incorporated herein by reference. The promoters used in the methods and compositions described herein can be the SLC26A4 promoter (e.g., the SLC26A4 promoter or enhancer-promoter provided in Table 2), a constitutive promoter (e.g., a promoter that is active in vivo in all circumstances), a core promoter or a minimal promoter. Constitutive promoters include the CAG promoter, the cytomegalovirus (CMV) promoter (e.g., the CMV immediate early enhancer and promoter, the CMV mini promoter, the minCMV promoter, the CMV-TATA+INR promoter or the min CMV-T6 promoter), the smCBA promoter (described in Haire et al., Invest. Opthalmol. Vis. Sci. 47:3745-3753, 2006), the CBA promoter, the CASI promoter, the dihydrofolate reductase (DHFR) promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, the β-globin promoter (e.g., the minimal β-globin promoter), the HSV promoter (e.g., the minimal HSV ICP0 promoter or the truncated HSV ICP0 promoter), the SV40 promoter (e.g., the SV40 minimal promoter) and the EF1α promoter. Constitutive promoters can also be referred to as ubiquitous promoters because they are capable of inducing the expression of polynucleotides in a wide range of cell and tissue types. Minimal promoters include the CMV minimal promoter (e.g., the minCMV promoter), the minimal β-globin promoter, the minimal HSV promoter (e.g., the minimal HSV ICP0 promoter) and the SV40 minimal promoter. Alternatively, promoters derived from viral genomes can also be used for the 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 the adenovirus late promoter, the vaccinia virus 7.5K promoter, the tk promoter of HSV, the mouse mammary tumor virus (MMTV) promoter, the LTR promoter of HIV, the Moloney virus promoter, the Epstein Barr virus (EBV) promoter and the Rous sarcoma virus (RSV) promoter.

[0186] Once a polynucleotide encoding a protein of interest is incorporated into mammalian cells, transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing mammalian cells to external chemical agents such as agents that modulate the binding of transcription factors and / or RNA polymerase to mammalian promoters and thereby regulate gene expression. Chemical agents can be used to facilitate the binding of RNA polymerase and / or transcription factors to mammalian promoters, for example, by removing repressor proteins that have bound to the promoter. Alternatively, chemical agents can be used to enhance the affinity of mammalian promoters for RNA polymerase and / or transcription factors such that in the presence of the chemical agent, the transcription rate of genes located downstream of the promoter is increased. Examples of chemical agents that enhance polynucleotide transcription through the above mechanisms include tetracycline and doxycycline. These agents are commercially available and can be administered to mammalian cells according to established protocols to promote gene expression.

[0187] The nucleic acid vectors described herein can include the woodchuck posttranscriptional 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 total amount of mRNA in the cell. Adding the WPRE to a vector can result in a significant improvement in the level of transgene expression driven by several different promoters in vitro and in vivo.

[0188] In some embodiments, the nucleic acid vectors described herein include a reporter sequence that can be used to verify the expression of a gene operably linked to the SLC26A4 promoter and / or SLC26A4 enhancer, for example, in cells and tissues (e.g., in cells that express SLC26A4 such as interdental cells, root cells, spiral prominence 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 other substances well known in the art. When associated with regulatory elements (such as promoters) that drive their expression, reporter gene sequences provide a signal that can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescent or other spectroscopic, fluorescence-activated cell sorting, and immunoassays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, in the case where the marker sequence is the LacZ gene, the presence of the signal-carrying vector is detected by an assay for β-galactosidase activity. In the case where the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visually detected by color or light production in a luminometer.

[0189] Methods for delivering exogenous polynucleotides to target cells

[0190] 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) that are subjected to an external electric field in this manner are then prone to uptake of exogenous polynucleotides. The electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection TM utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection TM and the protocols that can be used to perform this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005) and US2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0191] Additional techniques that can be used to transfect target cells include the squeeze-filtration method. This technique induces rapid mechanical deformation of the cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to the applied stress. The advantage of this technique is that a vector is not required to deliver the polynucleotide to the cells (such as human target cells). Squeeze-filtration 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.

[0192] Lipofection represents another technique that can be used to transfect target cells. This method involves loading polynucleotides into liposomes, which typically have cationic functional groups (such as quaternary amines or protonated amines) facing the exterior of the liposome. This promotes electrostatic interactions between the liposome and the cell due to the anionic nature of the cell membrane, ultimately resulting in the uptake of exogenous polynucleotides, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that utilize ionic interactions with the cell membrane to cause the uptake of foreign polynucleotides include contacting the cells with cationic polymer - polynucleotide complexes. Exemplary cationic molecules that associate with polynucleotides to confer a positive charge favorable for interaction with the cell membrane include activated dendrimers (described in, for example, Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE) - dextran, the use of which as transfection agents is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:I:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to direct the uptake of polynucleotides. This technique is described in detail, for example, in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0193] Another available tool for inducing the uptake of exogenous polynucleotides by target cells is laserfection, also known as optical transfection, a technique that involves exposing the cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow the polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to and in some cases found to be superior to electroporation.

[0194] 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 the gene intended for intracellular delivery is attached to the nanostructure surface. Then, a chip with an array of these needles is pressed against the cells or tissue. The cells pierced by the nanostructures can express one or more of the delivered genes. An example of this technique is described in Shalek et al., PNAS 107:1870 (2010), the disclosure of which is incorporated herein by reference.

[0195] Magnetofection can also be used to deliver polynucleotides to target cells. The principle of magnetofection is to associate the polynucleotides with cationic magnetic nanoparticles. The magnetic nanoparticles are made of fully biodegradable iron oxide and coated with specific cationic proprietary molecules that vary according to the application. Their association with gene carriers (DNA, siRNA, viral vectors, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interactions. Then, the magnetic particles are affected by an external magnetic field generated by a magnet and concentrated on the target cells. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0196] Another available tool for inducing the uptake of exogenous polynucleotides by target cells is sonoporation, a technique that involves using sound (usually at ultrasonic frequencies) to alter the permeability of the cytoplasmic membrane, rendering the cells permeable and allowing the polynucleotides to penetrate the cell membrane. This technique is described in detail in, for example, Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0197] Microvesicles represent another potential mediator that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles generated by co-overexpression of the glycoprotein VSV-G with, for example, a genome-modifying protein such as a nuclease can be used to efficiently deliver proteins into cells, which subsequently catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare cells for covalent incorporation of a polynucleotide of interest such as a gene or regulatory sequence into the genome. The use of such vesicles (also known as nanovesicles (Gesicles)) in the genetic modification of eukaryotic cells is described in detail in, 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 th Annual Meeting of the American Society of Gene and Cell Therapy); May 13, 2015, abstract No. 122.

[0198] Vectors for delivering exogenous polynucleotides to target cells

[0199] 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, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors for use in the compositions and methods described herein contain 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 higher sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) and / or an SLC26A4 promoter (e.g., a promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NOs: 1 and 20-27), a polynucleotide encoding an expression product operably linked thereto (e.g., a polynucleotide 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 mammalian cell genome. 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 within a cell), 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. Some vectors for expressing an expression product (e.g., a protein of interest) include plasmids containing regulatory sequences that direct gene transcription. Other useful vectors for expressing an expression product (e.g., a protein of interest) contain polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of the mRNA produced by gene transcription.These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRESs), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0200] Viral vectors for polynucleotide delivery

[0201] Viral genomes provide a rich source of vectors that can be used to efficiently deliver genes of interest into the genomes of target cells (e.g., mammalian cells, such as human cells). Viral genomes are vectors particularly useful for gene delivery because the polynucleotides contained within such genomes are generally 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 viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles 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, enveloped viruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, human papillomavirus, human foamy virus, and hepatitis viruses. Examples of retroviruses include avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoviruses, HTLV-BLV group, lentiviruses, alpha-retroviruses, gamma-retroviruses, foamy viruses (Coffin, J.M., 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 leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which regarding viral vectors for use in gene therapy is incorporated herein by reference.

[0202] AAV vectors for polynucleotide delivery

[0203] 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. In some embodiments, the rAAV vectors useful in the compositions and methods described herein are recombinant polynucleotide constructs that include (1) a promoter (e.g., the SLC26A4 promoter, such as the SLC26A4 promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NOs: 1 and 20-27), (2) a sequence to be expressed (e.g., a polynucleotide encoding a protein such as pannexin or Atoh1, or a polynucleotide that can be transcribed to produce an RNA molecule such as inhibitory RNA), and (3) viral sequences that facilitate the integration and expression of the sequence to be expressed. In some embodiments, the rAAV vector further includes 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 higher sequence identity) to any one of SEQ ID NOs: 4-9, 34 and 35). In other embodiments, the rAAV vectors useful in the compositions and methods described herein are recombinant polynucleotide constructs that include (1) 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 higher sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34 and 35), (2) a promoter (e.g., the SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or enhancer-promoter, minimal promoter, core promoter or constitutive promoter), (3) a sequence to be expressed (e.g., a polynucleotide encoding a protein such as pannexin or Atoh1, or a polynucleotide that can be transcribed to produce an RNA molecule such as inhibitory RNA), and (4) viral sequences that facilitate the integration and expression of the sequence to be expressed. The viral sequences can include those sequences of AAV required for the cis replication and packaging of DNA (e.g., functional ITRs) into virions.In typical applications, the sequence to be expressed encodes the wild-type form of a protein (such as the wild-type form of pendrin) that is mutated in a subject with a form of hereditary hearing loss and is expressed in inner ear cells that express SLC26A4, or a protein or RNA molecule (such as Atoh1) that can promote the differentiation of vestibular supporting cells into vestibular hair cells. Such rAAV vectors can also contain a marker gene or a reporter gene. Available rAAV vectors have one or more deletions in the AAVWT gene that are either complete or partial but still retain functional flanking ITR sequences. The AAV ITR can be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITR can be an AAV2 ITR. Methods of using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which regarding AAV vectors for gene delivery are incorporated herein by reference.

[0204] The polynucleotides and vectors described herein (e.g., SLC26A4 enhancer and / or SLC26A4 promoter operably linked to a polynucleotide encoding an expression product) can be incorporated into rAAV virions to facilitate introduction of the polynucleotide or vector into cells. The capsid proteins of AAV form the outer non-nucleic acid portion of the virion and are encoded by the AAVcap gene. The cap gene encodes the three viral capsid proteins VP1, VP2, and VP3 required for virion assembly. The construction of rAAV virions has been described, for example, in 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 Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which regarding AAV vectors for gene delivery are incorporated herein by reference.

[0205] rAAV virions for use in combination 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 that express SLC26A4, AAV1, AAV2, AAV2quad(Y-F), 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 and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference because they relate to AAV vectors for gene delivery.

[0206] Also useful in combination with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) that are pseudotyped with capsid genes derived from serotypes other than the given serotype (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques related to the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0207] AAV virions having mutations within the viral capsid can be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations that promote targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine scan mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV viral particles that can be used in the methods described herein include those capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0208] Pharmaceutical composition

[0209] 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 higher sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) and / or the SLC26A4 promoter (e.g., a promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NOs: 1 and 20-27) can be operably linked to a polynucleotide encoding an expression product (e.g., a transgene encoding a protein or RNA molecule of interest, such as an inhibitory RNA), and incorporated into a vector for administration to a patient, such as a human patient suffering from sensorineural hearing loss (e.g., pendrin-related hearing loss), vestibular dysfunction (e.g., vestibular dysfunction associated with vestibular hair cell damage or loss or pendrin-related vestibular dysfunction), or Meniere's disease. Pharmaceutical compositions containing a vector (such as a viral vector) 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 the desired form (e.g., in the form of a lyophilized preparation or an aqueous solution).

[0210] A mixture of a nucleic acid vector (e.g., a viral vector) containing an SLC26A4 enhancer as described herein operably linked to a polynucleotide encoding an expression product (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 higher sequence identity) with any one of SEQ ID NO: 4, 5, 8, 9, 34, and 35) and / or an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity with any one of SEQ ID NO: 1 and 20 - 27 (e.g., a promoter of formula B - A - C, 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 higher sequence identity) therewith)) can be prepared in water suitably mixed with one or more excipients, carriers, or diluents. The dispersion can also be prepared in glycerol, liquid polyethylene glycols, and their mixtures, and in oils. Under normal storage and use conditions, these preparations may contain preservatives to prevent the growth of microorganisms. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the preparation should be sterile and be fluid to the extent that easy injectability exists. The preparation can be stable under the conditions of manufacture and storage and can be preserved under conditions that prevent the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0211] For example, if desired, solutions containing the pharmaceutical compositions described herein may be appropriately buffered and first made isotonic with sufficient saline or dextrose to dilute the liquid diluent. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art from the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site. Depending on the disorder of the subject being treated, certain dosage variations will necessarily occur. For topical administration to the inner ear, the composition can be formulated to contain synthetic perilymph. Exemplary synthetic perilymph includes 20 - 200 mM NaCl, 1 - 5 mM KCl, 0.1 - 10 mM CaCl2, 1 - 10 mM glucose, and 2 - 50 mM HEPES, having a pH between about 6 and 9 and an osmolarity of about 300 mOsm / kg. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. In addition, for human administration, the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0212] Method of treatment

[0213] 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 topically to the middle ear or inner ear (e.g., to the perilymph or endolymph, such as to the oval window, round window, or semicircular canals (e.g., horizontal canal) or through the oval window, round window, or semicircular canals, or by transtympanic or intratympanic injection, e.g., to inner ear cells expressing SLC26A4), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. In any given case, the most suitable route of administration will depend on the particular composition being administered, the patient, the method of drug formulation, the method of administration (e.g., time of administration and route of administration), the age, weight, sex of the patient, the severity of the disease being treated, the diet of the patient, and the excretion rate of the patient. The composition can be administered once or more than once (e.g., once a year, twice a year, three times a year, once every two months, once a month, or once every two weeks).

[0214] Subjects treatable as described herein are subjects suffering from or at risk of developing sensorineural hearing loss. In some embodiments, the compositions described herein are used to treat pendrin-related hearing loss (e.g., DFNB4 or Pendred syndrome). DFNB4 and Pendred 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 higher sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) and / or an SLC26A4 promoter (e.g., a promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NOs: 1 and 20-27) operably linked to a polynucleotide encoding pendrin (e.g., a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11). The subject may have or be identified as having a mutation in SLC26A4 and may have severe, moderate, or mild hearing loss at the start of treatment or may 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, e.g., a subject carrying an SLC26A4 mutation associated with hearing loss but not yet exhibiting hearing impairment.

[0215] In some embodiments, the compositions described herein are used to treat a subject having Meniere's disease. Subjects having a mutation in SLC26A4 and subjects having Meniere's disease both have endolymphatic hydrops, and thus, a composition useful for treating a subject having a mutation in SLC26A4 (e.g., a nucleic acid vector comprising 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 higher sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) and / or an SLC26A4 promoter (e.g., a promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NOs: 1 and 20-27) operably linked to a polynucleotide encoding prestin (e.g., a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11)) may also be effective in reducing or ameliorating endolymphatic hydrops in a subject having Meniere's disease. Such treatment can be used to treat hearing loss, tinnitus, or vestibular dysfunction (e.g., vertigo) in a subject having Meniere's disease and can reduce a feeling of fullness or congestion in the ear.

[0216] In some embodiments, the compositions described herein are used to treat a subject having or at risk of developing vestibular dysfunction (e.g., vertigo, dizziness, imbalance, oscillopsia, balance disorder, or bilateral vestibulopathy). In some embodiments, the vestibular dysfunction is pendrin-related vestibular dysfunction associated with DFNB4 or Pendred syndrome (e.g., imbalance or loss of balance associated with DFNB4 or Pendred syndrome). Pendrin-related vestibular dysfunction can be treated by administering a nucleic acid vector comprising 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 higher sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) and / or an SLC26A4 promoter (e.g., a promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NOs: 1 and 20-27) operably linked to a polynucleotide encoding pendrin (e.g., a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11). 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 start of treatment or may be treated prior to the onset of symptoms (e.g., prophylactic treatment).

[0217] Vestibular dysfunction can also be caused by damage or loss of vestibular hair cells. Thus, 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 a disease or infection, head trauma, ototoxic drugs (e.g., vestibulotoxic 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 a gene mutation 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 an autoimmune response results in 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 and Behçet's disease. Some infectious disorders (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 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 administered as a prophylactic 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 exhibiting symptoms of vestibular dysfunction, or subjects exposed to risk factors for acquired vestibular dysfunction (e.g., disease or infection, head trauma, ototoxic drugs or aging). The compositions and methods described herein can also be used to treat subjects suffering from idiopathic vestibular dysfunction.

[0218] 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 have 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 who have abnormal vestibular hair cells (e.g., vestibular hair cells that do not function properly compared to normal vestibular hair cells), damaged vestibular hair cells (e.g., vestibular hair cell damage associated with trauma (e.g., head trauma), disease or infection, ototoxic drugs, or aging), or a reduced number of vestibular hair cells due to gene mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase vestibular hair cell maturation, which can lead to improved vestibular function. In some embodiments, the compositions and methods described herein promote or increase the maturation of regenerated vestibular hair cells.

[0219] The compositions and methods described herein can also be used to prevent or reduce vestibular dysfunction caused by ototoxic drug-induced vestibular hair cell damage or death (e.g., vestibular hair loss induced by vestibulotoxic drugs) in subjects who have received or are receiving or about to begin receiving ototoxic drug treatment. Ototoxic drugs are toxic to cells of the inner ear and can cause vestibular dysfunction (e.g., vertigo, dizziness, imbalance, bilateral vestibular lesions, or oscillopsia). Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin, and netilmicin), viomycin, antitumor drugs (e.g., platinum-containing chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin, or other chemotherapeutic agents such as mechlorethamine and vincristine), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, especially at high doses), and quinine. Some of these drugs, such as mechlorethamine, vincristine, gentamicin, streptomycin, and tobramycin, have been specifically identified as vestibulotoxic 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 subjects with aminoglycoside-induced bilateral vestibular lesions or oscillopsia).

[0220] Vestibular dysfunction associated with vestibular hair cell damage or loss (e.g., vestibular hair cell damage or loss associated with a disease or infection, head trauma, ototoxic drugs (e.g., vestibulotoxic drugs), or aging) can be treated by administering a nucleic acid vector comprising an 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 higher sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) or an SLC26A4 promoter (e.g., a promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NOs: 1 and 20-27) operably linked to a polynucleotide encoding Atoh1 (e.g., a polynucleotide encoding SEQ ID NO: 36 or SEQ ID NO: 38) or a polynucleotide encoding pannexin (e.g., a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11). 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.

[0221] The methods described herein may include screening a subject for one or more mutations in a gene known to be associated with hearing loss or vestibular dysfunction (e.g., SLC26A4) prior to treatment or administration with the compositions described herein. The subject may be screened for gene mutations using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein may also include assessing the hearing of a subject prior to treatment with or administration of the compositions described herein. Hearing may be assessed using standard tests such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions. These tests may also be used to assess the hearing of a subject after treatment or administration with the compositions described herein. In some embodiments, the methods described herein include assessing the vestibular function of a subject prior to treatment with or administration of the compositions described herein. Vestibular function may be assessed using standard tests such as oculomotor tests (e.g., electro-oculography (ENG) or video-oculography (VNG)), vestibulo-ocular reflex (VOR) tests (e.g., head impulse test (Halmagyi–Curthoys test) that may be performed at the bedside or using video head impulse test (VHIT)), or caloric reflex test), posturography, rotatory chair test, ECOG, vestibular evoked myogenic potential (VEMP), and specialized clinical balance tests such as those described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010). These tests may also be used to assess the vestibular function of a subject after treatment or administration with the compositions described herein.

[0222] A polynucleotide encoding an expression product operably linked to an SLC26A4 promoter (e.g., a promoter of formula B-A-C, 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 higher sequence identity) to any one of SEQ ID NO: 1 and 20 - 27) and / or an 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 higher sequence identity) to any one of SEQ ID NO: 4, 5, 8, 9, 34 and 35) for treating a subject as described herein can be a polynucleotide encoding pannexin (e.g., a polynucleotide encoding the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11), a polynucleotide encoding Atoh1 (e.g., a polynucleotide encoding the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 38), a polynucleotide encoding the wild-type form of a protein expressed in inner ear cells that express SLC26A4 and is mutated in a subject 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), transcription activator-like effector nuclease (TALEN) or zinc finger nuclease (ZFN) or guide RNA (gRNA)) or a microRNA. 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 pannexin, 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.

[0223] Treatment may include administering a composition containing a nucleic acid vector (e.g., an AAV vector) containing the SLC26A4 enhancer and / or SLC26A4 promoter described herein, in various unit doses. Each unit dose will typically 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 in the clinical art. The unit dose need not be administered as a single injection, but may comprise a continuous infusion over a set period of time. Administration may be carried out using a syringe pump to control the infusion rate so as to minimize damage to the inner ear (e.g., the cochlea and / or vestibular system). Where the nucleic acid vector is an AAV vector (e.g., AAV1, AAV2, AAV2quad(Y-F), 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, from about 1x10 9 vector genomes (VG) / mL to about 1x10 16 VG / mL (e.g., 1x10 9 VG / mL, 2x10 9 VG / mL, 3x10 9 VG / mL, 4x10 9 VG / mL, 5x10 9 VG / mL, 6x10 9 VG / mL, 7x10 9 VG / mL, 8x10 9 VG / mL, 9x10 9 VG / mL, 1x10 10 VG / mL, 2x10 10 VG / mL, 3x10 10 VG / mL, 4x10 10 VG / mL, 5x10 10 VG / mL, 6x10 10 VG / mL, 7x10 10 VG / mL, 8x10 10 VG / mL, 9x10 10 VG / mL, 1x10 11 VG / mL, 2x10 11 VG / mL, 3x10 11 VG / mL, 4x10 11 VG / mL, 5x10 11 VG / mL, 6x10 11 VG / mL, 7x10 11VG / mL, 8x10 11 VG / mL, 9x10 11 VG / mL, 1x10 12 VG / mL, 2x10 12 VG / mL, 3x10 12 VG / mL, 4x10 12 VG / mL, 5x10 12 VG / mL, 6x10 12 VG / mL, 7x10 12 VG / mL, 8x10 12 VG / mL, 9x10 12 VG / mL, 1x10 13 VG / mL, 2x10 13 VG / mL, 3x10 13 VG / mL, 4x10 13 VG / mL, 5x10 13 VG / mL, 6x10 13 VG / mL, 7x10 13 VG / mL, 8x10 13 VG / mL, 9x10 13 VG / mL, 1x10 14 VG / mL, 2x10 14 VG / mL, 3x10 14 VG / mL, 4x10 14 VG / mL, 5x10 14 VG / mL, 6x10 14 VG / mL, 7x10 14 VG / mL, 8x10 14 VG / mL, 9x10 14 VG / mL, 1x10 15 VG / mL, 2x10 15 VG / mL, 3x10 15 VG / mL, 4x10 15 VG / mL, 5x10 15 VG / mL, 6x10 15 VG / mL, 7x10 15 VG / mL, 8x10 15 VG / mL, 9x10 15 VG / mL, or 1x10 16Doses of (VG / mL) are 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). The AAV vector can be about 1x10 7 VG / ear to about 2x10 15 VG / ear (e.g., 1x10 7 VG / ear, 2x10 7 VG / ear, 3x10 7 VG / ear, 4x10 7 VG / ear, 5x10 7 VG / ear, 6x10 7 VG / ear, 7x10 7 VG / ear, 8x10 7 VG / ear, 9x10 7 VG / ear, 1x10 8 VG / ear, 2x10 8 VG / ear, 3x10 8 VG / ear, 4x10 8 VG / ear, 5x10 8 VG / ear, 6x10 8 VG / ear, 7x10 8 VG / ear, 8x10 8 VG / ear, 9x10 8 VG / ear, 1x10 9 VG / ear, 2x10 9 VG / ear, 3x10 9 VG / ear, 4x10 9 VG / ear, 5x10 9 VG / ear, 6x10 9 VG / ear, 7x10 9 VG / ear, 8x10 9 VG / ear, 9x10 9 VG / ear, 1x10 10 VG / ear, 2x10 10 VG / ear, 3x10 10 VG / ear, 4x10 10 VG / ear, 5x10 10 VG / ear, 6x10 10 VG / ear, 7x10 10 VG / ear, 8x1010 VG / ear, 9x10 10 VG / ear, 1x10 11 VG / ear, 2x10 11 VG / ear, 3x10 11 VG / ear, 4x10 11 VG / ear, 5x10 11 VG / ear, 6x10 11 VG / ear, 7x10 11 VG / ear, 8x10 11 VG / ear, 9x10 11 VG / ear, 1x10 12 VG / ear, 2x10 12 VG / ear, 3x10 12 VG / ear, 4x10 12 VG / ear, 5x10 12 VG / ear, 6x10 12 VG / ear, 7x10 12 VG / ear, 8x10 12 VG / ear, 9x10 12 VG / ear, 1x10 13 VG / ear, 2x10 13 VG / ear, 3x10 13 VG / ear, 4x10 13 VG / ear, 5x10 13 VG / ear, 6x10 13 VG / ear, 7x10 13 VG / ear, 8x10 13 VG / ear, 9x10 13 VG / ear, 1x10 14 VG / ear, 2x10 14 VG / ear, 3x10 14 VG / ear, 4x10 14 VG / ear, 5x10 14 VG / ear, 6x10 14 VG / ear, 7x10 14 VG / ear, 8x10 14 VG / ear, 9x10 14 VG / ear, 1x10 15 VG / ear or 2x10 15 A dose of VG / ear) is administered to the subject.

[0224] The compositions described herein are administered in an amount sufficient to improve or restore (e.g., salvage) 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 vestibulopathy, treat oscillopsia, inhibit or slow the progression of vestibular dysfunction, reduce a 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 the expression of an expression product in cells expressing SLC26A4 (e.g., interdental cells, spiral prominence cells, root cells, or vestibular supporting cells). Hearing can be evaluated using standard hearing tests (e.g., audiometry, ABR, electrocochleography (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 the hearing measurements obtained prior to 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 are also capable of being administered in an amount sufficient to delay or prevent the development of sensorineural hearing loss or deafness (e.g., in subjects carrying an SLC26A4 mutation but not showing hearing impairment at the time of treatment, or in subjects showing mild to moderate hearing loss at the time of treatment). Vestibular function can be evaluated using standard tests of balance and vertigo (e.g., oculomotor tests (e.g., ENG or VNG), VOR tests (e.g., head impulse test (Halmagyi-Curthoys test, e.g., VHIT) or caloric reflex test), posturography, rotatory chair test, ECOG, VEMP, and specialized clinical balance tests), 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 the measurements obtained prior to treatment. The compositions described herein are also capable of being 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 drugs, head trauma, or disease or infection) but do not show vestibular dysfunction (e.g., vertigo, dizziness, or imbalance) at the time of treatment, or in subjects showing mild to moderate vestibular dysfunction).Immunohistochemistry, western blot analysis, quantitative real-time PCR, or other methods known in the art for detecting proteins or mRNAs can be used to evaluate the expression of the protein encoded by the transgene operably linked to the SLC26A4 promoter and / or enhancer in a nucleic acid vector administered to a subject or to cells, and the expression may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to the expression prior to administration of the compositions described herein. Vestibular hair cell regeneration can be indirectly evaluated 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 prior to administration of the compositions described herein or compared to untreated subjects. The compositions and methods described herein can also reduce the toxicity associated with administration of the nucleic acid vector compared to the toxicity observed after administration of a nucleic acid vector that does not contain the SLC26A4 promoter and / or enhancer described herein (e.g., administration of a nucleic acid vector that expresses the same transgene using a ubiquitous promoter and / or does not use the 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 longer after administration of the compositions described herein. Depending on the dose and route of administration for treatment, the patient can be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer after administration of the composition. Based on the evaluation results, the patient may receive additional treatment.

[0225] Kit

[0226] The compositions described herein can be provided in a kit for treating sensorineural hearing loss or vestibular dysfunction. The composition can include one or more of the SLC26A4 enhancers 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 higher sequence identity) to any of SEQ ID NO: 4, 5, 8, 9, 34, and 35) and / or an SLC26A4 promoter (e.g., a promoter of formula B-A-C, 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 higher sequence identity) to any of SEQ ID NO: 1 and 20-27), a nucleic acid vector containing such polynucleotides, 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., prestin) or vestibular dysfunction (e.g., prestin 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(Y-F), AAV6, AAV8, AAV9, Anc80, Anc80L65, AAV-DJ, AAV-DJ / 9, 7m8, or PHP.B). The kit can also include package instructions that direct a user of the kit (such as a physician) to perform the methods described herein. The kit can optionally include a syringe or other device for administering the composition.

[0227] Examples

[0228] The following examples are provided to describe for the ordinary skilled person in the art how to use, prepare, and evaluate the compositions and methods described herein, and these examples are only intended as illustrations of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0229] Example 1. Transfection of the mouse cochlear lateral wall with AAV-DJ vectors containing various promoters and enhancers driving GFP expression

[0230] The lateral wall was dissected from the cochleae of 6- to 8-week-old male C57BL / 6J mice (000664, The Jackson Laboratory) for co-culture with AAV-DJ viral vectors containing a nuclear-targeted H2B-EGFP fusion transgene driven by multiple promoters (CMV and the various SLC26A4 promoters described herein, without the SLC26A4 enhancer) and AAV-DJ vectors containing an H2B-EGFP fusion transgene driven by the PEND3.1.8 promoter (SEQ ID NO:1) or the PEND3.1.6 promoter (SEQ ID NO:23) and various murine or human SLC26A4 enhancers. Figure 1A-1C Shows the general design of the transgenic plasmids used to generate the AAV viral vectors, and Figure 7 Shows the relative sizes and alignments of the various SLC26A4 promoters used in these experiments. After euthanizing the animals by CO2 asphyxiation, 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 a glass-bottom dish (10810-054, Matsunami Glass). The AAV was added to the medium in 250 μl of medium and left in the medium for 3 days, then washed with 2 ml of fresh medium prepared as described above. The cultured lateral walls were then maintained in culture for an additional 2 days (for a total of 5 days after adding the AAV to the medium).

[0231] At the end of the culture, 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 tissues were blocked with 10% normal donkey serum, 0.5% Triton X-100 in PBS at pH 7.4 for 1 hour at room temperature and then incubated overnight at 4°C with the primary antibody against pannexin (BiCell Scientific 20501) diluted 1:100 in 0.5% Triton X-100 in 1X PBS. The next day, after washing with PBS (3 times, 5 minutes), the tissues were incubated with the secondary antibody (1:500; Invitrogen A10042: donkey (host), rabbit IgG (target species) conjugated to Alexa Fluor 568) for 2 hours at room temperature. After secondary antibody incubation, the tissues were washed with PBS (3x, 5 minutes) and then mounted in Slowfade Diamond antifade mounting medium (DAKO) (ThermoFisher Molecular probes, s36963).

[0232] After fixation, the lateral walls were imaged on a Zeiss LSM 880 confocal microscope in the 488 (EGFP) and 568 (pannexin) 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 at the same laser power and gain in the study ( Figure 3A-3I all images in Figure 4A-4B were imaged with the same settings; Figure 5 all images in Figure 10A were imaged with the same settings; and

[0233] all images in Figure 6 were imaged with the same settings) to allow comparison between groups.

[0234] FromFigure 2A As can be seen, most of the cells expressing prestin are present in the spiral prominence and tight junctions of the stria vascularis. AAV expressing EGFP under the control of the ubiquitous CMV promoter produces EGFP expression throughout the lateral wall, which is not restricted to the prestin-expressing region, demonstrating the ubiquitous AAV-DJ tropism in the lateral wall with this promoter ( Figure 2B ). Replacing the CMV promoter with various SLC26A4 promoters disclosed herein results in EGFP expression being mainly restricted to cells that also express prestin, with little or no EGFP expression in other cells of the lateral wall ( Figure 3A-3I ).

[0235] Including certain SLC26A4 enhancer sequences (mouse E6 (SEQ ID NO:7), mouse E2 (SEQ ID NO:6), or human E6.4 (SEQ ID NO:5)) described herein fused to the 5' end of the PEND3.1.8 promoter (SEQ ID NO:1) in an AAV vector expressing EGFP results in increased EGFP expression without loss of specificity for prestin-expressing cells ( Figure 4A -4C). Interestingly, one other SLC26A4 enhancer (human E6.1; SEQ ID NO:4) results in decreased EGFP expression, although specificity for prestin-expressing cells is retained (Figure 4D). Two other enhancers (human E6.2 (SEQ ID NO:8) and E6.3 (SEQ ID NO:9)) result in loss of specificity for prestin-expressing cells and lead to EGFP expression throughout the lateral wall (Figures 4E-4F).

[0236] Including certain SLC26A4 enhancer sequences (mouse E6 (SEQ ID NO:7) or human E6.4 (SEQ ID NO:5)) described herein fused to the 5' end of the PEND3.1.6 promoter (SEQ ID NO:23) in an AAV vector expressing EGFP results in increased EGFP expression without loss of specificity for prestin-expressing cells ( Figure 5 A-5B).

[0237] Quantifying EGFP expression in prestin-positive and prestin-negative regions of the lateral wall using the PEND3.1.8 promoter with or without one of the mouse E6 or E2 enhancers or the human E6.1 or E6.4 enhancers is shown in Figure 6 In comparison to the PEND3.1.8 promoter alone, including the mouse E6 or E2 enhancer or the human E6.4 enhancer increased the percentage of prestin-expressing cells that also express EGFP ( Figure 6Panel A). The presence of the human E6.4 enhancer increased the number of cells expressing EGFP in the paneth cell-positive region of the lateral wall compared to the PEND3.1.8 promoter alone. Figure 6 Panel B).

[0238] In separate experiments, the quantification of EGFP expression from AAV vectors containing both the murine E2 and E6 SLC26A4 enhancers that were fused to each other and to the 5' end of the PEND3.1.8 promoter, generated from the transgenic plasmid P1708 Figure 9 ) was compared to EGFP expression from AAV vectors containing only one or the other of the E2 or E6 SLC26A4 enhancers. As Figure 10A can be seen, each of these AAV vectors induced specific expression in paneth cells, but expression was significantly lower in the AAV vector carrying only the E2 enhancer. As Figure 10B shown, inclusion of both enhancers did not significantly increase EGFP expression in cells expressing panethin compared to AAV vectors containing only the murine E6 enhancer. Compared to previous experiments using those same vectors, the seemingly lower GFP intensity observed in this experiment using AAV vectors containing only the murine E2 enhancer or only the murine E6 enhancer (compare Figure 4A with Figure 10A ) was due to the use of a lower laser power to visualize EGFP in the latter experiment.

[0239] Example 2. Single-cell RNA-seq analysis of murine lateral wall cells transfected with AAV-DJ vectors containing various promoters and enhancers

[0240] The lateral wall of the cochlea of 6-8-week-old male C57BL / 6J mice (000664, The Jackson Laboratory) was excised as described in Example 1 and transfected simultaneously with a combination of different AAV constructs, each containing a unique promoter or promoter-enhancer combination, and a unique barcode. Controls included constructs containing the ubiquitous Pgk promoter and constructs lacking a promoter. The promoterless was a filler sequence belonging to the ATP-binding cassette subfamily A member 4 (ABCA4) gene. The promoterless.minP was a shorter version of the same filler sequence with a minimal β-globin promoter sequence downstream. The AAV constructs containing promoters were prepared from transgenic plasmids with the general design shown in Figure 1A-1B . Seven days after AAV transfection, the cultured lateral wall explants were processed for scRNA-seq experiments. The tissues were pooled and dissociated into single cells using a combination of enzymatic digestion and mechanical disruption. After dissociation, the cell suspension was filtered to remove debris, and then the cells were washed and counted.

[0241] To identify different cell types in the lateral wall, targeted capture of 10,000 cells per sample was performed using the v3.1 chemistry with the high-throughput droplet microfluidics GemCode platform from 10x Genomics. Each droplet contains a single cell and a gel bead hybridized with an oligo(dT) primer encoding a unique cell barcode and a unique molecular identifier (UMI) in lysis buffer. After capturing mRNA with beads hybridized with oligo(dT) primers, the transcriptomes captured on the gel beads were pooled and reverse transcribed into cDNA. Reverse transcription and PCR amplification of cDNA and library preparation from the 3' end were performed according to the manufacturer's published protocol.

[0242] In addition to the above-mentioned full RNA library (for assessing the transcriptome of each cell), for each sample, a custom library was also generated to read out promoter-construct-specific expression by detecting the unique barcode assigned to each promoter-construct. The libraries were sequenced on an Illumina NovaSeq 6000. The reads were demultiplexed, aligned to the custom GRCm38 mm10 reference genome, and filtered; and cell barcodes and UMIs were quantified using the Cell Ranger pipeline (support.10xgenomics.com / single-cell-gene-expression / software / overview / welcome). Cell Ranger uses STAR (Dobin et al., Bioinformatics 29:15-21, 2013) for alignment and the manufacturer's software for all other steps.

[0243] Single-cell RNA-seq data was analyzed using Seurat v3 (Butler et al., Nat Biotechnol 36:411-420, 2018). Cells with fewer than 100 detected genes, more than 20% mitochondrial gene reads, and 40% ribosomal content reads were filtered out. Predicted doublets were excluded using Scrublet (Wollock et al., Cell Syst. 8:281-291, 2019e9). We used the default Seurat functions to select variable genes and cluster cells, and assigned cell types to each cluster based on the correlation of gene expression from the complete RNA library with a previously annotated dataset from the cochlea and based on the expression of known marker genes. Promoter-construct reads from each cell derived from the custom RNA library were added to the Seurat object with the matching cell ID, such that reads from the viral construct in each cell could be paired with its transcriptome. The percentage of cells positive (above the noise threshold) for each construct was calculated for each cell type. The average expression of each construct in the cell type was calculated using the CellRanger method for calculating average expression (support.10xgenomics.com / single-cell-gene-expression / software / pipelines / latest / algorithms / overview). Points were plotted for cell types with >2% of cells expressing the construct.

[0244] Figure 8 The results of this analysis are shown. The top 4 cell type clusters (spiral prominence / root cells / Boettcher’s cells; inner sulcus / outer sulcus / spiral prominence cells; interdental cells; and Reissner’s membrane epithelial cells) identified as described above are the cells expressing the highest levels of native pendrin. As Figure 8 can be seen, compared to the ubiquitously present Pgk promoter, the use of the pendrin promoter described herein restricted expression mainly to those top 4 cell clusters and supporting cells. Inclusion of the SLC26A4 enhancer and especially the human E6.4 or mouse E6 enhancer increased the percentage of cells expressing the construct and the overall expression level (as detected by barcoding) in these 4 cell clusters. Figure 8

[0245] Example 3. Administration of AAV-DJ vector containing the Pend3.1.8 promoter and the human E6.4 enhancer to Slc26a4 knockout mice

[0246] ​Based on the in vitro results using an AAV vector containing the E6.4 enhancer (SEQ ID NO:5) fused to the 5' end of the Pend3.1.8 minimal promoter as described in Example 1 (see Figure 4A , right panel), the same vector was tested to see if it would increase EGFP expression in the target cell types within both the cochlea and vestibule in vivo. Plasmid P1527 ( Figure 11 ) containing those elements was packaged into AAV-DJ. As a control, we used a plasmid containing an expression cassette encoding the ubiquitous CMV promoter (without any enhancer added) that drives the expression of nuclear-targeted EGFP packaged into AAV1.

[0247] The AAV-DJ vector produced with plasmid P1527 was locally administered via the bilateral posterior semicircular canals (IL) to neonatal Slc26a4 knockout mice at P3 age. These Slc26a4 KO mice were generated by CRISPR / Cas9-mediated deletion of exons 3-5 of the Slc26a4 gene on mouse chromosome 12 of mice with a C57BL / 6 background. The CMV-driven control AAV1 vector was injected into other P3-P4 age Slc26a4 KO mice via the bilateral posterior semicircular canals (IL). At P22 age, the control and test mice were sacrificed, 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 structure. EGFP fluorescence was detected in the nuclei of interdental cells (ID) and spiral prominence cells (SP) of the cochlea in the ears treated with AAV-DJ containing the E6.4 enhancer and the Pend3.1.8 promoter ( Figure 12 panels A-D). In contrast, treatment of ears with the control AAV1 containing the CMV promoter resulted in widespread non-specific expression throughout the cochlea ( Figure 12 panels E-F).

[0248] Example 4. Administration of a composition comprising a nucleic acid vector containing an SLC26A4 promoter and / or an SLC26A4 enhancer to a subject with sensorineural hearing loss

[0249] According to the methods disclosed herein, a skilled physician in the art can treat a patient suffering from hearing loss (e.g., pendrin-related hearing loss such as DFNB4 or Pendred syndrome), such as a human patient, to improve or restore hearing. To this end, a person skilled in the art can administer to a human patient a composition comprising an AAV vector (e.g., AAV1, AAV2, AAV2quad(Y-F), 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 an SLC26A4 promoter described herein (e.g., the SLC26A4 promoter of formula B-A-C described above, 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 higher sequence identity) with any one of SEQ ID NOs: 1 and 20-27, e.g., SEQ ID NO: 1) operably linked to a polynucleotide encoding an expression product (e.g., a wild-type version of pendrin, such as a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11) and optionally at least one SLC26A4 enhancer (e.g., one or more copies of 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 higher sequence identity) with any one of SEQ ID NOs: 4-9, 34 and 35), or an AAV vector containing 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 higher sequence identity) with any one of SEQ ID NOs: 4, 5, 8 or 9, e.g., SEQ ID NO: 4 or SEQ ID NO: 5) operably linked to a promoter (e.g., an SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or an enhancer-promoter, minimal promoter, core promoter or constitutive promoter) operably linked to a polynucleotide encoding an expression product (e.g., a wild-type version of pendrin, such as a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11).A composition comprising an AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or endolymph or through the round window membrane), to treat sensorineural hearing loss.

[0250] After administering the composition to the patient, those skilled in the art can monitor the improvement of the patient's response to the therapy by various methods. For example, a physician can monitor the patient's hearing after administering the composition by performing standard tests such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions. Compared with the hearing test results before administering the composition, it is found that the patient shows an improvement in hearing in one or more tests after administering the composition, indicating that the patient has a good response to the treatment. Subsequent doses can be determined and administered as needed.

[0251] Example 5. Administering a composition comprising a nucleic acid vector containing an SLC26A4 promoter and / or SLC26A4 enhancer to a subject with vestibular dysfunction

[0252] According to the methods disclosed herein, a skilled physician 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) in order to improve or restore vestibular function. To this end, a person skilled in the art can administer to a human patient a composition comprising an AAV vector containing a SLC26A4 promoter as described herein (e.g., the SLC26A4 promoter of formula B-A-C as described above, 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 higher sequence identity) with any one of SEQ ID NOs: 1 and 20-27, e.g., SEQ ID NO: 1) operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding the wild-type form of Atoh1, such as a polynucleotide encoding SEQ ID NO: 36 or SEQ ID NO: 38) and optionally at least one SLC26A4 enhancer (e.g., one or more copies of 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 higher sequence identity) with any one of SEQ ID NOs: 4-9, 34 and 35), such as AAV1, AAV2, AAV2quad(Y-F), 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), or an AAV vector containing at least one 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 higher sequence identity) with any one of SEQ ID NOs: 4, 5, 8 or 9, e.g., SEQ ID NO: 4 or SEQ ID NO: 5) operably linked to a promoter (e.g., the SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or an enhancer-promoter, minimal promoter, core promoter or constitutive promoter) operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding the wild-type form of Atoh1, such as a polynucleotide encoding SEQ ID NO: 36 or SEQ ID NO: 38).A composition containing an AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or endolymph, through the round window membrane, or injection into the semicircular canals) to treat vestibular dysfunction.

[0253] After administering the composition 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 patient's vestibular function by performing standard tests such as electrooculography, video nystagmography, VOR tests (e.g., head impulse test (Halmagyi–Curthoys test, e.g., VHIT) or caloric reflex test), rotary test, vestibular evoked myogenic potential, or computerized dynamic posturography. Compared with the test results obtained before administering the composition, it is found that the patient shows improvement in vestibular function in one or more tests after administering the composition, indicating that the patient has a good response to the treatment. Subsequent doses can be determined and administered as needed.

[0254] Example 6. Administering a composition containing a nucleic acid vector containing an SLC26A4 promoter and / or SLC26A4 enhancer to a subject with Meniere's disease

[0255] According to the methods disclosed herein, a skilled physician in the art can treat a patient (e.g., a human patient) suffering from Meniere's disease in order to reduce vertigo, improve hearing, reduce tinnitus, or reduce aural fullness. To this end, a person skilled in the art can administer to a human patient a composition comprising an AAV vector (e.g., AAV1, AAV2, AAV2quad(Y-F), 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 an SLC26A4 promoter as described herein (e.g., the SLC26A4 promoter of formula B-A-C as described above, 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 higher sequence identity) with any one of SEQ ID NOs: 1 and 20-27, e.g., SEQ ID NO: 1) operably linked to a polynucleotide encoding an expression product (e.g., a wild-type version of pendrin, such as a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11) and optionally at least one SLC26A4 enhancer (e.g., one or more copies of 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 higher sequence identity) with any one of SEQ ID NOs: 4-9, 34 and 35), or an AAV vector containing at least one 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 higher sequence identity) with any one of SEQ ID NOs: 4, 5, 8 or 9, e.g., SEQ ID NO: 4 or SEQ ID NO: 5) operably linked to a promoter (e.g., an SLC26A4 promoter, such as the SLC26A4 promoter provided in Table 2 or an enhancer-promoter, minimal promoter, core promoter or constitutive promoter) operably linked to a polynucleotide encoding an expression product (e.g., a wild-type version of pendrin, such as a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11).A composition containing an AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or endolymph, through the round window membrane, or injection into the semicircular canals) to treat Meniere's disease.

[0256] After administering the composition 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 various methods. For example, a physician can monitor the patient's vestibular function after administering the composition by performing standard tests such as electro-oculography, video nystagmography, VOR tests (e.g., head impulse test (Halmagyi–Curthoys test, e.g., VHIT) or caloric reflex test), rotary tests, vestibular evoked myogenic potentials, or computerized dynamic posturography; and can monitor the patient's hearing by performing standard tests such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions. The physician can also rely on the patient's reports regarding vertigo, tinnitus, and a feeling of fullness in the ear. It is found that the patient shows improved vestibular function or hearing in one or more tests or reports a reduction in vertigo, tinnitus, or a feeling of fullness in the ear after administering the composition compared to the test results obtained before administering the composition, indicating that the patient has a good response to the treatment. Subsequent doses can be determined and administered as needed.

[0257] Exemplary embodiments of the present invention are described in the paragraphs listed below.

[0258] E1. A nucleic acid vector containing an SLC26A4 promoter of formula 5'-B-A-C-3', wherein:

[0259] A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity);

[0260] B is absent or has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof containing 1 to 917 consecutive nucleotides starting from the 3' end of SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and

[0261] C is absent or 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:3 or a portion thereof that includes at least the first 159 contiguous nucleotides of SEQ ID NO:3.

[0262] wherein the SLC26A4 promoter is no longer than 1481 bases.

[0263] E2. The nucleic acid vector according to E1, wherein A has the sequence of SEQ ID NO:1.

[0264] E3. The nucleic acid vector according to E1 or E2, wherein B is absent.

[0265] E4. The nucleic acid vector according to E1 or E2, wherein B has the sequence of SEQ ID NO:2 or a portion thereof that includes from 1 to 917 contiguous nucleotides starting from the 3'-end of SEQ ID NO:2.

[0266] E5. The nucleic acid vector according to E4, wherein B has the sequence of a portion of SEQ ID NO:2 that includes from 1 to 917 contiguous nucleotides starting from the 3'-end of SEQ ID NO:2.

[0267] E6. The nucleic acid vector according to E5, wherein the portion of SEQ ID NO:2 is the first 307 contiguous nucleotides starting from the 3'-end of SEQ ID NO:2 (as shown in SEQ ID NO:14).

[0268] E7. The nucleic acid vector according to E4, wherein B has the sequence of SEQ ID NO:2.

[0269] E8. The nucleic acid vector according to any one of E1 - E7, wherein C is absent.

[0270] E9. The nucleic acid vector according to any one of E1 - E7, wherein C has the sequence of SEQ ID NO:3 or a portion thereof that includes at least the first 159 nucleotides of SEQ ID NO:3.

[0271] E10. The nucleic acid vector according to E9, wherein C has the sequence of a portion of SEQ ID NO:3 that includes at least the first 159 nucleotides of SEQ ID NO:3.

[0272] E11. The nucleic acid vector as described in E10, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:15, 16, 17, 18, and 19 respectively).

[0273] E12. The nucleic acid vector as described in E11, wherein the portion of SEQ ID NO:3 is the first 159 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:15).

[0274] E13. The nucleic acid vector as described in E11, wherein the portion of SEQ ID NO:3 is the first 324 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:16).

[0275] E14. The nucleic acid vector as described in E11, wherein the portion of SEQ ID NO:3 is the first 341 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:17).

[0276] E15. The nucleic acid vector as described in E11, wherein the portion of SEQ ID NO:3 is the first 716 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:18).

[0277] E16. The nucleic acid vector as described in E11, wherein the portion of SEQ ID NO:3 is the first 723 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:19).

[0278] E17. The nucleic acid vector as described in E9, wherein C has the sequence of SEQ ID NO:3.

[0279] E18. The nucleic acid vector as described in any one of E1, E3, and E8, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:1 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0280] E19. The nucleic acid vector as described in E13, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1.

[0281] E20. The nucleic acid vector according to any one of E1, E2, E4, E7, and E8, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0282] E21. The nucleic acid vector according to E15, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 20.

[0283] E22. The nucleic acid vector according to any one of E1-E3, E9, and E12, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 21 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0284] E23. The nucleic acid vector according to E17, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 21.

[0285] E24. The nucleic acid vector according to any one of E1, E2, E4-E6, and E8, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 22 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0286] E25. The nucleic acid vector according to E19, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 22.

[0287] E26. The nucleic acid vector according to any one of E1-E3, E9-E11, and E14, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 23 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0288] E27. The nucleic acid vector according to E21, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 23.

[0289] E28. The nucleic acid vector according to any one of E1-E3, E9-E11, and E16, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 24 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0290] E29. The nucleic acid vector according to E23, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 24.

[0291] E30. The nucleic acid vector according to any one of E1, E2, E4-E6, E9-E11, and E15, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 25 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0292] E31. The nucleic acid vector according to E25, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 25.

[0293] E32. The nucleic acid vector according to any one of E1, E2, E4-E6, E9-E11, and E13, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 26 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0294] E33. The nucleic acid vector according to E27, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 26.

[0295] E34. The nucleic acid vector according to any one of E1, E2, E4-E6, and E9-E12, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 27 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0296] E35. The nucleic acid vector according to E29, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 27.

[0297] E36. The nucleic acid vector as described in any one of E1 - E35, wherein the nucleic acid vector further comprises an SLC26A4 enhancer operably linked to the SLC26A4 promoter.

[0298] E37. The nucleic acid vector as described in E36, wherein the SLC26A4 enhancer has at least 85% sequence identity with any one of SEQ ID NO: 4 - 9, 34, and 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0299] E38. The nucleic acid vector as described in E37, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 4 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0300] E39. The nucleic acid vector as described in E38, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 4.

[0301] E40. The nucleic acid vector as described in E37, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 5 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0302] E41. The nucleic acid vector as described in E40, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 5.

[0303] E42. The nucleic acid vector as described in E37, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 6 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0304] E43. The nucleic acid vector as described in E42, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 6.

[0305] E44. The nucleic acid vector as described in E37, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:7 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0306] E45. The nucleic acid vector as described in E44, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:7.

[0307] E46. The nucleic acid vector as described in E37, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:8 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0308] E47. The nucleic acid vector as described in E46, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:8.

[0309] E48. The nucleic acid vector as described in E37, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:9 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0310] E49. The nucleic acid vector as described in E48, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:9.

[0311] E50. The nucleic acid vector as described in E37, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:34 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0312] E51. The nucleic acid vector as described in E50, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:34.

[0313] E52. The nucleic acid vector as described in E37, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0314] E53. The nucleic acid vector as described in E52, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 35.

[0315] E54. The nucleic acid vector as described in any one of E36 - E53, wherein the SLC26A4 enhancer is directly linked (e.g., fused) to the SLC26A4 promoter.

[0316] E55. The nucleic acid vector as described in any one of E36 - E53, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter through a nucleic acid linker of 1 to 500 nucleotides (e.g., 1 - 50, 1 - 100, 1 - 150, 1 - 200, 1 - 250, 1 - 300, 1 - 350, 1 - 400, 1 - 450, 1 - 500, 50 - 500, 100 - 500, 150 - 500, 200 - 500, 250 - 500, 300 - 500, 350 - 500, 400 - 500 or 450 - 500 nucleotides, such as about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 nucleotides).

[0317] E56. The nucleic acid vector as described in E55, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter through a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 - 10, 1 - 20, 1 - 30, 1 - 40, 1 - 50, 1 - 60, 1 - 70, 1 - 80, 1 - 90, 1 - 100, 10 - 100, 20 - 100, 30 - 100, 40 - 100, 50 - 100, 60 - 100, 70 - 100, 80 - 100 or 90 - 100 nucleotides, such as about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides).

[0318] E57. The nucleic acid vector according to any one of E36 - E56, wherein the SLC26A4 enhancer is located 5' to the SLC26A4 promoter.

[0319] E58. The nucleic acid vector according to any one of E36 - E56, wherein the SLC26A4 enhancer is located 3' to the SLC26A4 promoter.

[0320] E59. The nucleic acid vector according to any one of E1 - E58, wherein the SLC26A4 promoter is operably linked to a polynucleotide capable of being transcribed to produce an expression product.

[0321] E60. The nucleic acid vector according to E59, wherein the expression product is a heterologous expression product.

[0322] E61. The nucleic acid vector according to E59 or E60, wherein the expression product is an expression product endogenously expressed in cells expressing SLC26A4.

[0323] E62. The nucleic acid vector according to E61, wherein the expression product is an expression product endogenously expressed in inner ear cells expressing SLC26A4.

[0324] E63. The nucleic acid vector according to E62, wherein the expression product is an expression product endogenously expressed (e.g., expressed in at least one of these cell types) in interdental cells, spiral prominence cells, cochlear root cells, and / or vestibular supporting cells.

[0325] E64. The nucleic acid vector according to any one of E61 - E63, wherein the expression product is pannexin (e.g., mammalian pannexin).

[0326] E65. The nucleic acid vector according to E64, wherein the pannexin (the mammalian pannexin) is a wild - type isoform endogenously expressed in the mammalian inner ear.

[0327] E66. The nucleic acid vector according to E64 or E65, wherein the pannexin (the mammalian pannexin) has at least 85% sequence identity with SEQ ID NO:10 or SEQ ID NO:11 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0328] E67. The nucleic acid vector according to E66, wherein the pannexin (the mammalian pannexin) has the sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0329] E68. The nucleic acid vector according to any one of E59 - E63, wherein the expression product is Atoh1 (e.g., mammalian Atoh1).

[0330] E69. The nucleic acid vector according to E68, wherein the Atoh1 (the mammalian Atoh1 protein) is the wild - type isoform endogenously expressed in the mammalian inner ear.

[0331] E70. The nucleic acid vector according to any one of E68 or E69, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity with SEQ ID NO: 36 or SEQ ID NO: 38 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0332] E71. The nucleic acid vector according to E70, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO: 36 or SEQ ID NO: 38.

[0333] E72. The nucleic acid vector according to E59 or E60, wherein the expression product is a protein, short hairpin RNA (shRNA), antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR - associated protein 9 (Cas9), transcription activator - like effector nuclease (TALEN), or zinc - finger nuclease (ZFN) or guide RNA (gRNA)) or microRNA.

[0334] E73. The nucleic acid vector according to any one of E37 - E72, wherein the nucleic acid vector comprises two or more different SLC26A4 enhancers, and each enhancer is independently selected from enhancers having at least 85% sequence identity with any one of SEQ ID NO: 4 - 9, 34 and 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0335] E74. The nucleic acid vector according to E73, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NO: 4 - 9, 34 and 35.

[0336] E75. The nucleic acid vector as described in E73, wherein the nucleic acid vector comprises a first 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 higher sequence identity) with SEQ ID NO:6 and a second 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 higher sequence identity) with SEQ ID NO:7.

[0337] E76. The nucleic acid vector as described in E75, wherein the first enhancer has the sequence of SEQ ID NO:6 and the second enhancer has the sequence of SEQ ID NO:7.

[0338] E77. The nucleic acid vector as described in E75 or E76, wherein both the first enhancer and the second enhancer are located 5' of the SLC26A4 promoter.

[0339] E78. The nucleic acid vector as described in E77, wherein the first enhancer or the second enhancer is directly fused to the other of the first enhancer or the second enhancer that is directly fused to the SLC26A4 promoter (i.e., the enhancer and promoter elements are directly connected to each other without any intervening nucleic acid).

[0340] E79. The nucleic acid vector as described in E78, the nucleic acid vector comprises the sequence of SEQ ID NO:6, the sequence of SEQ ID NO:7 and the sequence of the SLC26A4 promoter in a 5' to 3' order (e.g., a polynucleotide sequence consisting of the polynucleotide sequence of SEQ ID NO:6 - SEQ ID NO:7 - SLC26A4 promoter).

[0341] E80. The nucleic acid vector as described in any one of E75 - E79, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1.

[0342] E81. The nucleic acid vector as described in any one of E37 - E74, wherein the nucleic acid vector comprises two or more copies of the SLC26A4 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 higher sequence identity) with any one of SEQ ID NO:4 - 9, 34 and 35.

[0343] E82. The nucleic acid vector as described in E81, wherein each of the two or more copies of the SLC26A4 enhancer has a sequence of any one of SEQ ID NO: 4-9, 34, and 35.

[0344] E83. A polynucleotide comprising an SLC26A4 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 higher sequence identity) with any one of SEQ ID NO: 4, 5, 8, 9, 34, and 35, the enhancer being operably linked to a promoter, wherein the enhancer is linked to the promoter directly or via a nucleic acid linker of 1 to 500 nucleotides (e.g., 1-50, 1-100, 1-150, 1-200, 1-250, 1-300, 1-350, 1-400, 1-450, 1-500, 50-500, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, or 450-500 nucleotides, such as about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides).

[0345] E84. The polynucleotide as described in E83, wherein the SLC26A4 enhancer is linked to the promoter via a nucleic acid linker of 1 to 100 nucleotides (e.g., 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, or 90-100 nucleotides, such as about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0346] E85. The polynucleotide as described in E83, wherein the SLC26A4 enhancer is directly linked (e.g., fused) to the promoter.

[0347] E86. The polynucleotide as described in any one of E83-E85, wherein the SLC26A4 enhancer is located 5' of the promoter.

[0348] E87. The polynucleotide according to any one of E83 - E85, wherein the SLC26A4 enhancer is located 3' of the promoter.

[0349] E88. The polynucleotide according to any one of E83 - E87, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:4 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0350] E89. The polynucleotide according to E88, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:4.

[0351] E90. The polynucleotide according to any one of E83 - E87, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:5 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0352] E91. The polynucleotide according to E90, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:5.

[0353] E92. The polynucleotide according to any one of E83 - E87, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:8 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0354] E93. The polynucleotide according to E92, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:8.

[0355] E94. The polynucleotide according to any one of E83 - E87, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:9 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0356] E95. The polynucleotide according to E94, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:9.

[0357] E96. The polynucleotide according to any one of E83 - E87, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 34 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0358] E97. The polynucleotide according to E96, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 34.

[0359] E98. The polynucleotide according to any one of E83 - E87, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0360] E99. The polynucleotide according to E98, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 35.

[0361] E100. The polynucleotide according to any one of E83 - E99, wherein the promoter is a minimal promoter, a core promoter or a constitutive promoter.

[0362] E101. The polynucleotide according to E100, 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 phosphoglycerate kinase (PGK) promoter, an EF1α promoter, a β - globin promoter, a CMV promoter, an HSV promoter or an SV40 promoter.

[0363] E102. The polynucleotide according to E101, 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 an SV40 minimal promoter.

[0364] E103. The polynucleotide according to E100, wherein the promoter is a minimal promoter.

[0365] E104. The polynucleotide according to any one of E83 - E99, wherein the promoter is a mammalian SLC26A4 promoter.

[0366] E105. The polynucleotide as described in E104, wherein the SLC26A4 promoter is a human or murine SLC26A4 promoter.

[0367] E106. The polynucleotide as described in E104 or E105, wherein the SLC26A4 promoter has the formula 5'-B-A-C-3', where:

[0368] A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity);

[0369] B is absent or has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and

[0370] C is absent or has at least 85% sequence identity with SEQ ID NO:3 or a portion thereof comprising at least the first 159 consecutive nucleotides of SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity),

[0371] wherein the SLC26A4 promoter is not longer than 1481 bases.

[0372] E107. The polynucleotide as described in E106, wherein A has the sequence of SEQ ID NO:1.

[0373] E108. The polynucleotide as described in E106 or E107, wherein B is absent. E109. The polynucleotide as described in E106 or E107, wherein B has the sequence of SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2.

[0374] E110. The polynucleotide as described in E109, wherein B has the sequence of a portion of SEQ ID NO:2, the portion comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2.

[0375] The polynucleotide as described in E110, wherein the portion of SEQ ID NO:2 is the first 307 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2 (as shown in SEQ ID NO:14).

[0376] The polynucleotide as described in E109, wherein B has the sequence of SEQ ID NO:2.

[0377] The polynucleotide as described in any one of E106 - E112, wherein C does not exist.

[0378] The polynucleotide as described in any one of E106 - E112, wherein C has the sequence of SEQ ID NO:3 or a portion thereof that includes at least the first 159 nucleotides of SEQ ID NO:3.

[0379] The polynucleotide as described in E114, wherein C has the sequence of a portion of SEQ ID NO:3, and the portion includes at least the first 159 nucleotides of SEQ ID NO:3.

[0380] The polynucleotide as described in E115, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:15, 16, 17, 18, and 19 respectively).

[0381] The polynucleotide as described in E116, wherein the portion of SEQ ID NO:3 is the first 159 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:15).

[0382] The polynucleotide as described in E116, wherein the portion of SEQ ID NO:3 is the first 324 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:16).

[0383] The polynucleotide as described in E116, wherein the portion of SEQ ID NO:3 is the first 341 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:17).

[0384] The polynucleotide as described in E116, wherein the portion of SEQ ID NO:3 is the first 716 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:18).

[0385] E121. The polynucleotide as described in E116, wherein the portion of SEQ ID NO:3 is the first 723 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:19).

[0386] E122. The polynucleotide as described in E114, wherein C has the sequence of SEQ ID NO:3.

[0387] E123. The polynucleotide as described in any one of E106, E108, and E113, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0388] E124. The polynucleotide as described in E123, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1.

[0389] E125. The polynucleotide as described in any one of E106, E107, E109, E112, and E113, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0390] E126. The polynucleotide as described in E125, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:20.

[0391] E127. The polynucleotide as described in any one of E106 - E108, E114, and E122, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:21 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0392] E128. The polynucleotide as described in E127, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:21.

[0393] A polynucleotide as described in any one of E106, E107, E109 - E111, and E113, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:22 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0394] E130. The polynucleotide as described in E129, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:22.

[0395] A polynucleotide as described in any one of E106 - E108, E114 - E116, and E119, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:23 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0396] E132. The polynucleotide as described in E131, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:23.

[0397] A polynucleotide as described in any one of E106 - E108, E114 - E116, and E121, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:24 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0398] E134. The polynucleotide as described in E133, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:24.

[0399] A polynucleotide as described in any one of E106, E107, E109 - E111, E114 - E116, and E120, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:25 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0400] The polynucleotide as described in E135, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:25.

[0401] The polynucleotide as described in any one of E106, E107, E109-E111, E114-E116 and E118, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:26 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0402] The polynucleotide as described in E137, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:26.

[0403] The polynucleotide as described in any one of E106, E107, E109-E111 and E114-E117, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:27 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0404] The polynucleotide as described in E139, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:27.

[0405] The polynucleotide as described in E104 or E105, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:28 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0406] The polynucleotide as described in E141, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:28.

[0407] The polynucleotide as described in E104 or E105, wherein the SLC26A4 promoter has at least 85% sequence identity with any one of SEQ ID NO:29-33 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0408] E144. The polynucleotide as described in E143, wherein the SLC26A4 promoter has the sequence of any one of SEQ ID NOs: 29 - 33.

[0409] E145. The polynucleotide as described in E104 or E105, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO: 41 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0410] E146. The polynucleotide as described in E145, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 41.

[0411] E147. The polynucleotide as described in any one of E83 - E146, wherein the promoter is operably linked to a polynucleotide capable of being transcribed to produce an expression product.

[0412] E148. The polynucleotide as described in E147, wherein the expression product is a heterologous expression product.

[0413] E149. The polynucleotide as described in E147 or E148, wherein the expression product is an expression product endogenously expressed in a cell that expresses SLC26A4.

[0414] E150. The polynucleotide as described in E149, wherein the expression product is an expression product endogenously expressed in an inner ear cell that expresses SLC26A4.

[0415] E151. The polynucleotide as described in E150, wherein the expression product is an expression product endogenously expressed (e.g., expressed in at least one of these cell types) in interdental cells, spiral prominence cells, cochlear root cells, and / or vestibular supporting cells.

[0416] E152. The polynucleotide as described in any one of E149 - E151, wherein the expression product is pannexin (e.g., mammalian pannexin).

[0417] E153. The polynucleotide as described in E152, wherein the pannexin (the mammalian pannexin) is a wild - type isoform endogenously expressed in the mammalian inner ear.

[0418] A polynucleotide as described in any one of E152 or E153, wherein the panprotein (the mammalian panprotein) has at least 85% sequence identity with SEQ ID NO:10 or SEQ ID NO:11 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0419] A polynucleotide as described in E154, wherein the panprotein (the mammalian panprotein) has the sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0420] A polynucleotide as described in any one of E147 - E151, wherein the expression product is Atoh1 (e.g., mammalian Atoh1).

[0421] A polynucleotide as described in E156, wherein the Atoh1 (the mammalian Atoh1 protein) is the wild - type isoform endogenously expressed in the mammalian inner ear.

[0422] A polynucleotide as described in any one of E156 or E157, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity with SEQ ID NO:36 or SEQ ID NO:38 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0423] A polynucleotide as described in E158, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO:36 or SEQ ID NO:38.

[0424] A polynucleotide as described in E147 or E148, wherein the expression product is a protein, short hairpin RNA (shRNA), antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR - associated protein 9 (Cas9), transcription activator - like effector nuclease (TALEN), or zinc - finger nuclease (ZFN)) or a guide RNA (gRNA)) or a microRNA.

[0425] E161. A polynucleotide as described in any one of E83 - E160, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, and each enhancer is independently selected from 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 higher sequence identity) with any one of SEQ ID NO: 4, 5, 8, 9, 34, and 35.

[0426] E162. The polynucleotide as described in E161, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NO: 4, 5, 8, 9, 34, and 35.

[0427] E163. A polynucleotide as described in any one of E83 - E162, wherein the polynucleotide comprises two or more copies of an SLC26A4 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 higher sequence identity) with any one of SEQ ID NO: 4, 5, 8, 9, 34, and 35.

[0428] E164. The polynucleotide as described in E163, wherein each of the two or more copies of the SLC26A4 enhancer has the sequence of one of SEQ ID NO: 4, 5, 8, 9, 34, and 35.

[0429] E165. A polynucleotide comprising an SLC26A4 promoter of the formula 5'-B-A-C-3', wherein the promoter is operably linked to a polynucleotide capable of being transcribed to produce an expression product, and wherein:

[0430] A 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: 1;

[0431] B is absent or 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: 2 or a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO: 2; and

[0432] C is absent or 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:3 or a portion thereof that includes at least the first 159 consecutive nucleotides of SEQ ID NO:3.

[0433] wherein the SLC26A4 promoter is no longer than 1481 bases.

[0434] E166. The polynucleotide according to E165, wherein A has the sequence of SEQ ID NO:1.

[0435] E167. The polynucleotide according to E165 or E166, wherein B is absent.

[0436] E168. The polynucleotide according to E165 or E166, wherein B has the sequence of SEQ ID NO:2 or a portion thereof that includes the sequence of 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2.

[0437] E169. The polynucleotide according to E168, wherein B has the sequence of a portion of SEQ ID NO:2, the portion including 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2.

[0438] E170. The polynucleotide according to E169, wherein the portion of SEQ ID NO:2 is the first 307 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2 (as shown in SEQ ID NO:14).

[0439] E171. The polynucleotide according to E168, wherein B has the sequence of SEQ ID NO:2.

[0440] E172. The polynucleotide according to any one of E165 - E171, wherein C is absent.

[0441] E173. The polynucleotide according to any one of E165 - E171, wherein C has the sequence of SEQ ID NO:3 or a portion thereof that includes at least the first 159 nucleotides of SEQ ID NO:3.

[0442] E174. The polynucleotide according to E173, wherein C has the sequence of a portion of SEQ ID NO:3, the portion including at least the first 159 nucleotides of SEQ ID NO:3.

[0443] The polynucleotide as described in E174, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716 or 723 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:15, 16, 17, 18 and 19 respectively).

[0444] The polynucleotide as described in E175, wherein the portion of SEQ ID NO:3 is the first 159 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:15).

[0445] The polynucleotide as described in E175, wherein the portion of SEQ ID NO:3 is the first 324 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:16).

[0446] The polynucleotide as described in E175, wherein the portion of SEQ ID NO:3 is the first 341 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:17).

[0447] The polynucleotide as described in E175, wherein the portion of SEQ ID NO:3 is the first 716 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:18).

[0448] The polynucleotide as described in E175, wherein the portion of SEQ ID NO:3 is the first 723 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:19).

[0449] The polynucleotide as described in E173, wherein C has the sequence of SEQ ID NO:3.

[0450] The polynucleotide as described in any one of E165, E167 and E172, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:1 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0451] The polynucleotide as described in E182, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1.

[0452] A polynucleotide as described in any one of E165, E166, E168, E171, and E172, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0453] E185. A polynucleotide as described in E184, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:20.

[0454] E186. A polynucleotide as described in any one of E165 - E167, E173, and E181, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:21 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0455] E187. A polynucleotide as described in E186, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:21.

[0456] E188. A polynucleotide as described in any one of E165, E166, E168 - E170, and E172, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:22 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0457] E189. A polynucleotide as described in E188, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:22.

[0458] E190. A polynucleotide as described in any one of E165 - 167, E173 - E175, and E178, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:23 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0459] E191. A polynucleotide as described in E190, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:23.

[0460] E192. A polynucleotide as described in any one of E165-167, E173-E175, and E180, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:24 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0461] E193. The polynucleotide as described in E192, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:24.

[0462] E194. A polynucleotide as described in any one of E165, E166, E168-E170, E173-E175, and E179, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:25 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0463] E195. The polynucleotide as described in E194, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:25.

[0464] E196. A polynucleotide as described in any one of E165, E166, E168-E170, E173-E175, and E177, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:26 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0465] E197. The polynucleotide as described in E196, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:26.

[0466] E198. A polynucleotide as described in any one of E165, E166, E168-E170, and E173-E176, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:27 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0467] E199. The polynucleotide as described in E198, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 27.

[0468] E200. The polynucleotide as described in any one of E165 - E199, wherein the nucleic acid vector further comprises an SLC26A4 enhancer operably linked to the SLC26A4 promoter.

[0469] E201. The polynucleotide as described in E200, wherein the SLC26A4 enhancer has at least 85% sequence identity with any one of SEQ ID NOs: 4 - 9, 34, and 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0470] E202. The polynucleotide as described in E201, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 4 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0471] E203. The polynucleotide as described in E202, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 4.

[0472] E204. The polynucleotide as described in E201, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 5 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0473] E205. The polynucleotide as described in E204, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 5.

[0474] E206. The polynucleotide as described in E201, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 6 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0475] E207. The polynucleotide as described in E206, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 6.

[0476] E208. The polynucleotide as described in E201, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:7 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0477] E209. The polynucleotide as described in E208, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:7.

[0478] E210. The polynucleotide as described in E201, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:8 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0479] E211. The polynucleotide as described in E210, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:8.

[0480] E212. The polynucleotide as described in E201, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:9 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0481] E213. The polynucleotide as described in E212, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:9.

[0482] E214. The polynucleotide as described in E201, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:34 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0483] E215. The polynucleotide as described in E214, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:34.

[0484] The polynucleotide as described in E201, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0485] The polynucleotide as described in E216, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:35.

[0486] The polynucleotide as described in any one of E200 - E217, wherein the SLC26A4 enhancer is directly linked (e.g., fused) to the SLC26A4 promoter.

[0487] The polynucleotide as described in any one of E200 - E217, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter through a nucleic acid linker of 1 to 500 nucleotides (e.g., 1 - 50, 1 - 100, 1 - 150, 1 - 200, 1 - 250, 1 - 300, 1 - 350, 1 - 400, 1 - 450, 1 - 500, 50 - 500, 100 - 500, 150 - 500, 200 - 500, 250 - 500, 300 - 500, 350 - 500, 400 - 500 or 450 - 500 nucleotides, such as about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 nucleotides).

[0488] The polynucleotide as described in E219, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter through a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 - 10, 1 - 20, 1 - 30, 1 - 40, 1 - 50, 1 - 60, 1 - 70, 1 - 80, 1 - 90, 1 - 100, 10 - 100, 20 - 100, 30 - 100, 40 - 100, 50 - 100, 60 - 100, 70 - 100, 80 - 100 or 90 - 100 nucleotides, such as about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides).

[0489] The polynucleotide according to any one of E200 - E220, wherein the SLC26A4 enhancer is located 5' to the SLC26A4 promoter.

[0490] E222. The polynucleotide according to any one of E200 - E220, wherein the SLC26A4 enhancer is located 3' to the SLC26A4 promoter.

[0491] E223. The polynucleotide according to any one of E165 - E222, wherein the expression product is a heterologous expression product.

[0492] E224. The polynucleotide according to any one of E165 - E222, wherein the expression product is an expression product endogenously expressed in cells expressing SLC26A4.

[0493] E225. The polynucleotide according to E224, wherein the expression product is an expression product endogenously expressed in inner ear cells expressing SLC26A4.

[0494] E226. The polynucleotide according to E225, wherein the expression product is an expression product endogenously expressed (e.g., expressed in at least one of these cell types) in interdental cells, spiral prominence cells, cochlear root cells, and / or vestibular supporting cells.

[0495] E227. The polynucleotide according to any one of E224 - E226, wherein the expression product is pendrin (e.g., mammalian pendrin).

[0496] E228. The polynucleotide according to E227, wherein the pendrin (the mammalian pendrin) is the wild - type isoform endogenously expressed in the mammalian inner ear.

[0497] E229. The polynucleotide according to E227 or E228, wherein the pendrin (the mammalian pendrin) has at least 85% sequence identity with SEQ ID NO:10 or SEQ ID NO:11 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0498] E230. The polynucleotide according to E229, wherein the pendrin (the mammalian pendrin) has the sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0499] E231. A polynucleotide as described in any one of E165 - E226, wherein the expression product is Atoh1 (e.g., mammalian Atoh1).

[0500] E232. The polynucleotide as described in E231, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild - type isoform endogenously expressed in the mammalian inner ear.

[0501] E233. The polynucleotide as described in any one of E231 or E232, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity with SEQ ID NO:36 or SEQ ID NO:38 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0502] E234. The polynucleotide as described in E233, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO:36 or SEQ ID NO:38.

[0503] E235. A polynucleotide as described in any one of E165 - E223, wherein the expression product is a protein, short hairpin RNA (shRNA), antisense oligonucleotide (ASO), a component of a gene - editing system (e.g., a nuclease such as Cas9, TALEN, or ZFN, or gRNA), or a microRNA.

[0504] E236. The polynucleotide as described in any one of E200 - E235, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, and each enhancer is independently selected from enhancers having at least 85% sequence identity with any one of SEQ ID NO:4 - 9, 34, and 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0505] E237. The polynucleotide as described in E236, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NO:4 - 9, 34, and 35.

[0506] The polynucleotide as described in E236, wherein the polynucleotide comprises a first enhancer having at least 85% sequence identity with SEQ ID NO: 6 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) and a second enhancer having at least 85% sequence identity with SEQ ID NO: 7 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0507] The polynucleotide as described in E238, wherein the first enhancer has the sequence of SEQ ID NO: 6 and the second enhancer has the sequence of SEQ ID NO: 7.

[0508] The polynucleotide as described in E238 or E239, wherein both the first enhancer and the second enhancer are located 5' of the SLC26A4 promoter.

[0509] The polynucleotide as described in E240, wherein the first enhancer or the second enhancer is directly fused to the other of the first enhancer or the second enhancer that is directly fused to the SLC26A4 promoter (i.e., the enhancer and the promoter element are directly connected to each other without any intervening nucleic acid).

[0510] The polynucleotide as described in E241, the nucleic acid vector comprises the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7 and the sequence of the SLC26A4 promoter in a 5' to 3' order (e.g., a polynucleotide sequence consisting of the polynucleotide sequence of SEQ ID NO: 6 - SEQ ID NO: 7 - SLC26A4 promoter).

[0511] The polynucleotide as described in any one of E238 - E242, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 1.

[0512] The polynucleotide as described in any one of E200 - E237, wherein the polynucleotide comprises two or more copies of the SLC26A4 enhancer having at least 85% sequence identity with any one of SEQ ID NO: 4 - 9, 34 and 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0513] The polynucleotide as described in E244, wherein each of the two or more copies of the SLC26A4 enhancer has a sequence of one of SEQ ID NOs: 4-9, 34, and 35.

[0514] E246. A polynucleotide comprising:

[0515] (a) an SLC26A4 promoter of the formula 5'-B-A-C-3', wherein:

[0516] A has at least 85% sequence identity with SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity);

[0517] B is absent or has at least 85% sequence identity with SEQ ID NO: 2 or a portion thereof comprising 1 to 917 consecutive nucleotides from the 3'-end of SEQ ID NO: 2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and

[0518] C is absent or has at least 85% sequence identity with SEQ ID NO: 3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO: 3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity); and

[0519] (b) An SLC26A4 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 higher sequence identity) with any one of SEQ ID NOs: 4-9, 34, and 35, wherein the enhancer is linked to the promoter directly or through a nucleic acid linker of 1 to 500 nucleotides (e.g., 1-50, 1-100, 1-150, 1-200, 1-250, 1-300, 1-350, 1-400, 1-450, 1-500, 50-500, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, or 450-500 nucleotides, such as about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides).

[0520] E247. The polynucleotide according to E246, wherein A has the sequence of SEQ ID NO: 1.

[0521] E248. The polynucleotide according to E246 or E247, wherein B is absent.

[0522] E249. The polynucleotide according to E246 or E247, wherein B has the sequence of SEQ ID NO: 2 or a sequence of a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO: 2.

[0523] E250. The polynucleotide according to E249, wherein B has the sequence of a portion of SEQ ID NO: 2, the portion comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO: 2.

[0524] E251. The polynucleotide according to E250, wherein the portion of SEQ ID NO: 2 is the first 307 consecutive nucleotides starting from the 3'-end of SEQ ID NO: 2 (as shown in SEQ ID NO: 14).

[0525] E252. The polynucleotide according to E249, wherein B has the sequence of SEQ ID NO: 2.

[0526] E253. The polynucleotide according to any one of E246-E252, wherein C is absent.

[0527] A polynucleotide according to any one of E246 - E252, wherein C has the sequence of SEQ ID NO:3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:3.

[0528] A polynucleotide according to E254, wherein C has a sequence that is a portion of SEQ ID NO:3, said portion comprising at least the first 159 nucleotides of SEQ ID NO:3.

[0529] A polynucleotide according to E255, wherein said portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:15, 16, 17, 18, and 19, respectively).

[0530] A polynucleotide according to E256, wherein said portion of SEQ ID NO:3 is the first 159 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:15).

[0531] A polynucleotide according to E256, wherein said portion of SEQ ID NO:3 is the first 324 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:16).

[0532] A polynucleotide according to E256, wherein said portion of SEQ ID NO:3 is the first 341 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:17).

[0533] A polynucleotide according to E256, wherein said portion of SEQ ID NO:3 is the first 716 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:18).

[0534] A polynucleotide according to E256, wherein said portion of SEQ ID NO:3 is the first 723 consecutive nucleotides of SEQ ID NO:3 (as shown in SEQ ID NO:19).

[0535] A polynucleotide according to E254, wherein C has the sequence of SEQ ID NO:3.

[0536] E263. A polynucleotide as described in any one of E246, E248, and E253, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0537] E264. The polynucleotide as described in E263, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1.

[0538] E265. A polynucleotide as described in any one of E246, E247, E249, E252, and E253, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0539] E266. The polynucleotide as described in E265, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:20.

[0540] E267. A polynucleotide as described in any one of E246 - E248, E254, and E262, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:21 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0541] E268. The polynucleotide as described in E267, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:21.

[0542] E269. A polynucleotide as described in any one of E246, E247, E249 - E251, and E253, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:22 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0543] E270. The polynucleotide as described in E269, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:22.

[0544] E271. A polynucleotide as described in any one of E246 - E248, E254 - E256, and E259, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:23 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0545] E272. The polynucleotide as described in E271, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:23.

[0546] E273. A polynucleotide as described in any one of E246 - E248, E254 - E256, and E261, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:24 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0547] E274. The polynucleotide as described in E273, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:24.

[0548] E275. A polynucleotide as described in any one of E246, E247, E249 - E251, E254 - E256, and E260, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:25 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0549] E276. The polynucleotide as described in E275, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:25.

[0550] E277. A polynucleotide as described in any one of E246, E247, E249 - E251, E254 - E256, and E258, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:26 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0551] The polynucleotide as described in E277, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:26.

[0552] The polynucleotide as described in any one of E246, E247, E249-E251 and E254-E257, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:27 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0553] The polynucleotide as described in E279, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:27.

[0554] The polynucleotide as described in any one of E246-E280, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:4 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0555] The polynucleotide as described in E281, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:4.

[0556] The polynucleotide as described in any one of E246-E280, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:5 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0557] The polynucleotide as described in E283, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:5.

[0558] The polynucleotide as described in any one of E246-E280, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:6 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0559] E286. A polynucleotide as described in E285, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 6.

[0560] E287. A polynucleotide as described in any one of E246 - E280, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 7 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0561] E288. A polynucleotide as described in E287, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 7.

[0562] E289. A polynucleotide as described in any one of E246 - E280, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 8 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0563] E290. A polynucleotide as described in E289, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 8.

[0564] E291. A polynucleotide as described in any one of E246 - E280, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 9 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0565] E292. A polynucleotide as described in E291, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 9.

[0566] E293. A polynucleotide as described in any one of E246 - E280, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 34 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0567] The polynucleotide as described in E293, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:34.

[0568] E295. The polynucleotide as described in any one of E246 - E280, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0569] E296. The polynucleotide as described in E295, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:35.

[0570] E297. The polynucleotide as described in any one of E246 - E296, wherein the SLC26A4 enhancer is directly linked (e.g., fused) to the SLC26A4 promoter.

[0571] E298. The polynucleotide as described in any one of E246 - E296, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter through a nucleic acid linker of 1 to 500 nucleotides (e.g., 1 - 50, 1 - 100, 1 - 150, 1 - 200, 1 - 250, 1 - 300, 1 - 350, 1 - 400, 1 - 450, 1 - 500, 50 - 500, 100 - 500, 150 - 500, 200 - 500, 250 - 500, 300 - 500, 350 - 500, 400 - 500 or 450 - 500 nucleotides, e.g., about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 nucleotides).

[0572] A polynucleotide as described in E298, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter by a nucleic acid linker of 1 to 100 nucleotides (e.g., 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, or 90-100 nucleotides, such as approximately 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0573] E300. A polynucleotide as described in any one of E246-E299, wherein the SLC26A4 enhancer is located 5' to the SLC26A4 promoter.

[0574] E301. A polynucleotide as described in any one of E246-E299, wherein the SLC26A4 enhancer is located 3' to the SLC26A4 promoter.

[0575] E302. A polynucleotide as described in any one of E246-E301, wherein the SLC26A4 promoter is operably linked to a polynucleotide capable of being transcribed to produce an expression product.

[0576] E303. A polynucleotide as described in E302, wherein the expression product is a heterologous expression product.

[0577] E304. A polynucleotide as described in E302, wherein the expression product is an expression product endogenously expressed in a cell that expresses SLC26A4.

[0578] E305. A polynucleotide as described in E304, wherein the expression product is an expression product endogenously expressed in an inner ear cell that expresses SLC26A4.

[0579] E306. A polynucleotide as described in E305, wherein the expression product is an expression product endogenously expressed (e.g., expressed in at least one of these cell types) in interdental cells, spiral prominence cells, cochlear root cells, and / or vestibular supporting cells.

[0580] E307. A polynucleotide as described in any one of E304-E306, wherein the expression product is pannexin (e.g., mammalian pannexin).

[0581] The polynucleotide as described in E307, wherein the pan protein (the mammalian pan protein) is a wild-type isoform endogenously expressed in the mammalian inner ear.

[0582] The polynucleotide as described in E307 or E308, wherein the pan protein (the mammalian pan protein) has at least 85% sequence identity with SEQ ID NO:10 or SEQ ID NO:11 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0583] The polynucleotide as described in E309, wherein the pan protein (the mammalian pan protein) has the sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0584] The polynucleotide as described in any one of E302 - E306, wherein the expression product is Atoh1 (e.g., mammalian Atoh1).

[0585] The polynucleotide as described in E311, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the mammalian inner ear.

[0586] The polynucleotide as described in any one of E311 or E312, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity with SEQ ID NO:36 or SEQ ID NO:38 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0587] The polynucleotide as described in E313, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO:36 or SEQ ID NO:38.

[0588] The polynucleotide as described in E302 or E303, wherein the expression product is a protein, short hairpin RNA (shRNA), antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease, such as Cas9, TALEN, or ZFN, or gRNA), or a microRNA.

[0589] E316. A polynucleotide as described in any one of E246 - E315, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, and each enhancer is independently selected from 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 higher sequence identity) with any one of SEQ ID NO: 4 - 9, 34, and 35.

[0590] E317. The polynucleotide as described in E316, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NO: 4 - 9, 34, and 35.

[0591] E318. The polynucleotide as described in E316, wherein the polynucleotide comprises a first 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 higher sequence identity) with SEQ ID NO: 6 and a second 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 higher sequence identity) with SEQ ID NO: 7.

[0592] E319. The polynucleotide as described in E316, wherein the first enhancer has the sequence of SEQ ID NO: 6, and the second enhancer has the sequence of SEQ ID NO: 7.

[0593] E320. The polynucleotide as described in E318 or E319, wherein both the first enhancer and the second enhancer are located 5' of the SLC26A4 promoter.

[0594] E321. The polynucleotide as described in E320, wherein the first enhancer or the second enhancer is directly fused to the other of the first enhancer or the second enhancer that is directly fused to the SLC26A4 promoter (i.e., the enhancer and the promoter element are directly connected to each other without any intervening nucleic acid).

[0595] E322. The polynucleotide as described in E321, wherein the nucleic acid vector contains the sequence of SEQ ID NO:6, the sequence of SEQ ID NO:7, and the sequence of the SLC26A4 promoter in the 5'-to-3' order (for example, a polynucleotide sequence composed of the polynucleotide sequence of SEQ ID NO:6-SEQ ID NO:7-SLC26A4 promoter).

[0596] E323. The polynucleotide as described in any one of E318-E322, wherein the SLC26A4 promoter has the nucleotide sequence of SEQ ID NO:1.

[0597] E324. The polynucleotide as described in any one of E246-E317, wherein the polynucleotide contains two or more copies of an SLC26A4 enhancer having at least 85% sequence identity (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) with any one of SEQ ID NO:4-9, 34, and 35.

[0598] E325. The polynucleotide as described in E324, wherein each of the two or more copies of the SLC26A4 enhancer has the sequence of one of SEQ ID NO:4-9, 34, and 35.

[0599] E326. A nucleic acid vector, which contains the polynucleotide as described in any one of E83-E325.

[0600] E327. A nucleic acid vector, which contains a polynucleotide that contains an SLC26A4 enhancer having at least 85% sequence identity (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) with any one of SEQ ID NO:4, 5, 8, 9, 34, and 35.

[0601] E328. The nucleic acid vector as described in E327, wherein the SLC26A4 enhancer has at least 85% sequence identity (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity) with SEQ ID NO:4.

[0602] E329. The nucleic acid vector as described in E328, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:4.

[0603] E330. The nucleic acid vector as described in E327, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:5 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0604] E331. The nucleic acid vector as described in E330, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:5.

[0605] E332. The nucleic acid vector as described in E327, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:8 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0606] E333. The nucleic acid vector as described in E332, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:8.

[0607] E334. The nucleic acid vector as described in E327, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:9 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0608] E335. The nucleic acid vector as described in E334, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:9.

[0609] E336. The nucleic acid vector as described in E327, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO:34 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0610] E337. The nucleic acid vector as described in E336, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:34.

[0611] The nucleic acid vector as described in E327, wherein the SLC26A4 enhancer has at least 85% sequence identity with SEQ ID NO: 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0612] The nucleic acid vector as described in E338, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 35.

[0613] The nucleic acid vector as described in any one of E327 - E339, wherein the SLC26A4 enhancer is operably linked to a promoter.

[0614] The nucleic acid vector as described in E340, wherein the SLC26A4 enhancer is linked to the promoter by a nucleic acid linker of 1 to 500 nucleotides (e.g., 1 - 50, 1 - 100, 1 - 150, 1 - 200, 1 - 250, 1 - 300, 1 - 350, 1 - 400, 1 - 450, 1 - 500, 50 - 500, 100 - 500, 150 - 500, 200 - 500, 250 - 500, 300 - 500, 350 - 500, 400 - 500 or 450 - 500 nucleotides, such as about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 nucleotides).

[0615] The nucleic acid vector as described in E341, wherein the SLC26A4 enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 - 10, 1 - 20, 1 - 30, 1 - 40, 1 - 50, 1 - 60, 1 - 70, 1 - 80, 1 - 90, 1 - 100, 10 - 100, 20 - 100, 30 - 100, 40 - 100, 50 - 100, 60 - 100, 70 - 100, 80 - 100 or 90 - 100 nucleotides, such as about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides).

[0616] The nucleic acid vector as described in E340, wherein the SLC26A4 enhancer is directly linked (e.g., fused) to the promoter.

[0617] E344. The nucleic acid vector according to any one of E340 - E343, wherein the SLC26A4 enhancer is located 5' of the promoter.

[0618] E345. The nucleic acid vector according to any one of E340 - E343, wherein the SLC26A4 enhancer is located 3' of the promoter.

[0619] E346. The nucleic acid vector according to any one of E340 - E345, wherein the promoter is a minimal promoter, a core promoter, or a constitutive promoter.

[0620] E347. The nucleic acid vector according to E346, 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 phosphoglycerate kinase (PGK) promoter, an EF1α promoter, a β - globin promoter, a CMV promoter, an HSV promoter, or an SV40 promoter.

[0621] E348. The nucleic acid vector according to E347, 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 an SV40 minimal promoter.

[0622] E349. The nucleic acid vector according to E346, wherein the promoter is a mammalian promoter.

[0623] E350. The nucleic acid vector according to any one of E340 - E345, wherein the promoter is a mammalian SLC26A4 promoter.

[0624] E351. The nucleic acid vector according to E350, wherein the SLC26A4 promoter is a human or murine SLC26A4 promoter.

[0625] E352. The nucleic acid vector according to E350 or E351, wherein the SLC26A4 promoter has the formula 5'-B - A - C - 3', wherein:

[0626] A has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity);

[0627] B is absent or 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:2 or a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2; and

[0628] C is absent or 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:3 or a portion thereof comprising at least the first 159 consecutive nucleotides of SEQ ID NO:3,

[0629] wherein the SLC26A4 promoter is no longer than 1481 bases.

[0630] E353. The nucleic acid vector according to E352, wherein A has the sequence of SEQ ID NO:1.

[0631] E354. The nucleic acid vector according to E352 or E353, wherein B is absent.

[0632] E355. The nucleic acid vector according to E352 or E353, wherein B has the sequence of SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2.

[0633] E356. The nucleic acid vector according to E355, wherein B has the sequence of a portion of SEQ ID NO:2, the portion comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2.

[0634] E357. The nucleic acid vector according to E356, wherein the portion of SEQ ID NO:2 is the first 307 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2 (as shown in SEQ ID NO:14).

[0635] E358. The nucleic acid vector according to E355, wherein B has the sequence of SEQ ID NO:2.

[0636] E359. The nucleic acid vector according to any one of E352-E358, wherein C is absent.

[0637] The nucleic acid vector according to any one of E352 - E358, wherein C has the sequence of SEQ ID NO:3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:3.

[0638] The nucleic acid vector according to E360, wherein C has a sequence that is a portion of SEQ ID NO:3, said portion comprising at least the first 159 nucleotides of SEQ ID NO:3.

[0639] The nucleic acid vector according to E361, wherein said portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:15, 16, 17, 18, and 19, respectively).

[0640] The nucleic acid vector according to E362, wherein said portion of SEQ ID NO:3 is the first 159 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:15).

[0641] The nucleic acid vector according to E362, wherein said portion of SEQ ID NO:3 is the first 324 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:16).

[0642] The nucleic acid vector according to E362, wherein said portion of SEQ ID NO:3 is the first 341 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:17).

[0643] The nucleic acid vector according to E362, wherein said portion of SEQ ID NO:3 is the first 716 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:18).

[0644] The nucleic acid vector according to E362, wherein said portion of SEQ ID NO:3 is the first 723 consecutive nucleotides of SEQ ID NO:3 (shown as SEQ ID NO:19).

[0645] The nucleic acid vector according to E360, wherein C has the sequence of SEQ ID NO:3.

[0646] E369. A nucleic acid vector as described in any one of E352, E354, and E359, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0647] E370. A nucleic acid vector as described in E369, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1.

[0648] E371. A nucleic acid vector as described in any one of E352, E353, E355, E358, and E359, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0649] E372. A nucleic acid vector as described in E371, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:20.

[0650] E373. A nucleic acid vector as described in any one of E352-E354, E360, and E368, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:21 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0651] E374. A nucleic acid vector as described in E373, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:21.

[0652] E375. A nucleic acid vector as described in any one of E352, E353, E355-E357, and E359, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:22 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0653] E376. A nucleic acid vector as described in E375, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:22.

[0654] E377. A nucleic acid vector as described in any one of E352-E354, E360-E362, and E365, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:23 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0655] E378. A nucleic acid vector as described in E377, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:23.

[0656] E379. A nucleic acid vector as described in any one of E352-E354, E360-E362, and E367, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:24 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0657] E380. A nucleic acid vector as described in E379, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:24.

[0658] E381. A nucleic acid vector as described in any one of E352, E353, E355-E357, E360-E362, and E364, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:25 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0659] E382. A nucleic acid vector as described in E381, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:25.

[0660] E383. A nucleic acid vector as described in any one of E352, E353, E355-E357, E360-E362, and E364, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:26 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0661] The nucleic acid vector as described in E383, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:26.

[0662] The nucleic acid vector as described in any one of E352, E353, E355 - E357 and E360 - E363, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:27 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0663] The nucleic acid vector as described in E385, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:27.

[0664] The nucleic acid vector as described in E350 or E351, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:28 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0665] The nucleic acid vector as described in E387, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:28.

[0666] The nucleic acid vector as described in E350 or E351, wherein the SLC26A4 promoter has at least 85% sequence identity with any one of SEQ ID NO:29 - 33 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0667] The nucleic acid vector as described in E389, wherein the SLC26A4 promoter has the sequence of any one of SEQ ID NO:29 - 33.

[0668] The nucleic acid vector as described in E350 or E351, wherein the SLC26A4 promoter has at least 85% sequence identity with SEQ ID NO:41 (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0669] E392. The nucleic acid vector as described in E391, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 41.

[0670] E393. The nucleic acid vector as described in any one of E340 - E392, wherein the promoter is operably linked to a polynucleotide capable of being transcribed to produce an expression product.

[0671] E394. The nucleic acid vector as described in E393, wherein the expression product is a heterologous expression product.

[0672] E395. The nucleic acid vector as described in E393, wherein the expression product is an expression product endogenously expressed in cells expressing SLC26A4.

[0673] E396. The nucleic acid vector as described in E395, wherein the expression product is an expression product endogenously expressed in inner ear cells expressing SLC26A4.

[0674] E397. The nucleic acid vector as described in E396, wherein the expression product is an expression product endogenously expressed (e.g., expressed in at least one of these cell types) in interdental cells, spiral prominence cells, cochlear root cells, and / or vestibular supporting cells.

[0675] E398. The nucleic acid vector as described in any one of E395 - E397, wherein the expression product is pannexin (e.g., mammalian pannexin).

[0676] E399. The nucleic acid vector as described in E398, wherein the pannexin (the mammalian pannexin) is the wild - type isoform endogenously expressed in the mammalian inner ear.

[0677] E400. The nucleic acid vector as described in any one of E398 or E399, wherein the pannexin (the mammalian pannexin) has at least 85% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 11 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0678] E401. The nucleic acid vector as described in E400, wherein the pannexin (the mammalian pannexin) has the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

[0679] E402. The nucleic acid vector as described in any one of E393 - E397, wherein the expression product is Atoh1 (e.g., mammalian Atoh1).

[0680] E403. The nucleic acid vector as described in E402, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform that is endogenously expressed in the mammalian inner ear.

[0681] E404. The nucleic acid vector as described in any one of E402 or E403, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity with SEQ ID NO: 36 or SEQ ID NO: 38 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0682] E405. The nucleic acid vector as described in E404, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO: 36 or SEQ ID NO: 38.

[0683] E406. The nucleic acid vector as described in E393 or E394, wherein the expression product is a protein, short hairpin RNA (shRNA), antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), transcription activator-like effector nuclease (TALEN), or zinc finger nuclease (ZFN) or guide RNA (gRNA)) or a microRNA.

[0684] E407. The nucleic acid vector as described in any one of E327 - E406, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, and each enhancer is independently selected from enhancers having at least 85% sequence identity with any one of SEQ ID NO: 4, 5, 8, 9, 34, and 35 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity).

[0685] E408. The nucleic acid vector as described in E407, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NO: 4, 5, 8, 9, 34, and 35.

[0686] E409. A nucleic acid vector as described in any one of E327 - E408, wherein the polynucleotide comprises two or more copies of an SLC26A4 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 higher sequence identity) with any one of SEQ ID NO: 4, 5, 8, 9, 34, and 35.

[0687] E410. A nucleic acid vector as described in E409, wherein each of the two or more copies of the SLC26A4 enhancer has the sequence of one of SEQ ID NO: 4, 5, 8, 9, 34, and 35.

[0688] E411. A nucleic acid vector as described in any one of E1 - E82 and E326 - E410, wherein the nucleic acid vector is a viral vector, plasmid, cosmid, or artificial chromosome.

[0689] E412. A nucleic acid vector as described in any one of E1 - E82 and E326 - E411, wherein the nucleic acid vector is a viral vector.

[0690] E413. A nucleic acid vector as described in E412, wherein the viral vector is an adeno - associated virus (AAV) viral vector, an adenovirus viral vector, or a lentivirus viral vector.

[0691] E414. A nucleic acid vector as described in E413, wherein the viral vector is an AAV vector.

[0692] E415. A nucleic acid vector as described in E414, wherein the AAV vector has an AAV1, AAV2, AAV2quad(Y - F), 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.

[0693] E416. A composition comprising a nucleic acid vector as described in any one of E1 - E82 and E326 - E415 and a pharmaceutically acceptable carrier, diluent, or excipient.

[0694] E417. A cell comprising a polynucleotide as described in any one of E83 - E325 or a nucleic acid vector as described in any one of E1 - E82 and E326 - E415.

[0695] E418. The cell as described in E417, wherein the cell is a cell expressing SLC26A4.

[0696] E419. The cell as described in E418, wherein the cell is an inner ear cell expressing SLC26A4.

[0697] E420. The cell as described in any one of E417-E419, wherein the cell is a mammalian cell.

[0698] E421. The cell as described in E420, wherein the mammalian cell is a human cell.

[0699] E422. The cell as described in any one of E417-E431, wherein the cell is an interdental cell, a spiral prominence cell, a cochlear root cell or a vestibular supporting cell.

[0700] E423. A method for expressing an expression product in a cell, the method comprising the step of contacting the cell with the nucleic acid vector as described in any one of E1-E82 and E326-E415 or the composition as described in E416.

[0701] E424. The method as described in E423, wherein the cell is an inner ear cell.

[0702] E425. The method as described in E423 or E424, wherein the cell is a cell expressing SLC26A4.

[0703] E426. The cell as described in any one of E423-E425, wherein the cell is an inner ear cell expressing SLC26A4.

[0704] E427. The method as described in E426, wherein the inner ear cell expressing SLC26A4 is an interdental cell, a spiral prominence cell, a cochlear root cell or a vestibular supporting cell.

[0705] E428. The method as described in any one of E423-E427, wherein the cell is a mammalian cell.

[0706] E429. The method as described in E428, wherein the mammalian cell is a human cell.

[0707] E430. The method as described in any one of E423-E429, wherein the contacting is carried out in a subject (e.g., in vivo).

[0708] E431. A method of treating a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss or deafness), the method comprising the step of administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid carrier as described in any one of E1-E82 and E326-E415 or the composition as described in E416.

[0709] E432. The method according to E431, wherein the hearing loss is pendrin-related hearing loss.

[0710] E433. The method according to E432, wherein the expression product is pendrin.

[0711] E434. The method according to E432 or E433, wherein the pendrin-related hearing loss is hearing loss associated with Pendred syndrome or DFNB4.

[0712] E435. A method of treating a subject in need thereof for hearing loss associated with Meniere's disease, the method comprising the step of administering to the inner ear of the subject an effective amount of a nucleic acid carrier as described in any one of E1-E82 and E326-E415 or the composition as described in E416.

[0713] E436. A method of treating a subject in need thereof for tinnitus associated with Meniere's disease, the method comprising the step of administering to the inner ear of the subject an effective amount of a nucleic acid carrier as described in any one of E1-E82 and E326-E415 or the composition as described in E416.

[0714] E437. The method according to E435 or E436, wherein the expression product is pendrin.

[0715] E438. A method of treating a subject in need thereof for vestibular dysfunction associated with Meniere's disease, the method comprising the step of administering to the inner ear of the subject an effective amount of a nucleic acid carrier as described in any one of E1-E82 and E326-E415 or the composition as described in E416.

[0716] E439. The method according to E438, wherein the expression product is pendrin or Atoh1.

[0717] E440. The method according to E438 or E439, wherein the vestibular dysfunction is vertigo.

[0718] E441. A method of treating a subject having 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 carrier as described in any one of E1-E82 and E326-E415 or the composition as described in E416.

[0719] E442. The method according to E441, wherein the vestibular dysfunction is a pendrin-related vestibular dysfunction.

[0720] E443. The method according to E442, wherein the expression product is pendrin.

[0721] E444. The method according to E442 or E443, wherein the pendrin-related vestibular dysfunction is a vestibular dysfunction associated with Pendred syndrome or DFNB4.

[0722] E445. The method according to E441, wherein the expression product is pendrin or Atoh1.

[0723] E446. 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 carrier according to any one of E1-E82 and E326-E415 or the composition according to E416.

[0724] E447. The method according to E446, wherein the expression product is Atoh1.

[0725] E448. The method according to E446 or E447, wherein the contacting is in vivo (e.g., in a subject).

[0726] E449. The method according to E448, wherein the subject has vestibular dysfunction or is at risk of developing vestibular dysfunction.

[0727] E450. A method for inducing or increasing the regeneration of vestibular hair cells 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 carrier according to any one of E1-E82 and E326-E415 or the composition according to E416.

[0728] E451. The method according to E450, wherein the expression product is Atoh1.

[0729] E452. The method according to E450 or E451, wherein the subject has vestibular dysfunction or is at risk of developing vestibular dysfunction.

[0730] E453. A method for improving the function of cells expressing SLC26A4, the method comprising the step of contacting the cells expressing SLC26A4 with the nucleic acid carrier according to any one of E1-E82 and E326-E415 or the composition according to E416.

[0731] E454. The method according to E453, wherein the contacting is carried out in vivo (e.g., in a subject).

[0732] E455. The method according to E454, wherein the subject has hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction or is at risk of developing hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction.

[0733] E456. The method according to any one of E441 - E455, wherein the vestibular dysfunction is vertigo, dizziness, imbalance (e.g., loss of balance or balance disorder), oscillopsia or bilateral vestibular lesions.

[0734] E457. The method according to any one of E441 - E456, wherein the vestibular dysfunction is associated with damage or loss of vestibular hair cells.

[0735] E458. The method according to E457, wherein the damage or loss of vestibular hair cells is associated with: age (the vestibular dysfunction is age - related vestibular dysfunction), exposure to ototoxic (e.g., vestibulotoxic) 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).

[0736] E459. The method according to E458, wherein the ototoxic drugs are aminoglycosides (aminoglycoside antibiotics such as gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin and netilmicin), viomycin, antitumor drugs (e.g., platinum - containing chemotherapeutic agents such as cisplatin, carboplatin or oxaliplatin, or another chemotherapeutic agent such as nitrogen mustard or vincristine), loop diuretics (e.g., ethacrynic acid or furosemide), salicylates or quinine.

[0737] E460. The method according to any one of E431 - E437, wherein the method further comprises evaluating the hearing of the subject before administering the nucleic acid vector or composition.

[0738] E461. The method according to any one of E431 - E437 and E460, wherein the method further comprises evaluating the hearing of the subject after administering the nucleic acid vector or composition.

[0739] E462. The method according to any one of E438 - E459, wherein the method further comprises evaluating the vestibular function of the subject before administering the nucleic acid vector or composition.

[0740] E463. The method according to any one of E438-E459 and E462, wherein the method further comprises evaluating the vestibular function of the subject after administering the nucleic acid vector or composition.

[0741] E464. The method according to any one of E423-E463, wherein the nucleic acid vector or composition is administered locally.

[0742] E465. The method according to E464, wherein the nucleic acid vector or composition is administered to the inner ear.

[0743] E466. The method according to E464, wherein the nucleic acid vector or composition is administered to the middle ear.

[0744] E467. The method according to E464, wherein the nucleic acid vector or composition is administered through the tympanic membrane or into the tympanic cavity.

[0745] E468. The method according to E464, wherein the nucleic acid vector or composition is administered into the perilymph.

[0746] E469. The method according to E464, wherein the nucleic acid vector or composition is administered into the endolymph.

[0747] E470. The method according to E464, wherein the nucleic acid vector or composition is administered to or through the oval window.

[0748] E471. The method according to E464, wherein the nucleic acid vector or composition is administered to or through the round window.

[0749] E472. The method according to E464, wherein the nucleic acid vector or composition is administered to the semicircular canals.

[0750] E473. The method according to any one of E423-E472, 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 the expression product in cells 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 a feeling of ear fullness, increase vestibular hair cell regeneration, induce or increase the differentiation of vestibular supporting cells into vestibular hair cells, increase or induce the maturation of hair cells (e.g., the maturation of regenerated hair cells), or improve vestibular supporting cell function.

[0751] E474. The method according to any one of E423-E473, wherein the subject is a human subject.

[0752] E475. A kit, the kit comprising the polynucleotide according to any one of E83-E325, the nucleic acid vector according to any one of E1-E82 and E326-E415, or the composition according to E416.

[0753] Other embodiments

[0754] Without departing from the scope and spirit of the present invention, various modifications and variations of the present invention will be apparent to those skilled in the art. Although the present invention has been described in connection with specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described ways of carrying out the present invention will be apparent to those skilled in the art and are intended to be included within the scope of the present invention. Other embodiments are within the scope of the claims.

Claims

1. A nucleic acid vector, the nucleic acid vector comprising an SLC26A4 promoter of the following formula: 5'-B-A-C-3', wherein: A has at least 85% sequence identity with SEQ ID NO:1; B is absent or has at least 85% sequence identity with SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:2; and C is absent or has at least 85% sequence identity with SEQ ID NO:3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:3, wherein the SLC26A4 promoter is not longer than 1481 bases.

2. The nucleic acid vector according to claim 1, wherein A has the sequence of SEQ ID NO:

1.

3. The nucleic acid vector according to claim 1 or 2, wherein B is absent.

4. The nucleic acid vector according to claim 1 or 2, wherein B has the sequence of SEQ ID NO:2 or a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:

2.

5. The nucleic acid vector according to claim 4, wherein B has the sequence of a portion of SEQ ID NO:2, the portion comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:

2.

6. The nucleic acid vector according to claim 5, wherein the portion of SEQ ID NO:2 is the first 307 consecutive nucleotides starting from the 3'-end of SEQ ID NO:

2.

7. The nucleic acid vector according to claim 4, wherein B has the sequence of SEQ ID NO:

2.

8. The nucleic acid vector according to any one of claims 1-7, wherein C is absent.

9. The nucleic acid vector according to any one of claims 1-7, wherein C has the sequence of SEQ ID NO:3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:

3.

10. The nucleic acid vector according to claim 9, wherein C has the sequence of a portion of SEQ ID NO:3, the portion comprising at least the first 159 nucleotides of SEQ ID NO:

3.

11. The nucleic acid vector according to claim 10, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716 or 723 consecutive nucleotides of SEQ ID NO:

3.

12. The nucleic acid vector according to claim 9, wherein C has the sequence of SEQ ID NO:

3.

13. The nucleic acid vector according to any one of claims 1-12, the nucleic acid vector further comprising an SLC26A4 enhancer operably linked to the SLC26A4 promoter.

14. The nucleic acid vector according to claim 13, wherein the SLC26A4 enhancer has at least 85% sequence identity with any one of SEQ ID NO:4-7, 34 and 35.

15. The nucleic acid vector according to claim 14, wherein the SLC26A4 enhancer has the sequence of any one of SEQ ID NO: 4-7, 34, and 35.

16. The nucleic acid vector according to any one of claims 13-15, wherein the SLC26A4 enhancer is directly linked to the SLC26A4 promoter.

17. The nucleic acid vector according to any one of claims 13-15, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter through a nucleic acid linker of 1 to 500 nucleotides.

18. The nucleic acid vector according to claim 17, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter through a nucleic acid linker of 1 to 100 nucleotides.

19. The nucleic acid vector according to any one of claims 13-18, wherein the SLC26A4 enhancer is located 5' of the SLC26A4 promoter.

20. The nucleic acid vector according to any one of claims 13-19, wherein the vector comprises two or more different SLC26A4 enhancers, and each SLC26A4 enhancer is independently selected from enhancers having at least 85% sequence identity with any one of SEQ ID NO: 4-7, 34, and 35.

21. The nucleic acid vector according to claim 20, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of any one of SEQ ID NO: 4-7, 34, and 35.

22. The nucleic acid vector according to claim 20, wherein the vector comprises a first enhancer having at least 85% sequence identity with SEQ ID NO: 6 and a second enhancer having at least 85% sequence identity with SEQ ID NO:

7.

23. The nucleic acid vector according to claim 22, wherein the first enhancer has the sequence of SEQ ID NO: 6, and the second enhancer has the sequence of SEQ ID NO:

7.

24. The nucleic acid vector according to claim 23, wherein the first enhancer or the second enhancer is directly fused to the other of the first enhancer or the second enhancer that is directly fused to the SLC26A4 promoter.

25. The nucleic acid vector according to claim 24, wherein the nucleic acid vector comprises SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 1 in a 5' to 3' order.

26. The nucleic acid vector according to any one of claims 13-21, wherein the vector comprises two or more copies of an SLC26A4 enhancer having at least 85% sequence identity with any one of SEQ ID NO: 4-7, 34, and 35.

27. The nucleic acid vector according to claim 26, wherein each copy of the two or more copies of the SLC26A4 enhancer has the sequence of any one of SEQ ID NO: 4-7, 34, and 35.

28. The nucleic acid vector according to any one of claims 1-27, wherein the SLC26A4 promoter is operably linked to a polynucleotide capable of being transcribed to produce an expression product.

29. The nucleic acid vector according to claim 28, wherein the expression product is a heterologous expression product.

30. The nucleic acid vector according to claim 28, wherein the expression product is an expression product endogenously expressed in inner ear cells that express SLC26A4.

31. The nucleic acid vector according to claim 30, wherein the inner ear cells that express SLC26A4 are interdental cells, spiral prominence cells, cochlear root cells, or vestibular supporting cells.

32. The nucleic acid vector according to any one of claims 28, 30, and 31, wherein the expression product is mammalian pantophysin.

33. The nucleic acid vector according to claim 32, wherein the mammalian pantophysin is a wild-type isoform endogenously expressed in the ear of a mammal.

34. The nucleic acid vector according to claim 33, wherein the mammalian pantophysin has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:

11.

35. The nucleic acid vector according to any one of claims 28, 30, and 31, wherein the expression product is mammalian Atoh1 protein.

36. A polynucleotide comprising an SLC26A4 enhancer having at least 85% sequence identity to any one of SEQ ID NOs: 4, 5, 34, and 35, the enhancer being operably linked to a promoter, wherein the enhancer is directly or indirectly linked to the promoter through a nucleic acid linker of 1 to 500 nucleotides.

37. The polynucleotide according to claim 36, wherein the enhancer is linked to the promoter through a nucleic acid linker of 1 to 100 nucleotides.

38. The polynucleotide according to claim 36, wherein the enhancer is directly linked to the promoter.

39. The polynucleotide according to any one of claims 36-38, wherein the enhancer is located 5' of the promoter.

40. The polynucleotide according to any one of claims 36-39, wherein the enhancer has the sequence of SEQ ID NO:

4.

41. The polynucleotide according to any one of claims 36-39, wherein the enhancer has the sequence of SEQ ID NO:

5.

42. A polynucleotide comprising: a. The SLC26A4 promoter of the following formula: 5'-B-A-C-3', wherein: A has at least 85% sequence identity to SEQ ID NO: 1; B is absent or has at least 85% sequence identity to SEQ ID NO: 2 or a portion thereof comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2; and C is absent or has at least 85% sequence identity to SEQ ID NO: 3 or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO: 3; and b. An SLC26A4 enhancer having at least 85% sequence identity to any one of SEQ ID NOs: 4-7, 34, and 35, wherein the enhancer is directly or connected to the promoter through a nucleic acid linker of 1 to 500 nucleotides.

43. The polynucleotide according to claim 42, wherein A has the sequence of SEQ ID NO:

1.

44. The polynucleotide according to claim 42 or 43, wherein B is absent.

45. The polynucleotide according to claim 42 or 43, wherein B has the sequence of SEQ ID NO: 2 or a sequence of a portion thereof comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:

2.

46. The polynucleotide according to claim 45, wherein B has a sequence of a portion of SEQ ID NO: 2, the portion comprising 1 to 917 consecutive nucleotides starting from the 3'-end of SEQ ID NO:

2.

47. The polynucleotide according to claim 46, wherein the portion of SEQ ID NO: 2 is the first 307 consecutive nucleotides starting from the 3'-end of SEQ ID NO:

2.

48. The polynucleotide according to claim 45, wherein B has the sequence of SEQ ID NO:

2.

49. The polynucleotide according to any one of claims 42-48, wherein C is absent.

50. The polynucleotide according to any one of claims 42-48, wherein C has the sequence of SEQ ID NO: 3 or a sequence of a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:

3.

51. The polynucleotide according to claim 50, wherein C has a sequence of a portion of SEQ ID NO: 3, the portion comprising at least the first 159 nucleotides of SEQ ID NO:

3.

52. The polynucleotide according to claim 51, wherein the portion of SEQ ID NO: 3 is the first 159, 324, 341, 716, or 723 consecutive nucleotides of SEQ ID NO:

3.

53. The polynucleotide according to claim 50, wherein C has the sequence of SEQ ID NO:

3.

54. The polynucleotide according to claim 49, wherein B is absent and C is absent.

55. The polynucleotide according to any one of claims 42-54, wherein the SLC26A4 promoter is no longer than 1481 bases.

56. The polynucleotide according to any one of claims 42-55, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:

4.

57. The polynucleotide according to any one of claims 42-55, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:

5.

58. A polynucleotide as claimed in any one of claims 42 - 55, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, each of which is independently selected from enhancers having at least 85% sequence identity with any one of SEQ ID NOs: 4 - 7, 34, and 35.

59. The polynucleotide as claimed in claim 58, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of any one of SEQ ID NOs: 4 - 7, 34, and 35.

60. The polynucleotide as claimed in claim 58, the polynucleotide comprising a first enhancer having at least 85% sequence identity with SEQ ID NO: 6 and a second enhancer having at least 85% sequence identity with SEQ ID NO:

7.

61. The nucleic acid vector as claimed in claim 60, wherein the first enhancer has the sequence of SEQ ID NO: 6 and the second enhancer has the sequence of SEQ ID NO:

7.

62. The nucleic acid vector as claimed in claim 61, wherein the first enhancer or the second enhancer is directly fused to the other of the first enhancer or the second enhancer that is directly fused to the SLC26A4 promoter.

63. The nucleic acid vector as claimed in claim 62, the nucleic acid vector comprising SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 1 in a 5' to 3' order.

64. The polynucleotide as claimed in any one of claims 36 - 63, wherein the promoter is operably linked to a polynucleotide capable of being transcribed to produce an expression product.

65. The polynucleotide as claimed in claim 64, wherein the expression product is a heterologous expression product.

66. The polynucleotide as claimed in claim 64, wherein the expression product is an expression product that is endogenously expressed in inner ear cells that express SLC26A4.

67. The polynucleotide as claimed in claim 66, wherein the inner ear cells that express SLC26A4 are interdental cells, spiral prominence cells, cochlear root cells, or vestibular supporting cells.

68. The polynucleotide as claimed in any one of claims 64, 66, and 67, wherein the expression product is a mammalian pantophysin.

69. The polynucleotide as claimed in claim 68, wherein the mammalian pantophysin is a wild - type isoform that is endogenously expressed in the ear of a mammal.

70. The polynucleotide as claimed in claim 69, wherein the mammalian pantophysin has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:

11.

71. The polynucleotide as claimed in any one of claims 64 - 67, wherein the expression product is a mammalian Atoh1 protein.

72. A nucleic acid vector comprising the polynucleotide as claimed in any one of claims 36 - 71.

73. The nucleic acid vector as claimed in any one of claims 1 - 35 and 72, wherein the vector is a viral vector.

74. The nucleic acid vector according to claim 73, wherein the viral vector is an adeno-associated viral vector.

75. A composition comprising the nucleic acid vector according to any one of claims 1-35 and 72, and a pharmaceutically acceptable carrier, diluent or excipient.

76. A method for expressing an expression product in inner ear cells, the method comprising contacting the inner ear cells with the nucleic acid vector according to any one of claims 1-35 and 72 or the composition according to claim 75.

77. The method according to claim 70, wherein the contacting is carried out in a subject.

78. A method for treating a subject suffering from or at risk of developing pendrin-related hearing loss, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid vector according to any one of claims 1-35 and 72, wherein the expression product is pendrin.

79. The method according to claim 72, wherein the pendrin-related hearing loss is Pendred syndrome or DFNB4.

80. A method for treating a subject in need thereof for hearing loss associated with Meniere's disease, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid vector according to any one of claims 1-35 and 72, wherein the expression product is pendrin.

81. A method for treating a subject suffering from or at risk of developing pendrin-related vestibular dysfunction, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid vector according to any one of claims 1-35 and 72, wherein the expression product is pendrin.

82. A method for treating a subject in need thereof for vestibular dysfunction associated with Meniere's disease, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid vector according to any one of claims 1-35 and 72, wherein the expression product is pendrin or Atoh1.

83. A method for treating a subject suffering from or at risk of developing vestibular dysfunction associated with damage or loss of vestibular hair cells, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid vector according to claims 1-35 and 72, wherein the expression product is pendrin or Atoh1.

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