SLC26A4 regulatory elements and uses thereof

Abstract

The present disclosure provides an SL26A4 promoter and an SLC26A4 enhancer, as well as vectors containing them, that can be used to restrict gene expression to SLC26A4-expressing cells and increase gene expression in these cells, such as interdental cells, root cells, spiral ridge cells, and vestibular supporting cells. The SLC26A4 enhancer and SLC26A4 promoter described herein can be operably linked to a polynucleotide, such as a transgene, that encodes an expression product and used to treat subjects with hearing loss or vestibular dysfunction or at risk of developing such a condition.

Description

[Technical Field]

[0001] The present disclosure relates to SLC26A4 regulatory elements and uses thereof. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML copy was created on September 25, 2023, has the file name "51124-101WO3_Sequence_Listing_9_25_23.xml", and is 73,879 bytes in size. [Background technology]

[0002] Hearing loss is the most common human sensory deficit, affecting approximately 15% of school-age children and one in three by age 65. Hereditary hearing loss occurs in approximately 1 in 500 newborns each year in the United States. Approximately 80% of congenital cases are caused by mutations found 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 can be lost or disrupted, and these patients may develop prelingual or postlingual nonsyndromic hearing loss or may be born with hearing loss that progresses over time to severe hearing impairment. Pendrin is expressed in specialized epithelial cells of the inner ear (the cochlea, vestibular labyrinth, and endolymphatic sac, as well as nonsensory epithelial cells of the saccule, utricle, and ampulla), the thyroid gland (thyroid cells), the kidney (renal aggregate type B intercalary cells), the airways, mammary glands, salivary ducts, and the apical membrane of the liver. Within the inner ear, pendrin regulates pH and fluid absorption by exchanging chloride and bicarbonate anions between the epithelium and the endolymphatic compartment. Currently, no curative therapy exists for this population. Therefore, there is a need for therapeutic agents that restore and / or reverse the progression of hearing loss in patients with these mutations. Summary of the Invention

[0003] The present invention provides compositions and methods for promoting expression of a gene of interest in a specific cell type, e.g., a gene endogenously expressed in an SLC26A4-expressing cell. 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, e.g., an inhibitory RNA molecule) to induce expression of the expression product in an SLC26A4-expressing cell. 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-associated 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-associated vestibular dysfunction or vestibular dysfunction associated with damage or loss of vestibular hair cells).

[0004] In a first aspect, the present invention provides a nucleic acid vector containing an SLC26A4 promoter of the formula 5'-BAC-3', wherein 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 more sequence identity) to SEQ ID NO: 1, and 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 more sequence identity) to SEQ ID NO: 2, or a portion thereof comprising 1 to 917 contiguous nucleotides from the 3' end of SEQ ID NO: 2. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), 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 more sequence identity) to SEQ ID NO:3 or a portion thereof comprising at least the first 159 contiguous nucleotides of SEQ ID NO:3, and the SLC26A4 promoter is 1481 bases or less.

[0005] In another aspect, the invention provides a polynucleotide containing the SLC26A4 promoter of the formula 5'-BAC-3' operably linked to a polynucleotide that can be transcribed to generate an expression product, wherein 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 more sequence identity) to SEQ ID NO:1, and B is absent or has at least 85% sequence identity with SEQ ID NO:2, or a portion thereof comprising 1 to 917 contiguous nucleotides from the 3' end of SEQ ID NO:2. or more sequence identity) to SEQ ID NO:3 or a portion thereof comprising at least the first 159 contiguous nucleotides of SEQ ID NO:3; and the SLC26A4 promoter is 1481 bases or less.

[0006] In another aspect, the present invention provides 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35 operably linked to a promoter, wherein the enhancer is located directly or within 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 500, 1 to 500, 1 to 150, 1 to 200, 1 to 350, 1 to 450, 1 to 500, 1 to 500, 1 to 1500, 1 to 2000, 1 to 3500, 1 to 4500, 1 to 5000, 1 to 5000, 1 to 1500, 1 to 2000, 1 to 3500, 1 to 45 ...0, 1 to 2000, 1 to 35000, 1 to 35000, 1 to 4500, 1 to 5000, 1 to and the nucleic acid linker is about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides.

[0007] In another aspect, the invention provides a nucleic acid vector containing 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 more 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.

[0008] In another aspect, the invention provides (a) an SLC26A4 promoter of the formula 5'-BAC-3', wherein 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 more sequence identity) to SEQ ID NO: 1, and B is absent or has at least 85% sequence identity (e.g., 85% or more) to SEQ ID NO: 2, or a portion thereof comprising 1 to 917 contiguous nucleotides from the 3' end of SEQ ID NO: 2. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), and 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 more sequence identity) with SLC26A, SLC26B, SLC26C, SLC26D, SLC26E, SLC26F, SLC26G, SLC26H, SLC26I, SLC26IH ... and a 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 more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35, wherein the enhancer is directly attached to the promoter or is attached to a region of 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 40 In some embodiments, the SLC26A4 promoter is 1,481 bases or less in length.

[0009] In another aspect, the present invention provides a nucleic acid vector containing a polynucleotide according to any of the previous aspects. 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 phosphoglycerol 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 CMV mini promoter, a minCMV promoter, a CMV-TATA+INR promoter, a minCMV-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.

[0010] In some embodiments of any of the foregoing aspects, the promoter is a minimal promoter. In some embodiments, the SLC26A4 promoter is a human or mouse SLC26A4 promoter. In some embodiments, the SLC26A4 promoter has the formula 5'-BAC-3', where 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 more sequence identity) to SEQ ID NO: 1, and B is absent or has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 99%, or more sequence identity) to SEQ ID NO: 2, or a portion thereof comprising 1 to 917 contiguous nucleotides from the 3' end of SEQ ID NO: 2. 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 more sequence identity) to SEQ ID NO:3 or a portion thereof comprising at least the first 159 contiguous nucleotides of SEQ ID NO:3, and the SLC26A4 promoter is 1481 bases or less.

[0011] In some embodiments of any of the aforementioned aspects, A has the sequence of SEQ ID NO:1. In some embodiments of any of the aforementioned aspects, B is absent. 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 contiguous nucleotides from the 3' end of SEQ ID NO:2. In some embodiments, B has the sequence of a portion of SEQ ID NO:2 comprising 1 to 917 contiguous nucleotides from the 3' end of SEQ ID NO:2. In some embodiments, the portion of SEQ ID NO:2 is the first 307 contiguous nucleotides (set forth in SEQ ID NO:14) from the 3' end of SEQ ID NO:2. In some embodiments, B has the sequence of SEQ ID NO:2.

[0012] In some embodiments of any of the aforementioned aspects, C is absent. In some embodiments of any of the aforementioned aspects, both B and C are absent. 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 the sequence of a portion of SEQ ID NO:3 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 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NOs:15, 16, 17, 18, and 19, respectively). In some embodiments, the portion of SEQ ID NO:3 has the sequence of SEQ ID NO:15. In some embodiments, the portion of SEQ ID NO:3 has the sequence of SEQ ID NO:16. In some embodiments, the portion of SEQ ID NO:3 has the sequence of SEQ ID NO:17. In some embodiments, the portion of SEQ ID NO:3 has the sequence of SEQ ID NO:18. In some embodiments, the 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.

[0013] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 1. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 1.

[0014] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 20. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 20.

[0015] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 21. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 21.

[0016] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 22. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 22.

[0017] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 23. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 23.

[0018] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 24. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 24.

[0019] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 25. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 25.

[0020] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 26. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 26.

[0021] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 27. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 27.

[0022] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 28. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 28.

[0023] 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 more sequence identity) to 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.

[0024] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 41. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 41.

[0025] In some embodiments of any of the aforementioned aspects, the SLC26A4 promoter is operably linked to an SLC26A4 enhancer. 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 more sequence identity) to any one of SEQ ID NOs: 4-9, 34.

[0026] In some embodiments of any of the aforementioned aspects, the SLC26A4 enhancer is directly linked to (eg, fused to) the promoter. In some embodiments of any of the foregoing aspects, the SLC26A4 enhancer is a sequence of 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to and is attached to the promoter via a nucleic acid linker of 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.

[0027] In some embodiments of any of the aforementioned aspects, the SLC26A4 enhancer is linked to the promoter via a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 nucleotides, e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0028] In some embodiments of any of the aforementioned aspects, the SLC26A4 enhancer is located 5' to the promoter. In some embodiments of any of the aforementioned aspects, the SLC26A4 enhancer is located 3' to the promoter.

[0029] In some embodiments of any of the aforementioned aspects, the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product. In some embodiments of any of the aforementioned aspects, the expression product is a heterologous expression product.

[0030] In some embodiments of any of the foregoing aspects, the expression product is an expression product endogenously expressed in an SLC26A4-expressing cell. In some embodiments, the expression product is an expression product endogenously expressed in an SLC26A4-expressing inner ear cell. In some embodiments, the expression product is an expression product endogenously expressed in an interdental cell, a spiral eminence cell, a cochlear root cell, and / or a vestibular supporting cell (e.g., expressed in at least one of these cell types).

[0031] In some embodiments of any of the foregoing aspects, the expression product is pendrin (e.g., a mammalian pendrin protein). In some embodiments, the pendrin (e.g., a mammalian pendrin protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal. In some embodiments, the pendrin (mammalian pendrin protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:8 or SEQ ID NO:10. In some embodiments, the pendrin (mammalian pendrin protein) has the sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0032] In some embodiments of any of the foregoing aspects, the expression product is Atoh1 (e.g., mammalian Atoh1). In some embodiments, the Atoh1 (e.g., mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the mammalian inner ear. In some embodiments, the Atoh1 (mammalian Atoh1 protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:36 or SEQ ID NO:38. In some embodiments, the Atoh1 (mammalian Atoh1 protein) has the sequence of SEQ ID NO:36 or SEQ ID NO:38.

[0033] In some embodiments of any of the foregoing aspects, the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0034] In some embodiments of any of the foregoing aspects, the nucleic acid vector or polynucleotide contains two or more different SLC26A4 enhancers, each enhancer 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 more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35. In some embodiments, each different SLC26A4 enhancer independently selected from enhancers having the sequence of one of SEQ ID NOs: 4-9, 34, and 35. In some embodiments, a nucleic acid vector or polynucleotide contains two or more different SLC26A4 enhancers, a 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 more sequence identity) to SEQ ID NO: 6, and a 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 more sequence identity) to SEQ ID NO: 7. In some embodiments, a 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, the enhancers of SEQ ID NO:6 and SEQ ID NO:7 are both located 5' of the SLC26A4 promoter, e.g., the SLC26A4 promoter of SEQ ID NO:1. In some embodiments, the enhancers of SEQ ID NO:6 and SEQ ID NO:7 are both 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'→3' order: SEQ ID NO:6-SEQ ID NO:7-SLC26A4 promoter. In one particular embodiment, the nucleic acid vector or polynucleotide is directly linked in the 5'→3' order: SEQ ID NO:6-SEQ ID NO:7-SLC26A4 promoter.

[0035] In some embodiments of any of the foregoing aspects, the nucleic acid vector or polynucleotide contains 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 more sequence identity) to 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.

[0036] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 4. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 4.

[0037] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 5. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 5.

[0038] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 6. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 6.

[0039] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 7. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 7.

[0040] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 8. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 8.

[0041] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 9. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 9.

[0042] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 34. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 34.

[0043] In some embodiments of any of the aforementioned 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 more sequence identity) to SEQ ID NO: 35. In some embodiments, the SLC26A4 enhancer has the sequence of SEQ ID NO: 35.

[0044] In some embodiments of any of the foregoing aspects, the nucleic acid vector is a viral vector, a plasmid, a cosmid, or an artificial chromosome. In some embodiments, the nucleic acid vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, an adenoviral 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(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S capsid. In some embodiments, the AAV vector has an AAV1 capsid. In some embodiments, the AAV vector has an AAV2 capsid. In some embodiments, the AAV vector has an AAV2quad(YF) capsid. In some embodiments, the AAV vector has an AAV6 capsid. In some embodiments, the AAV vector has an AAV8 capsid. In some embodiments, the AAV vector has an AAV9 capsid. In some embodiments, the AAV vector has an Anc80 capsid. In some embodiments, the AAV vector has an Anc80L65 capsid. In some embodiments, the AAV vector has a DJ capsid. In some embodiments, the AAV vector has a DJ / 9 capsid. In some embodiments, the AAV vector has a 7m8 capsid. In some embodiments, the AAV vector has a PHP.B capsid. In some embodiments, the AAV vector has a PHP.S capsid. In some embodiments, the AAV vector has a PHP.eB capsid. In some embodiments, the AAV vector has an AAV3 capsid. In some embodiments, the AAV vector has an AAV4 capsid. In some embodiments, the AAV vector has an AAV5 capsid. In some embodiments, the AAV vector has an AAV7 capsid.

[0045] In another aspect, the invention provides a composition containing the nucleic acid vector of any of the foregoing aspects and embodiments, hi some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0046] In another aspect, the invention provides a cell containing a polynucleotide or nucleic acid vector of any of the foregoing aspects and embodiments. In some embodiments, the cell is an SLC26A4-expressing cell. In some embodiments, the cell is an SLC26A4-expressing inner ear cell. 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 ridge cell, a cochlear root cell, or a vestibular supporting cell.

[0047] In another aspect, the invention provides a method for expressing an expression product in a cell, comprising contacting the cell with a nucleic acid vector or composition of any of the preceding aspects and embodiments. In some embodiments, the cell is an inner ear cell. In some embodiments, the cell is a SLC26A4-expressing cell. In some embodiments, the cell is a SLC26A4-expressing inner ear cell. In some embodiments, the SLC26A4-expressing inner ear cell is an interdental cell, a spiral ridge 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 occurs inside a subject (e.g., in vivo).

[0048] In another aspect, the invention provides a method of treating a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss), comprising administering to the subject's inner ear a therapeutically effective amount of a nucleic acid vector or composition of any of the aforementioned aspects and embodiments. In some embodiments, the hearing loss is pendrin-associated hearing loss. In some embodiments of any of the aforementioned aspects, the expression product is pendrin (e.g., a wild-type isoform of mammalian pendrin). In some embodiments, the pendrin-associated hearing loss is hearing loss associated with Pendred syndrome or DFNB4.

[0049] In another aspect, the invention provides a method of treating hearing loss associated with Meniere's disease in a subject in need thereof, comprising administering to the inner ear of the subject an effective amount of a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments of any of the foregoing aspects, the expression product is pendrin (e.g., the wild-type isoform of mammalian pendrin).

[0050] In another aspect, the invention provides a method of treating tinnitus associated with Meniere's disease in a subject in need thereof, comprising administering to the inner ear of the subject an effective amount of a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments of any of the foregoing aspects, the expression product is pendrin (e.g., the wild-type isoform of mammalian pendrin).

[0051] In another aspect, the invention provides a method of treating vestibular dysfunction associated with Meniere's disease in a subject in need thereof, comprising administering to the subject's inner ear an effective amount of a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments of any of the foregoing aspects, the expression product is pendrin (e.g., mammalian pendrin) or Atoh1 (e.g., the wild-type isoform of mammalian Atoh1). In some embodiments, the vestibular dysfunction is vertigo.

[0052] In another aspect, the invention provides a method of treating a subject having or at risk of developing a vestibular dysfunction, comprising administering to the subject's inner ear a therapeutically effective amount of a nucleic acid vector or composition of any of the aforementioned aspects and embodiments. In some embodiments, the vestibular dysfunction is a pendrin-associated vestibular dysfunction. In some embodiments of any of the aforementioned aspects, the expression product is pendrin (e.g., a wild-type isoform of mammalian pendrin). In some embodiments, the pendrin-associated vestibular dysfunction is a vestibular dysfunction associated with Pendred syndrome or DFNB4. In some embodiments of any of the aforementioned aspects, the expression product is pendrin (e.g., a mammalian pendrin) or Atoh1 (e.g., a wild-type isoform of mammalian Atoh1).

[0053] In another aspect, the invention provides a method of inducing or increasing vestibular hair cell regeneration (i.e., inducing or increasing differentiation of vestibular supporting cells into vestibular hair cells), comprising contacting vestibular supporting cells with a nucleic acid vector or composition described in any of the foregoing aspects and embodiments. In some embodiments of any of the foregoing aspects, the expression product is Atoh1 (e.g., a wild-type isoform of mammalian Atoh1). In some embodiments, the contacting is performed in vivo (e.g., within a subject). In some embodiments, the subject has or is at risk of developing a vestibular dysfunction.

[0054] In another aspect, the invention provides a method of inducing or increasing maturation of vestibular hair cells (e.g., regenerated vestibular hair cells), comprising contacting vestibular supporting cells with a nucleic acid vector or composition described in any of the foregoing aspects and embodiments. In some embodiments of any of the foregoing aspects, the expression product is Atoh1 (e.g., a wild-type isoform of mammalian Atoh1). In some embodiments, the contacting occurs in vivo (e.g., within a subject). In some embodiments, the subject has or is at risk of developing a vestibular dysfunction.

[0055] In another aspect, the present invention provides a method for improving the function of an SLC26A4-expressing cell, comprising contacting the SLC26A4-expressing cell with a nucleic acid vector or composition of any of the foregoing aspects and embodiments. In some embodiments, the contacting is performed in vivo (e.g., inside a subject). In some embodiments, the subject has or is at risk of developing hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction.

[0056] In some embodiments, the vestibular dysfunction is vertigo, dizziness, imbalance (e.g., balance impairment or equilibrium disorder), oscillopsia, or bilateral vestibular dysfunction. 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 aging (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 agent is an aminoglycoside (an aminoglycoside antibiotic, e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin, or netilmicin), a viomycin, an anti-tumor agent (e.g., a platinum-containing chemotherapy agent such as cisplatin, carboplatin, or oxaliplatin, or other chemotherapy agent such as nitrogen mustard or vincristine), a loop diuretic (e.g., ethacrynic acid or furosemide), a salicylate, or quinine.

[0057] In some embodiments of any of the aforementioned aspects, the method further includes assessing the subject's hearing prior to administering the nucleic acid vector or composition. In some embodiments of any of the aforementioned aspects, the method further includes assessing the hearing of the subject after administering the nucleic acid vector or composition.

[0058] In some embodiments of any of the aforementioned aspects, the method further includes assessing vestibular function of the subject prior to administering the nucleic acid vector or composition. In some embodiments of any of the aforementioned aspects, the method further includes assessing vestibular function of the subject after administering the nucleic acid vector or composition.

[0059] 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 transtympanically 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 through the oval window. In some embodiments, the nucleic acid vector or composition is administered to or through the round window. In some embodiments, the nucleic acid vector or composition is administered to a semicircular canal.

[0060] In some embodiments of any of the aforementioned aspects, the nucleic acid vector or composition is administered in an amount sufficient to prevent or alleviate hearing loss, delay the onset of hearing loss, slow the progression of hearing loss, improve hearing, increase or induce expression of an expression product in SLC26A4-expressing cells, 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 aural fullness, increase vestibular hair cell regeneration, induce or increase differentiation of vestibular supporting cells into vestibular hair cells, increase or induce maturation of vestibular hair cells (e.g., maturation of regenerated vestibular hair cells), or improve the function of SLC26A4-expressing cells (e.g., SLC26A4-expressing inner ear cells).

[0061] In some embodiments of any of the aforementioned aspects, the subject is a human subject. In another aspect, the invention provides a kit comprising a polynucleotide, nucleic acid vector, or composition of any of the foregoing aspects and embodiments.

[0062] definition As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0063] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., a nucleic acid vector containing an SLC26A4 promoter and / or an SLC26A4 enhancer) to a subject by any effective route. Exemplary administration routes are described herein below.

[0064] As used herein, the phrase "administering to the inner ear" refers to providing or imparting a therapeutic agent described herein to a subject by any route that allows for transduction of inner ear cells. Exemplary routes of administration to the inner ear include administration into the perilymph or endolymph, e.g., into or through the oval window, round window, or semicircular canal (e.g., the horizontal semicircular canal), or by transtympanic or intratympanic injection, e.g., into SLC26A4-expressing inner ear cells.

[0065] As used herein, the term "cell type" refers to a group of cells that share a statistically separable phenotype based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation properties. Cells of a common cell type may include those isolated from a common tissue (e.g., epithelial, nervous, connective, or muscle tissue) and / or those isolated from a common organ, tissue system, blood vessel, or other structure and / or region in an organism.

[0066] As used herein, the terms "conservative mutation," "conservative substitution," and "conservative amino acid substitution" refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk. These properties are summarized in Table 1 below for each of the 20 naturally occurring amino acids.

[0067] [Table 1]

[0068] From this table, it can be seen that conservative amino acid families include: (i) G, A, V, L, and I, (ii) D and E, (iii) C, S, and T, (iv) H, K, and R, (v) N and Q, and (vi) F, Y, and W. Thus, a conservative variation or substitution is one that replaces one amino acid with a member of the same amino acid family (e.g., replacing Ser with Thr or Lys with Arg).

[0069] 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. Accordingly, "effective amount" or its synonyms will depend on the context in which it is applied. For example, in the context of treating hearing loss (e.g., hearing loss associated with DFNB4 or Pendred syndrome), it is the amount of a composition, vector construct, or viral vector sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors, such as the given drug, pharmaceutical formulation, route of administration, type of disease or disorder, and characteristics of the subject or host being treated (e.g., age, sex, weight), but can nevertheless be routinely determined by one of skill in the art. Also, as used herein, a "therapeutically effective amount" of a composition, vector construct, or viral vector of the present disclosure is an amount that produces a beneficial or desired result in a subject compared to a control. A therapeutically effective amount of a composition, vector construct, or viral vector of the present disclosure, as defined herein, can be readily determined by one of ordinary skill in the art by routine methods known in the art. Dosage regimens may be adjusted to provide the optimal therapeutic response.

[0070] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or human cell, e.g., a cell such as a human cochlear supporting cell).

[0071] As used herein, the term "express" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., 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.

[0072] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell such as a human cochlear supporting cell). Exogenous material includes materials provided from an external source to an organism or culture extracted therefrom.

[0073] As used herein, the term "heterologous" refers to a combination of elements that do not occur in nature. For example, a heterologous transgene refers to a transgene that is not naturally expressed by a promoter to which it is operably linked.

[0074] As used herein, the terms "increasing" and "decreasing" refer to modulating the function, expression, or activity of a metric to a greater or lesser extent, respectively, relative to a reference. For example, after administration of a composition according to the methods described herein, the amount of a marker for an indicator described herein (e.g., transgene expression level or auditory brainstem response) in a subject can be increased or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more compared to the amount of the marker before administration. Generally, the metric is measured after administration at a time when administration has had the recited effect, for example, at least 1 week, 1 month, 3 months, or 6 months after the treatment regimen has begun.

[0075] As used herein, "locally" or "local administration" refers to administration at a particular site in the body where a local, rather than a systemic, effect is intended. Examples of local administration include epidermal administration, inhalation administration, intra-articular administration, intrathecal administration, intravaginal administration, intravitreal administration, intrauterine administration, intralesional administration, lymph node administration, intratumoral administration, direct administration to the inner or middle ear (e.g., injection through the oval or round window membrane, or transtympanic or intratympanic injection), and administration to a mucous membrane of a subject, where administration is intended to produce a local, rather than a systemic, effect.

[0076] As used herein, the term "operably linked" refers to a first molecule attached to a second molecule, the molecules being positioned so 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 be adjacent or non-adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if it regulates the transcription of the transcribable polynucleotide molecule of interest in a cell. In addition, two portions of a transcriptional regulatory element are operably linked to each other if they are joined such that the transcriptional activation functionality of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other by a linker polynucleotide (e.g., an intervening non-coding polynucleotide) or without any intervening nucleotides.

[0077] As used herein, the term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule into which additional DNA segments can be ligated. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Certain plasmids are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain plasmids are capable of directing the expression of genes to which they are operably linked.

[0078] As used herein, the term "polynucleotide" refers to a polymer of nucleosides. Typically, polynucleotides are composed of nucleosides found naturally in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. The term encompasses molecules containing nucleosides or nucleoside analogs with chemically or biologically modified bases, modified backbones, etc., whether or not found in natural nucleic acids, and such molecules may be preferred for certain applications. When the present application refers to a polynucleotide, it is understood that both DNA and RNA, and in each case, both single- and double-stranded forms (and the complement of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information (i.e., the series of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise specified.

[0079] As used herein, a "polynucleotide that can be transcribed to produce" an RNA or protein refers to a polynucleotide that can direct the production of an RNA or protein, e.g., through transcription or translation. Such a polynucleotide can be incorporated into a nucleic acid vector or expression cassette and operably linked, for example, 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., pendrin or Atoh1) or an RNA molecule (e.g., an inhibitory RNA molecule such as an siRNA, shRNA, or miRNA).

[0080] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase promotes transcription of the transgene. "Percent sequence identity (%)" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, 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 may alternatively be expressed 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: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and Y is the total number of nucleic acids in B. It will be recognized that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0081] As used herein, the terms "pendrin" and "SLC26A4" refer to the protein encoded by the SLC26A4 gene and the gene encoding this protein, respectively. SLC26A4 is a member of solute transporter 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 nucleic acids encoding same, respectively, such as variant proteins having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or greater) to the amino acid sequence of the wild-type pendrin protein (e.g., SEQ ID NO: 4 or SEQ ID NO: 5), or the nucleic acid sequence of the wild-type SLC26A4 gene (e.g., , SEQ ID NO:6 or SEQ ID NO:7) or a codon-optimized sequence thereof, provided that the encoded pendrin analog retains the therapeutic function of wild-type (WT) pendrin (e.g., the ability to transport negatively charged ions such as chloride, iodide, and bicarbonate across cell membranes).

[0082] As used herein, the term "SLC26A4 enhancer" refers to a polynucleotide that is operably linked to a promoter (e.g., an SLC26A4 promoter, a minimal promoter, a core promoter, or a constitutive promoter) and is capable of controlling gene expression in an SLC26A4-expressing cell. SLC26A4 enhancers for use in the compositions and methods described herein have 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 greater) to any one of SEQ ID NOs: 4 to 9, 34, and 35. The SLC26A4 enhancer described herein is operably linked to a promoter that is operably linked to a polynucleotide encoding an expression product, and can increase the expression level of the expression product in SLC26A4-expressing cells and / or increase the number of SLC26A4-expressing cells in which the expression product is expressed.

[0083] As used herein, the term "SLC26A4 promoter" refers to a polynucleotide or variant thereof that is capable of expressing a transgene specifically in SLC26A4-expressing cells, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to an SLC26A4 promoter described herein (e.g., an SLC26A4 promoter of a formula BAC described herein, or an SLC26A4 promoter or enhancer promoter provided in Table 2). An SLC26A4 promoter for use in the compositions and methods described herein may have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to 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 more sequence identity) to any one of SEQ ID NOs: 28-33 and 41.

[0084] As used herein, the term "SLC26A4-expressing cells" refers to cell types in the body that are known to endogenously express SLC26A4. SLC26A4-expressing cells include breast cells (adipocytes, vascular cells, luminal epithelial cells, fibroblasts, dendritic cells, macrophages, basal myoepithelial cells, pericytes, and smooth muscle cells); esophagus (adipocytes, lymphocytes, vascular cells, epithelial cells of the esophageal mucosa (basal, suprabasal, and squamous), fibroblasts (mucosal and muscle), mucous cells, myofibroblasts of the esophageal mucosa, neurons of the esophageal muscular layer, Schwann cells, myocytes (smooth muscle), immune cells (dendritic cells, macrophages, T cells, mast cells), pericytes, and smooth muscle cells); heart (adipocytes, lymphocytes, vascular cells, fibroblasts, Schwann cells, myocytes (cardiac and cytoplasmic), immune cells (dendritic cells, macrophages, T cells, mast cells), pericytes, and smooth muscle cells); lung (lymphocytes, vascular cells, epithelial cells (alveolar) These include cells in the skeletal muscle (lymphocytes, vascular cells, fibroblasts, dendritic cells, macrophages, satellite cells, myocytes (skeletal, cytoplasmic), pericytes, and smooth muscle cells); prostate (lymphocytes, vascular cells, epithelial cells (Hillock, luminal), fibroblasts, dendritic cells, macrophages, myocytes (smooth muscle)); skin (vascular cells, epithelial cells (basal keratinocytes, mature keratinocytes, suprabasal keratinocytes), fibroblasts, sebocytes, and sweat gland cells); kidney (cortical and medullary cells (interstitial cells)); and thyroid (follicular and parafollicular cells) and inner ear cells expressing SLC26A4.

[0085] As used herein, the term "SLC26A4-expressing inner ear cells" refers to cells in the inner ear that endogenously express SLC26A4. SLC26A4-expressing cells in the ear are found in both the cochlea and the vestibule. Cochlear SLC26A4-expressing cells include root cells, spindle cells, inner sulcus cells, outer sulcus cells, spiral ridge cells, interdental cells, macrophages, Reissner's membrane, Deiters cells, vascular cells, marginal cells, and intermediate cells. Vestibular SLC26A4-expressing cells include non-sensory epithelial cells. Additional SLC26A4-expressing inner ear cells include endolymphatic sac mitochondria-rich cells of the endolymphatic sac and endolymphatic duct.

[0086] As used herein, the term "pendrin-associated hearing loss" refers to diseases or conditions characterized by hearing loss associated with mutations in SLC26A4, such as DFNB4, which is characterized by prelingual or postlingual hearing loss that may be accompanied by enlargement of the vestibular aqueduct, and Pendred syndrome, which is characterized by an enlarged thyroid gland (called goiter), severe to complete hearing loss (often present from birth), and other abnormalities of the inner ear, including enlargement of the vestibular aqueduct.

[0087] As used herein, the term "pendrin-associated vestibular dysfunction" refers to a disease or condition 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.

[0088] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, administered to a subject, such as a mammal (e.g., a human), to prevent, treat, or manage a particular disease or condition that is or may affect the subject.

[0089] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, and other significant complications, commensurate with a reasonable benefit / risk ratio.

[0090] 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., from the placenta or dermis), pancreatic juice, chorionic villus samples, and cells).

[0091] As used herein, the terms "subject" and "patient" refer to an animal (e.g., a mammal such as a human). A subject treated according to the methods described herein may have been diagnosed with hearing loss (e.g., pendrin-associated hearing loss), vestibular dysfunction (e.g., pendrin-associated vestibular dysfunction or vestibular dysfunction associated with loss of vestibular hair cells), or Meniere's disease, or may be at risk for developing these conditions (e.g., a mutation in SLC26A4, or exposure to an insult that may cause vestibular hair cell damage or death, e.g., exposure to ototoxic drugs, head trauma, or aging). Diagnosis may be performed by any method or technique known in the art. One skilled in the art will understand that a subject treated according to the present disclosure may have undergone standard testing, or may have not been tested but may have been identified as at risk due to the presence of one or more risk factors associated with the disease or condition.

[0092] As used herein, the terms "transcriptional regulatory element" and "regulatory sequence" refer to polynucleotides that, at least in part, control the transcription of a gene of interest. Transcriptional regulatory elements can include promoters, enhancers, and other polynucleotides (e.g., polyadenylation signals) that control or help control gene transcription. Examples of transcriptional regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0093] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, imparefection, etc.

[0094] As used herein, the terms "transduction" and "transducing" refer to a method of introducing a vector construct, or a portion thereof, into a cell. When the vector construct is contained in a viral vector, such as, for example, an AAV vector, transduction refers to viral infection of the cell and subsequent transfer and integration of the vector construct, or a portion thereof, into the cellular genome.

[0095] As used herein, "treatment" and "treating," with respect to a disease or condition, refer to an approach for obtaining a beneficial or desired result, e.g., a clinical result. Beneficial or desired results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable; diminution of the extent of the disease or condition; stabilization of the disease, disorder, or condition status (i.e., not worsening); prevention of the progression of the disease or condition; delaying or slowing the progression of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or complete). "Ameliorating" or "alleviating" a disease or condition means reducing the severity and / or undesirable clinical signs of the disease, disorder, or condition and / or slowing or prolonging the time course of progression compared to the severity or time course in the absence of treatment. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0096] 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). Various vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in Gellissen, "Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems" (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use in the compositions and methods described herein contain polynucleotide sequences and additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of mammalian cells. Particular vectors that can be used to express the transgenes described herein include vectors containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for transgene expression contain polynucleotide sequences that enhance the translation rate of the transgene or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of genes incorporated into the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0097] As used herein, the term "wild type" refers to the most frequently occurring genotype for a particular gene in a given organism. [Brief explanation of the drawings]

[0098] [Figure 1A] Figure 1A shows the general design of various plasmids used to generate AAV-DJ vectors for nuclear-targeted expression of enhanced green fluorescent protein (EGFP) via fusion with H2B. The general design of the plasmid shows that expression of the H2B-EGFP fusion protein is driven by a promoter without the addition of any SLC26A4 enhancer. The general design shown in Figure 1A also includes the barcode used in the scRNA-seq experiments described herein. [Figure 1B] Figure 1B shows the general design of various plasmids used to generate AAV-DJ vectors for nuclear-targeted expression of enhanced green fluorescent protein (EGFP) via fusion with H2B. The general design of the plasmid is shown in which expression of the H2B-EGFP fusion protein is driven by the SLC26A4 promoter disclosed herein, which includes the SLC26A4 enhancer fused to the 5' end of the promoter. The general design shown in Figure 1B also includes the barcode utilized in the scRNA-seq experiments described herein. [Figure 1C]

[0023] Figure 1 depicts the general design of various plasmids used to generate AAV-DJ vectors for expressing nuclear-targeted enhanced green fluorescent protein (EGFP) via fusion with H2B. The general design of the plasmid is shown, in which expression of the H2B-EGFP fusion protein is driven by the SLC26A4 promoter disclosed herein, which contains two SLC26A4 enhancers (enhancer #1 and enhancer #2) fused to each other and to the 5' end of the promoter. [Figure 2A] 1 is a series of photomicrographs depicting pendrin immunostaining. 2 is a photomicrograph showing the orientation of cultured lateral wall and cochlear dissections stained with an antibody specific for pendrin. [Figure 2B]A series of photomicrographs depicting pendrin immunostaining. Whole-mount views of a mouse lateral wall transfected with an AAV-DJ viral vector expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of the ubiquitous CMV promoter, counterstained with an antibody specific for pendrin. The first (left) image shows only the pendrin channel, the second (center) image shows both the pendrin and GFP channels, and the third (right) image shows only the GFP channel. [Figure 3A]

[0039] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, after 5 days of culture, counterstained with an antibody specific for pendrin, Pend3.1 (SEQ ID NO: 40). Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 3B]

[0039] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, after 5 days of culture, counterstained with an antibody specific for pendrin. Pend3.1.1 (SEQ ID NO: 27). Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 3C]

[0039] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, after 5 days of culture, counterstained with an antibody specific for pendrin. Pend3.1.3 (SEQ ID NO: 25). Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 3D]

[0049] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, after 5 days of culture, counterstained with an antibody specific for pendrin. Pend3.1.4 (SEQ ID NO: 24). Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 3E]

[0039] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, after 5 days of culture. Pend3.1.5 (SEQ ID NO: 21). Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 3F]

[0039] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, after 5 days of culture, counterstained with an antibody specific for pendrin. Pend3.1.6 (SEQ ID NO: 23). Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 3G]

[0039] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, after 5 days of culture. Pend3.1.7 (SEQ ID NO: 22). Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 3H]

[0039] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, after 5 days of culture, counterstained with an antibody specific for pendrin. Pend3.1.8 (SEQ ID NO: 1). Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 3I]

[0049] Figure 1 shows whole-mount views of mouse lateral walls transfected with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of various human SLC26A4 promoters disclosed herein, counterstained with an antibody specific for pendrin, after 5 days of culture. Pend3.1.9 (SEQ ID NO: 20) human core promoter. Scale bar: 100 μm. In each figure, the top image shows the pendrin and GFP channels, the middle image shows only the pendrin channel, and the bottom image shows only the GFP channel. [Figure 4A] Whole-mount views of mouse lateral walls transfected with an AAV-DJ viral vector expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of the Pene3.1.8 promoter (SEQ ID NO: 1) and the mouse or human SLC26A4 enhancer (as indicated) are shown after 5 days of culture. Left panel: mouse E2 enhancer (mE2; SEQ ID NO: 6); middle panel: mouse E6 enhancer (mE6; SEQ ID NO: 7); right panel: human 6.4 enhancer (hE6.4; SEQ ID NO: 5); scale bar: 100 μm. The top row of images in each panel shows the pendrin and GFP channels; the second row shows only the pendrin channel; the third row shows only the GFP channel; and the fourth row shows only the GFP channel with gain adjustment. [Figure 4B]Whole-mount views of mouse lateral walls transfected with an AAV-DJ viral vector expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of the Pene3.1.8 promoter (SEQ ID NO: 1) and the mouse or human SLC26A4 enhancer (as indicated) are shown after 5 days of culture. The left panel shows the human 6.1 enhancer (mE6.1; SEQ ID NO: 4); the middle panel shows the human 6.2 enhancer (hE6.2; SEQ ID NO: 8); and the right panel shows the human 6.3 enhancer (hE6.3; SEQ ID NO: 9). Scale bar: 100 μm. The top row of images in each panel shows the pendrin and GFP channels; the second row shows only the pendrin channel; the third row shows only the GFP channel; and the fourth row shows only the GFP channel with gain adjustment. [Figure 5] Whole-mount views of mouse lateral walls transfected with an AAV-DJ viral vector expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B, under the control of the Pene3.1.6 promoter alone (SEQ ID NO: 23), and in combination with the mouse or human SLC26A4 enhancer as indicated, after 5 days of culture, counterstained with an antibody specific for pendrin: left panel—human 6.4 enhancer (hE6.4; SEQ ID NO: 5); middle panel—mouse E6 enhancer (mE6; SEQ ID NO: 7); right panel—Pend3.1.6 without any enhancer. Scale bar: 100 μm. The top row of images in each figure shows the pendrin and GFP channels; the second row shows only the pendrin channel; the third row shows only the GFP channel; and the fourth row shows only the GFP channel with gain adjustment. [Figure 6]A series of bar graphs showing quantification of GFP signal in whole mounts of mouse lateral wall shown in Figure 3H (Pend3.1.8 promoter only), the left, middle, and right panels of Figure 4A (Pend3.1.8 promoter + mouse E2 enhancer (left panel); Pend3.1.8 promoter + mouse E6 enhancer (center panel); Pend3.1.8 promoter + human 6.4 enhancer (right panel)), and the left panel of Figure 4B (Pend3.1.8 promoter + human 6.1 enhancer) (n = 3-5 / group; mean ± SEM). Panel A shows the percentage of pendrin-positive cells that are also GFP-positive. Panel B shows the number of GFP-positive cells in the pendrin-positive region of the lateral wall. Panel C shows the number of GFP-positive cells outside the pendrin-positive region of the lateral wall. Panel D shows the average intensity of GFP signal in pendrin-positive cells. [Figure 7] Figure 7 shows the sequence overlap of the various SLC26A4 promoters described herein and how they overlap with the genomic sequence of the human SLC26A4 gene. Figure 7 also shows the correlation of these promoters with various regions of SEQ ID NOS: 1-3. [Figure 8]

[0023] Figure 1 shows the results of single-cell RNA-seq analysis of various promoters disclosed herein, alone or in combination with various enhancers disclosed herein, in various cell types present in the mouse lateral wall. Controls include the ubiquitous Pgk promoter and a construct lacking any promoter (promoterless). These results also include results for the Pend3.1.8 promoter in combination with the human 5.1 enhancer (SEQ ID NO: 34) ("hE5.1+3.1.8") and in combination with the human 5.4 enhancer (SEQ ID NO: 35) ("hE5.4+3.1.8"). [Figure 9] The plasmid map of P1708 is shown. [Figure 10A]Figure 1 shows the results of transfecting mouse lateral wall with AAV-DJ viral vectors expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of the Pend3.1.8 promoter (SEQ ID NO: 1) and one or both of the mouse E2 and E6 SLC26A4 enhancers, counterstained with a pendrin-specific antibody, after 5 days of culture. Whole-mount views of the mouse lateral wall for each AAV-DJ vector are shown: left panel—mouse E2 (SEQ ID NO: 6) and mouse E6 enhancer (SEQ ID NO: 7); center panel—mouse E2 enhancer; right panel—mouse E6 enhancer. [Figure 10B]

[0039] Figure 1 shows the results of transfecting mouse lateral wall with an AAV-DJ viral vector expressing an enhanced green fluorescent protein (EGFP) transgene targeted to the nucleus by fusion with H2B and under the control of the Pend3.1.8 promoter (SEQ ID NO: 1) and one or both of the mouse E2 and E6 SLC26A4 enhancers, counterstained with a pendrin-specific antibody, after 5 days of culture. Figure 2 shows a violin plot depicting GFP intensity in cells expressing pendrin after transfection with an AAV-DJ vector containing mouse E2 and E6 and an AAV-DJ vector containing only the mouse E6 enhancer. [Figure 11] The plasmid map of P1517 is shown. [Figure 12]

[0049] Figure 12 is a series of images at different magnifications of the same field depicting EGFP expression in the cochlea after in vivo administration to pendrin knockout mice of either an AAV-DJ vector (Figure 12, panels A-D) containing an EGFP expression cassette encoding the E6.4 enhancer (SEQ ID NO:5) directly fused to the 5' end of the human SLC26A4 minimal promoter (SEQ ID NO:1) (plasmid P1527; see Figure 11); or an AAV1 vector (Figure 12, panels E-F) containing an EGFP expression cassette driven by the CMV promoter. Panels A-E of Figure 12 are photomicrographs taken at minimum magnification. The area indicated by the box labeled "B" in panel A of Figure 12 is shown at a higher magnification than in panel B of Figure 12. The boxes labeled "C" and "D" in panel B of Figure 12 are shown at a higher magnification than in panels C and D of Figure 12, respectively. The area indicated by the box labeled "F" in panel E of Figure 12 is shown at a higher magnification than panel F of Figure 12. To better illustrate the outline of the cochlea, the field was counterstained with an antibody against Kcnj10. RM - Reissner's membrane; ID - interdental cells; SP - spiral ridge. Scale bar = 100 μm. DETAILED DESCRIPTION OF THE INVENTION

[0099] Described herein are compositions and methods for specifically inducing gene expression in SLC26A4-expressing cells or subpopulations thereof (e.g., SLC26A4-expressing inner ear cells, e.g., interdental cells, spiral ridge cells, root cells, and vestibular supporting cells). The invention features SLC26A4 promoters capable of inducing expression of a transgene in SLC26A4-expressing cells (e.g., SLC26A4-expressing inner ear cells) and SLC26A4 enhancers operably linked to the promoter to induce or increase transgene expression in SLC26A4-expressing cells. 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 SLC26A4-expressing cells in which transgene expression can be detected. In some embodiments, the SLC26A4 enhancer can reduce or minimize off-target expression in non-SLC26A4-expressing cells (e.g., when operably linked to a constitutive promoter). Thus, the SLC26A4 promoters and enhancers 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 SLC26A4-expressing cells 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, as well as nucleic acid vectors containing one or more SLC26A4 enhancers operably linked to a promoter operably linked to a polynucleotide encoding an expression product (e.g., an SLC26A4 promoter, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 2), and methods of using these vectors to treat hearing loss (e.g., pendrin-associated hearing loss), vestibular dysfunction (e.g., pendrin-associated vestibular dysfunction, or imbalance or loss of balance associated with damage or loss of vestibular hair cells), or Meniere's disease.

[0100] Pendrin Pendrin is an anion exchange protein encoded by the SLC26A4 gene and is a member of the solute transporter family 26. Mutations in SLC26A4 are associated with both nonsyndromic and syndromic hearing loss. Dozens of SLC26A4 mutations have been identified in subjects with nonsyndromic hearing loss (hearing loss without signs or symptoms affecting other parts of the body), called DFNB4. This form of hearing loss can be prelingual or postlingual, and subjects with DFNB4 often have enlarged vestibular aqueducts. More than 150 mutations in SLC26A4 have been associated with Pendred syndrome, which is characterized by an enlarged thyroid gland (goiter), hearing loss, and other abnormalities of the inner ear, including enlarged vestibular aqueducts. Pendred syndrome is the most common form of syndromic hearing loss, and subjects with Pendred syndrome often experience hearing loss beginning at birth or by age 3, which worsens over time, with some progressing to complete hearing loss. There is no cure for pendrin-related hearing loss, and supportive care is usually aimed at improving hearing, such as with hearing aids.

[0101] Gene therapy has recently emerged as an attractive therapeutic approach for treating hearing loss, particularly hearing loss caused by mutations in genes expressed in the inner ear, due to the potential for improving or restoring hearing by delivering wild-type versions of mutated genes. 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 cells that endogenously express the gene. This is challenging for genes such as SLC26A4, which are expressed in a collection of different cell types within the cochlea and vestibule.

[0102] The present invention is based, in part, on the discovery of SLC26A4 promoters and enhancers that can be used to induce gene expression in SLC26A4-expressing cells while minimizing off-target expression in non-SLC26A4-expressing cells. The SLC26A4 promoter can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding pendrin) and, optionally, to an SLC26A4 enhancer (e.g., an SLC26A4 enhancer described herein). The SLC26A4 enhancer can be operably linked to a promoter, e.g., an SLC26A4 promoter, a constitutive promoter, a core promoter, or a minimal promoter operably linked to a polynucleotide encoding an expression product (e.g., pendrin). The SLC26A4 promoters and enhancers described herein can be used to induce expression of an expression product in SLC26A4-expressing cells while simultaneously reducing or eliminating off-target expression in non-SLC26A4-expressing cells (e.g., cochlear hair cells). An SLC26A4 enhancer can also be used to increase the expression level of an expression product in SLC26A4-expressing cells and to increase the number of SLC26A4-expressing cells 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 pendrin, Atoh1, or another protein endogenously expressed in SLC26A4-expressing cells, or an RNA molecule such as an inhibitory RNA molecule) in SLC26A4-expressing cells (e.g., SLC26A4-expressing inner ear cells such as interdental cells, spiral ridge cells, root cells, or vestibular supporting cells) to treat a subject who has or is at risk of developing hearing loss (e.g., sensorineural hearing loss) or hearing loss (e.g., pendrin-related hearing loss), a subject who has or is at risk of developing vestibular dysfunction (e.g., pendrin-related vestibular dysfunction or vestibular dysfunction associated with loss of vestibular hair cells), or a subject who has Meniere's disease.The discovery of SLC26A4 enhancers and promoters that can improve cell-type specific expression may improve the safety and efficacy of gene therapy by reducing toxicity resulting from off-target expression.

[0103] The compositions and methods described herein can include an SLC26A4 promoter. In some embodiments, the SLC26A4 promoter has the formula BAC, where 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 more) to SEQ ID NO: 1, and 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 more) to SEQ ID NO: 2, or a portion thereof comprising 1 to 917 contiguous nucleotides starting from the 3' end of SEQ ID NO: 2. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), and 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 more sequence identity) to 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 contiguous nucleotides starting from the 5' end of SEQ ID NO:3), and the SLC26A4 promoter is 1481 bases or less. In some embodiments, C is absent and the SLC26A4 promoter has the formula BA, where 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 more sequence identity) to SEQ ID NO:1, and B has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or a portion thereof comprising 1 to 917 contiguous nucleotides starting from the 3' end of SEQ ID NO:2.In some embodiments, B is absent and the SLC26A4 promoter has the formula AC, where 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 more sequence identity) to SEQ ID NO:1, and C has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3, or a portion thereof comprising at least the first 159 nucleotides of SEQ ID NO:3 (i.e., the first 159 contiguous nucleotides starting from the 5' end of SEQ ID NO:3). 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 to SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity). 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 includes 1 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, or 800 to 917 contiguous nucleotides of SEQ ID NO:2, starting from the 3' end of SEQ ID NO:2 (e.g., the portion of SEQ ID NO:2 includes 1 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, or 800 to 917 contiguous nucleotides of SEQ ID NO:2, starting from the 3' end of SEQ ID NO:2 in an SLC26A4 promoter having the formula BAC, or BA). , 750, 800, 825, 850, 875, 900, or 917 consecutive nucleotides of SEQ ID NO:2, starting from the end of the sequence.In some embodiments, the portion of SEQ ID NO:2 is the first 307 contiguous nucleotides from the 3' end of SEQ ID NO:2 (nucleotides 611-917 of SEQ ID NO:3, as set forth in SEQ ID NO:14). In some embodiments, the portion of SEQ ID NO:3 includes 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 contiguous nucleotides 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 contiguous nucleotides (set forth in SEQ ID NOs:15, 16, 17, 18, and 19, respectively) starting from the 5' end of SEQ ID NO:3.

[0104] In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1, or has the sequence of SEQ ID NO: 1 (formula BAC, where B and C are absent). In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 20, or has the sequence of SEQ ID NO: 20 (formula BAC, 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 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 21, or has the sequence of SEQ ID NO: 21 (formula BAC, 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 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 22, or has the sequence of SEQ ID NO: 22 (formula BAC, where C is absent, B has the sequence of the first 307 contiguous nucleotides from the 3' end of SEQ ID NO: 2 (set forth 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 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 23, or has the sequence of SEQ ID NO: 23 (formula BAC, where B is absent, C has the sequence of the first 341 contiguous nucleotides from the 5' end of SEQ ID NO: 3 (set forth 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 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 24, or has the sequence of SEQ ID NO: 24 (formula BAC, where B is absent, C has the sequence of the first 723 contiguous nucleotides from the 5' end of SEQ ID NO: 3 (set forth 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 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 25, or has the sequence of SEQ ID NO: 25 (formula BAC, where B has the first 307 contiguous nucleotides from the 3' end of SEQ ID NO: 2 (set forth in SEQ ID NO: 14), A has the sequence of SEQ ID NO: 1, and C has the sequence of the first 716 contiguous nucleotides from the 5' end of SEQ ID NO: 3 (set forth in SEQ ID NO: 18)).In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 26, or has the sequence of SEQ ID NO: 26 (formula BAC, where B has the first 307 contiguous nucleotides from the 3' end of SEQ ID NO: 2 (set forth in SEQ ID NO: 14), A has the sequence of SEQ ID NO: 1, and C has the sequence of the first 324 contiguous nucleotides from the 5' end of SEQ ID NO: 3 (set forth in SEQ ID NO: 16)). In some embodiments, the SLC26A4 promoter has at least 85% sequence identity to SEQ ID NO:27 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), or has the sequence of SEQ ID NO:27 (formula BAC, where B has the first 307 contiguous nucleotides from the 3' end of SEQ ID NO:2 (set forth in SEQ ID NO:14), A has the sequence of SEQ ID NO:1, and C has the sequence of the first 159 contiguous nucleotides from the 5' end of SEQ ID NO:3 (set forth in SEQ ID NO:15)). The above-mentioned SLC26A4 promoter sequences are shown in Table 2 below.

[0105] [Table 2-1]

[0106] [Table 2-2]

[0107] [Table 2-3]

[0108] [Table 2-4]

[0109]

Table 2-5

[0110]

Table 2-6

[0111]

Table 2-7

[0112]

Table 2-8

[0113]

Table 2-9

[0114]

Table 2-10

[0115]

Table 2-11

[0116]

Table 2-12

[0117]

Table 2-13

[0118]

Table 2-14

[0119] In some embodiments, an SLC26A4 promoter described herein (e.g., an SLC26A4 promoter of the formula BAC 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 pendrin or Atoh1, a polynucleotide encoding a protein or RNA molecule endogenously expressed in an SLC26A4-expressing cell, or a polynucleotide encoding an inhibitory RNA molecule). A nucleic acid vector comprising the SLC26A4 promoter operably linked to a polynucleotide encoding pendrin can be used to treat pendrin-associated hearing loss (e.g., hearing loss associated with DFNB4 or Pendred syndrome), pendrin-associated vestibular dysfunction (e.g., vestibular dysfunction associated with DFNB4 or Pendred syndrome), or Meniere's disease. A nucleic acid vector containing the SLC26A4 promoter operably linked to a polynucleotide encoding Atoh1 can treat vestibular dysfunction associated with damage or loss of vestibular hair cells (e.g., damage or loss of vestibular hair cells due to aging, disease or infection, head trauma, or exposure to ototoxic agents). In some embodiments, the SLC26A4 promoter is operably linked to an enhancer, such as the SLC26A4 enhancer described herein below.

[0120] The compositions and methods described herein can include an SLC26A4 enhancer listed in Table 3 (e.g., any one of SEQ ID NOS: 4-9, 34, and 35), or a variant thereof, such as a polynucleotide sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOS: 4-9, 34, and 35. In some embodiments, an SLC26A4 enhancer for use 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, an SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO: 4. In some embodiments, an SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO: 5. In some embodiments, an SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO: 6. In some embodiments, an SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO: 7. In some embodiments, an SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO: 8. In some embodiments, an SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO: 9. In some embodiments, an SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO: 34. In some embodiments, an SLC26A4 enhancer for use 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, e.g., 2, 3, 4, 5, or 6 different enhancer sequences, of the SLC26A4 enhancers listed in Table 3). 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, can be positioned directly next to each other (e.g., can be linked without any intervening sequence between the enhancer sequences, e.g., the 3' end of a first enhancer is positioned directly before the 5' end of a second enhancer), or can be separated by 1 to 500 nucleotides (e.g., The enhancer sequences can be linked by a nucleic acid linker, such as a nucleic acid linker comprising 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500 nucleotides (e.g., the nucleic acid linker can be located between the enhancer sequences contained in the composition or between at least two of the enhancer sequences in the composition). In some embodiments in which the composition contains two or more SLC26A4 enhancers, the composition contains two different SLC26A4 enhancers. In some embodiments in which 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 in which 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.Both are located 5' of the SLC26A4 promoter, with one enhancer directly linked to the other enhancer, for example, in the following order: 5'-mE2-mE6-SLC26A4 promoter-3'. In one specific embodiment, the 5'-mE2-mE6-SLC26A4 promoter-3' polynucleotide sequence is, in 5'→3' order, SEQ ID NO:6-SEQ ID NO:7-SEQ ID NO:1.

[0121] Exemplary SLC26A4 enhancers are shown in Table 3.

[0122] [Table 3-1]

[0123] [Table 3-2]

[0124] [Table 3-3]

[0125] [Table 3-4]

[0126] [Table 3-5]

[0127] The enhancer sequence described herein can be included in a nucleic acid vector and operably linked to a promoter (e.g., an SLC26A4 promoter such as the SLC26A4 promoter of formula BAC, 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., the SLC26A4 promoter of any one of SEQ ID NOs: 28-33 and 41, a minimal promoter, a core promoter, or a constitutive promoter), and the promoter itself can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein of interest such as pendrin or Atoh1, or an RNA molecule such as an inhibitory RNA), to specifically express the expression product in SLC26A4-expressing cells (e.g., in SLC26A4-expressing inner ear cells, e.g., interdental cells, root cells, spiral ridge 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 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 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).

[0128] In some embodiments, a polynucleotide encoding wild-type pendrin, or a variant thereof, e.g., a polynucleotide encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) pendrin (e.g., SEQ ID NO: 10 or SEQ ID NO: 11). The promoter sequence may be a SLC26A4 promoter described herein (e.g., a promoter of the formula BAC, e.g., a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 17, or a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, or more sequence identity) to SEQ ID NO: 17. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity), which may optionally be operably linked to a SLC26A4 enhancer (e.g., a SLC26A4 enhancer shown in Table 3), or one or more SLC26A4 enhancers described herein (e.g., a promoter having at least 85% sequence identity to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35). The enhancer may be operably linked to a promoter (e.g., an SLC26A4 promoter, a minimal promoter, a core promoter, or a constitutive promoter, such as the SLC26A4 promoter or enhancer-promoter provided in Table 2) operably linked to a promoter (e.g., one or more copies of an enhancer having sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).In some embodiments, a polynucleotide sequence encoding a pendrin 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: 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, iodide, and bicarbonate across cell membranes). Not more than 10% of the amino acids in the pendrin protein may be substituted with conservative amino acid substitutions. In some embodiments, a polynucleotide sequence encoding pendrin is any polynucleotide sequence that, due to redundancy in the genetic code, encodes SEQ ID NO: 10 or SEQ ID NO: 11. A polynucleotide sequence encoding pendrin may be partially or fully codon-optimized for expression (e.g., in human SLC26A4-expressing inner ear cells). Pendrin proteins can also be encoded by polynucleotides having single nucleotide polymorphisms (SNPs) known to be non-pathogenic in human subjects (e.g., SNPs that do 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. The Gjb2 protein can be a human Gjb2 protein or a homolog of the human Gjb2 protein from another mammalian species (e.g., mouse, rat, cow, horse, goat, sheep, donkey, cat, dog, rabbit, guinea pig, or other mammal). Exemplary pendrin amino acid and polynucleotide sequences are provided below in Table 4.A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer described herein (e.g., an SLC26A4 enhancer having at least 85% sequence identity to any one of SEQ ID NOS: 4, 5, 8, 9, 34, and 35) operably linked to a promoter (e.g., an SLC26A4 promoter, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter shown in Table 2) operably linked to a polynucleotide encoding pendrin, or an SLC26A4 promoter described herein (e.g., a promoter of the formula BAC, e.g., an enhancer-promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 1 A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer described herein (e.g., an SLC26A4 enhancer in Table 3) operably linked to a polynucleotide encoding pendrin, an SLC26A4 promoter having a sequence identity (e.g., 6%, 97%, 98%, 99% or more) with a polynucleotide encoding pendrin, can be administered to a subject for the treatment, alleviation, or prevention of pendrin-associated hearing loss, e.g., hearing loss in a subject with DFNB4 or Pendred syndrome, or pendrin-associated vestibular dysfunction, e.g., 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 for the treatment of 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 (damage or loss of vestibular hair cells associated with head trauma, disease or infection, ototoxic drugs, or aging, e.g., age-related vestibular dysfunction, ototoxic drug-induced vestibular dysfunction, disease- or infection-related vestibular dysfunction, or head trauma-related vestibular dysfunction).

[0129] [Table 4-1]

[0130] [Table 4-2]

[0131] [Table 4-3]

[0132] [Table 4-4]

[0133] Expression of exogenous polynucleotides in mammalian cells The compositions and methods described herein can be used to induce expression of a protein or RNA molecule of interest by administering a nucleic acid vector containing at least one SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SLC26A4, SLC26A4, SLC26A4 promoters, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 2) operably linked to a polynucleotide encoding an expression product (e.g., a protein or RNA molecule of interest, e.g., an inhibitory RNA), or, optionally, an SLC26A4 enhancer (e.g., an SLC26A4 enhancer in Table 3). By administering a nucleic acid vector containing the SLC26A4 promoter (e.g., an SLC26A4 promoter of the formula BAC, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOS: 1 and 20-27), operably linked, i.e., operably linked to a polynucleotide encoding an expression product (e.g., a protein of interest or an RNA molecule such as an inhibitory RNA molecule), it is possible to induce or increase expression of an exogenous polynucleotide (e.g., a gene endogenously expressed in SLC26A4-expressing cells, such as a polynucleotide encoding pendrin) specifically in SLC26A4-expressing cells (e.g., SLC26A4-expressing inner ear cells such as interdental cells, root cells, spiral ridge cells, and vestibular supporting cells). A wide range of methods have been established for the delivery of proteins into mammalian cells and for the stable expression of polynucleotides encoding proteins in mammalian cells.

[0134] A nucleic acid vector (e.g., an AAV vector) described herein (e.g., a nucleic acid vector containing at least one SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 4, 5, 8, or 9) operably linked to a promoter (e.g., an SLC26A4 promoter, a minimal promoter, a core promoter, or a constitutive promoter, such as the SLC26A4 promoter or enhancer-promoter provided in Table 2). Alternatively, a nucleic acid vector containing an SLC26A4 promoter (e.g., an SLC26A4 promoter of the formula BAC, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1 and 20-27) optionally operably linked to an SLC26A4 enhancer (e.g., an SLC26A4 enhancer in Table 3) can be used to express a polynucleotide in one or more SLC26A4-expressing cells (e.g., SLC26A4-expressing inner ear cells). Exemplary polynucleotides that can be expressed using the nucleic acid vectors described herein include polynucleotides encoding proteins expressed in healthy SLC26A4-expressing cells, such as pendrin and Atoh1, polynucleotides that correspond to the wild-type form of genes that are endogenously expressed in SLC26A4-expressing inner ear cells and that are mutated in subjects with hearing loss, hearing loss, tinnitus, or vestibular dysfunction, and other polynucleotides that can be expressed in SLC26A4-expressing inner ear cells to treat hearing loss, hearing loss, tinnitus, or vestibular dysfunction.The nucleic acid vectors described herein can 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 SLC26A4-expressing cells (e.g., SLC26A4-expressing inner ear cells such as interdental cells, root cells, spiral ridge cells, and vestibular supporting cells).

[0135] In some embodiments, a polynucleotide encoding wild-type Atoh1, or a variant thereof, e.g., a polynucleotide encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) Atoh1 (e.g., SEQ ID NO: 36 or SEQ ID NO: 38). The promoter sequence may be a SLC26A4 promoter described herein (e.g., a promoter of the formula BAC, e.g., a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 17, or a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, or more sequence identity) to SEQ ID NO: 17. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity), which may optionally be operably linked to a SLC26A4 enhancer (e.g., a SLC26A4 enhancer shown in Table 3), or one or more SLC26A4 enhancers described herein (e.g., a promoter having at least 85% sequence identity to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35). The enhancer may be operably linked to a promoter (e.g., an SLC26A4 promoter, a minimal promoter, a core promoter, or a constitutive promoter, such as the SLC26A4 promoter or enhancer-promoter provided in Table 2) operably linked to a promoter (e.g., one or more copies of an enhancer having sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).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 proliferation). Up to 10% of the amino acids in the Atoh1 protein may be substituted with 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 due to redundancy in the genetic code. The polynucleotide sequence encoding Atoh1 may be partially or fully codon-optimized for expression (e.g., in human SLC26A4-expressing inner ear cells, such as vestibular supporting cells). The Atoh1 protein can also be encoded by a polynucleotide having a single nucleotide polymorphism (SNP) known to be non-pathogenic in human subjects (e.g., an SNP that does not result in 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 a homolog of a human Atoh1 protein from another mammalian species (e.g., mouse, rat, cow, 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. 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 described herein (e.g., the SLC26A4 enhancer of any one of SEQ ID NOS: 4, 5, 8, 9, 34, and 35) operably linked to a promoter (e.g., an SLC26A4 promoter, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter shown in Table 2) operably linked to a polynucleotide encoding Atoh1, or an SLC26A4 promoter described herein (e.g., a promoter of the formula BAC, e.g., a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer described herein (e.g., an SLC26A4 enhancer in Table 3) operably linked to a polynucleotide encoding Atoh1 (e.g., an SLC26A4 promoter having a sequence identity (e.g., 6%, 97%, 98%, 99% or more) with a polynucleotide encoding Atoh1 can be administered to a subject to treat, alleviate, or prevent vestibular dysfunction (e.g., imbalance or loss of balance, dizziness, or vertigo) associated with vestibular hair cell damage or loss (vestibular hair cell damage or loss associated with head trauma, disease or infection, ototoxic drugs, or aging, e.g., age-related vestibular dysfunction, ototoxic drug-induced vestibular dysfunction, disease- or infection-related vestibular dysfunction, or head trauma-related vestibular dysfunction).

[0136] [Table 5-1]

[0137] [Table 5-2]

[0138] A polynucleotide encoding a protein of interest One platform that can be used to achieve therapeutically effective intracellular concentrations of a protein of interest in mammalian cells is by stable expression of a gene encoding the protein of interest (e.g., by integration into the nuclear or mitochondrial genome of the mammalian cell, or by episomal concatemer formation in the nucleus of the mammalian cell). A gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein. To introduce an exogenous gene into mammalian cells, the gene can be incorporated into a vector. Vectors can be introduced into cells by various methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and encapsulation of the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in further detail, for example, in Green, et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014); and Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0139] A target protein can also be introduced into mammalian cells by targeting a vector containing a gene encoding the target protein to cell membrane phospholipids. For example, the vector can be targeted to phospholipids on the extracellular surface of the cell membrane by binding the vector molecule to the VSV-G protein, a viral protein that has affinity for all cell membrane phospholipids. Such constructs can be produced using methods well known to those skilled in the art.

[0140] Recognition and binding of a polynucleotide encoding a protein of interest by mammalian RNA polymerase is important for gene expression. Therefore, a polynucleotide may contain sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and promote the assembly of a transcription complex at the transcription start site. Such sequence elements include, for example, mammalian promoters, whose sequences can be recognized and bound by specific transcription initiation factors and ultimately by RNA polymerase. Examples of mammalian promoters are described in Smith, et al., Mol. Sys. Biol., 3:73 (published online), the disclosure of which is incorporated herein by reference. The promoter used in the methods and compositions described herein can be an SLC26A4 promoter (e.g., an SLC26A4 promoter or enhancer-promoter listed in Table 2), a constitutive promoter (e.g., a promoter active in vivo in all circumstances), a core promoter, or a minimal promoter. Constitutive promoters include the CAG promoter, cytomegalovirus (CMV) promoter (e.g., the CMV immediate-early enhancer and promoter, CMV mini promoter, minCMV promoter, CMV-TATA+INR promoter, or min CMV-T6 promoter), smCBA promoter (described in Haire et al., Invest. Opthalmol. Vis. Sci. 47:3745-3753, 2006), CBA promoter, CASI promoter, dihydrofolate reductase (DHFR) promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, β-globin promoter (e.g., the minimal β-globin promoter), HSV promoter (e.g., the minimal HSV ICP0 promoter or truncated HSV ICP0 promoter), SV40 promoter (e.g., the SV40 minimal promoter), and EF1α promoter. Constitutive promoters are also sometimes referred to as ubiquitous promoters due to their ability to direct expression of polynucleotides in a wide range of cell and tissue types.Minimal promoters include a CMV minimal promoter (e.g., minCMV promoter), a minimal β-globin promoter, a minimal HSV promoter (e.g., a minimal HSV ICP0 promoter), and an SV40 minimal promoter. Alternatively, promoters derived from viral genomes can be used for stable expression of polynucleotides in mammalian (e.g., human) cells. Examples of functional viral promoters that can be used for expression of polynucleotides in primate (e.g., human) cells include the adenovirus late promoter, the vaccinia virus 7.5K promoter, the HSV tk promoter, the mouse mammary tumor virus (MMTV) promoter, the HIV LTR promoter, the Moloney virus promoter, the Epstein-Barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter.

[0141] Once a polynucleotide encoding a protein of interest has been introduced into a mammalian cell, transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the mammalian cell to an external chemical reagent, such as a drug that modulates gene expression by modulating the binding of transcription factors and / or RNA polymerase to the mammalian promoter. The chemical reagent can function to promote the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example, by removing promoter-bound repressor proteins. Alternatively, the chemical reagent can serve to increase the affinity of the mammalian promoter for RNA polymerase and / or transcription factors, thereby increasing the transcription rate of genes located downstream of the promoter in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available and can be administered to mammalian cells to promote gene expression according to established protocols.

[0142] The nucleic acid vectors described herein may include a woodchuck post-transcriptional regulatory element (WPRE). The WPRE acts at the mRNA level by facilitating nuclear export of transcripts and / or increasing the efficiency of polyadenylation of nascent transcripts, thereby increasing the total amount of mRNA in the cell. The addition of a WPRE to a vector can result in substantial improvements in the level of transgene expression from several different promoters, both in vitro and in vivo.

[0143] In some embodiments, the nucleic acid vectors described herein include a reporter sequence, which may be useful, for example, for verifying expression of a gene operably linked to the SLC26A4 promoter and / or enhancer in cells and tissues (e.g., SLC26A4-expressing cells such as interdental cells, root cells, spiral ridge cells, and vestibular supporting cells). Reporter sequences that may be provided within a transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When combined with regulatory elements, such as promoters, that drive their expression, reporter sequences produce signals that can be detected by conventional means, such as enzyme assays, radioassays, colorimetric assays, fluorescent assays or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunoassays such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemical staining. For example, if the marker sequence is the LacZ gene, the presence of a vector bearing a signal is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the presence of a vector bearing a signal can be visually measured by color or light production in a luminometer.

[0144] Methods for delivery of exogenous polynucleotides to target cells Techniques that can be used to introduce polynucleotides, e.g., 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 target cells. Mammalian cells, such as human cells, exposed to an external electric field in this manner are then susceptible to uptake of exogenous polynucleotides. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection™ and protocols useful for carrying out this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0145] Additional techniques useful for transfecting target cells include squeeze-poration. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technique is advantageous in that no vector is required for the delivery of polynucleotides into cells, such as human target cells. Squeeze-poration 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.

[0146] Lipofection is another technique useful for transfecting target cells. This method involves loading polynucleotides into liposomes, which often present cationic functional groups, such as quaternary amines or protonated amines, toward the outer surface 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 the exogenous polynucleotide, 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. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous polynucleotides involves contacting cells with cationic polymer-polynucleotide complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge favorable for interaction with cell membranes include activated dendrimers (e.g., as described in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran; their use as transfection agents is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to guide the uptake of polynucleotides. This technology is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0147] Another useful tool for inducing the uptake of exogenous polynucleotides by target cells is laser transfection, also known as optical transfection, which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells, allowing polynucleotides to penetrate the cell membrane.The biological activity of this technique is similar to that of electroporation, and in some cases, is found to be superior to that of electroporation.

[0148] Impalefection is another technique that can be used to deliver genetic material to target cells. This technique relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-shaped nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing genes intended for intracellular delivery is attached to the surface of the nanostructures. A chip with an array of these needles is then pressed against cells or tissues. Cells stimulated by the nanostructures can express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107:1870 (2010), the disclosure of which is incorporated herein by reference.

[0149] Magnetofection can also be used to deliver polynucleotides to target cells. The principle of magnetofection is to bind polynucleotides to cationic magnetic nanoparticles. The magnetic nanoparticles are made entirely of biodegradable iron oxide and are coated with specific cationic specific molecules that vary depending on the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on target cells under the influence of an external magnetic field generated by a magnet. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0150] Another useful tool for inducing the uptake of exogenous polynucleotide by target cell is sonoporation, which is a technique that involves using sound waves (typically ultrasonic frequency) to permeabilize cell plasma membrane, so as to make cell permeable, allowing polynucleotide to penetrate the cell membrane.This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0151] Microvesicles represent another potential vehicle that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of glycoprotein VSV-G and a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins to cells and then catalyze site-specific cleavage of endogenous polynucleotide sequences, thereby preparing the genome of the cell for covalent integration of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also known as gesicles, for genetically modifying eukaryotic cells is described in detail, for example, in Quinn et al., "Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract] In: Methylation changes in early embryonic genes in cancer [abstract]," in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0152] Vectors for delivering exogenous polynucleotides to target cells In addition to achieving high transcription and translation rates, stable expression of exogenous polynucleotides in mammalian cells can be achieved by integrating the polynucleotide into the nuclear genome of the mammalian cell. Various 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 may contain at least one SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35), and / or a polynucleotide encoding an expression product (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). The BAC promoter comprises an SLC26A4 promoter (e.g., a promoter of a BAC, 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 more sequence identity) to any one of SEQ ID NOS: 1 and 20-27), operably linked to a non-transgenic polynucleotide, and additional sequence elements used for expression of these agents and / or integration of these polynucleotide sequences into the genome of a mammalian cell.Vectors that can 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 capable of autonomous replication within a cell), cosmids (e.g., pWE vectors or sCos vectors), artificial chromosomes (e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), and viral vectors. Particular vectors that can be used to express expression products (e.g., proteins of interest) include plasmids containing regulatory sequences that direct gene transcription. Other vectors useful for expressing expression products (e.g., proteins of interest) contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, an internal ribosome entry site (IRES), and a polyadenylation signal site to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use in conjunction 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.

[0153] Viral Vectors for Polynucleotide Delivery Viral genomes provide a rich source of vectors that can be used to efficiently deliver genes of interest into the genomes of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specific transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., Retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, mutant vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include avian leukosis / sarcoma virus, avian C virus, mammalian C virus, mammalian B virus, mammalian D virus, oncoretrovirus, HTLV-BLV complex, lentivirus, alpharetrovirus, gammaretrovirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor viruses, bovine leukemia viruses, feline leukemia viruses, feline sarcoma viruses, avian leukemia viruses, human T-cell leukemia viruses, baboon endogenous viruses, gibbon leukemia viruses, Mason-Pfizer monkey viruses, simian immunodeficiency viruses, simian sarcoma viruses, Rous sarcoma viruses, and lentiviruses. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.

[0154] AAV vectors for polynucleotide delivery In some embodiments, the polynucleotides of the compositions and methods described herein are incorporated into rAAV vectors and / or virions to facilitate their introduction into cells. In some embodiments, rAAV vectors useful in the compositions and methods described herein are recombinant polynucleotide constructs that include: (1) a promoter (e.g., an SLC26A4 promoter, e.g., an SLC26A4 promoter of formula BAC, 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 more 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 pendrin or Atoh1, or a polynucleotide that can be transcribed to produce an RNA molecule such as an 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 comprises at least one SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35).In other embodiments, an rAAV vector useful in the compositions and methods described herein is a recombinant polynucleotide construct comprising: (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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35); (2) a promoter (e.g., an SLC26A4 promoter, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 2); (3) a sequence to be expressed (e.g., a polynucleotide encoding a protein such as pendrin or Atoh1, or a polynucleotide that can be transcribed to produce an RNA molecule such as an inhibitory RNA); and (4) viral sequences that facilitate integration and expression of the sequence to be expressed. The viral sequence may include AAV sequences required in cis for DNA replication and packaging into virions (e.g., functional ITRs). In an exemplary application, the expressed sequence encodes a wild-type protein expressed in SLC26A4-expressing inner ear cells, such as the wild-type form of pendrin, which is mutated in subjects with a form of inherited hearing loss, or a protein or RNA molecule capable of promoting the differentiation of vestibular supporting cells into vestibular hair cells, such as Atoh1. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors lack one or more AAV WT genes, in whole or in part, but retain functional flanking ITR sequences. AAV ITRs may be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs may be AAV2 ITRs.Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0155] To facilitate the introduction of a polynucleotide or vector into a cell, the polynucleotides and vectors described herein (e.g., the SLC26A4 enhancer and / or SLC26A4 promoter operably linked to a polynucleotide encoding an expression product) can be incorporated into rAAV viral particles. The capsid protein of AAV constitutes the outer, non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for viral particle assembly. Construction of rAAV viral particles is described, for example, in US Pat. No. 5,173,414; US Pat. No. 5,139,941; US ​​Pat. No. 5,863,541; US ​​Pat. No. 5,869,305; US Pat. No. 6,057,152; and US Pat. No. 6,376,237; as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0156] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a wide variety of AAV serotypes, including AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S. When directed to SLC26A4-expressing cells, AAV1, AAV2, AAV2quad(YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, AAV-DJ, AAV-DJ / 9, 7m8, and PHP.B may be particularly useful. Serotypes evolved for retinal transduction may also be used in the methods and compositions described herein. The construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0157] Pseudotyped rAAV vectors are also useful in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with capsid genes derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques for constructing and using pseudotyped rAAV viral particles are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0158] AAV virions with mutations in the virion capsid can be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations to facilitate targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV viral particles that can be used in the methods described herein include capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0159] Pharmaceutical Composition The SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) and / or the SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1 and 20 to 27) described herein may be used. A promoter of formula BAC, such as a polynucleotide having 3%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a polynucleotide encoding an expression product (e.g., a transgene encoding an RNA molecule, such as a protein of interest or an inhibitory RNA), can be operably linked to a polynucleotide encoding an expression product and incorporated into a vehicle for administration to a patient, such as a human patient suffering from sensorineural hearing loss (e.g., pendrin-associated hearing loss), vestibular dysfunction (e.g., vestibular dysfunction associated with damage or loss of vestibular hair cells, or pendrin-associated vestibular dysfunction), or Meniere's disease. Pharmaceutical compositions containing vectors, such as viral vectors, containing the SLC26A4 enhancer and / or SLC26A4 promoter described herein operably linked to a polynucleotide encoding an expression product can be prepared using methods known in the art. For example, such compositions can be prepared using, for example, a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Remington: The Science and Practice of Pharmacology 22, incorporated herein by reference). nd edition, Allen, L. Ed. (2013)) can be used to prepare the desired form, for example, a lyophilized formulation or an aqueous solution.

[0160] a SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35), and / or a SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) described herein operably linked to a polynucleotide encoding an expression product. For example, mixtures of nucleic acid vectors (e.g., viral vectors) containing a promoter of formula BAC, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOS: 1 and 20-27, can be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In all cases, the formulation may be sterile and fluid to the extent that easy syringability exists. The formulation may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0161] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, suitable sterile aqueous media will be known to those skilled in the art in light of this disclosure. For example, a single dosage may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous fluid or injected at the proposed injection site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition may be formulated to contain synthetic perilymph solution. An exemplary synthetic perilymph solution contains 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl, 1-10 mM glucose, and 2-50 mM HEPES, with a pH of about 6-9 and an osmolality of about 300 mOsm / kg. The individual responsible for administration will, in any event, determine the appropriate dose for the individual subject. Furthermore, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards required by FDA Office of Biologics standards.

[0162] Treatment method The compositions described herein can be administered to a subject having or at risk of developing sensorineural hearing loss, vestibular dysfunction, or Meniere's disease by a variety of routes, including local administration to the middle or inner ear (e.g., administration into the perilymph or endolymph, e.g., administration into or via the oval window, round window, or semicircular canal (e.g., the horizontal semicircular canal), or administration by transtympanic or intratympanic injection, e.g., to SLC26A4-expressing inner ear cells), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most suitable route for administration in any given case will depend on the particular composition being administered, the patient, the pharmaceutical formulation, the method of administration (e.g., time and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's rate of excretion. The composition may be administered once or multiple times (eg, once a year, twice a year, three times a year, every other month, once a month, or every other week).

[0163] Subjects that may be treated as described herein are those who have or are at risk of developing sensorineural hearing loss. In some embodiments, the compositions described herein are used to treat pendrin-associated hearing loss (e.g., DFNB4 or Pendred syndrome). DFNB4 and Pendred syndrome are characterized by the presence of 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35), and / or a polynucleotide encoding pendrin (e.g., a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11). The subject can be treated by administering a nucleic acid vector containing an SLC26A4 promoter described herein (e.g., a promoter of a BAC, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOS: 1 and 20-27) operably linked to a polynucleotide encoding the SLC26A4 promoter. The subject can have a mutation in SLC26A4 or can be identified as having a mutation in SLC26A4 and can have severe, moderate, or mild hearing loss when treatment is initiated or can be treated (e.g., prophylactically) at the onset of symptoms. In some embodiments, the compositions are administered as a preventative treatment to subjects at risk of developing hearing loss, for example, subjects who carry a mutation in SLC26A4 associated with hearing loss but have not yet exhibited hearing loss.

[0164] In some embodiments, the compositions described herein are used to treat subjects with Meniere's disease. Both subjects with a mutation in SLC26A4 and subjects with Meniere's disease have endolymphatic hydrops. Therefore, compositions that can be used to treat subjects with a mutation in SLC26A4 include 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) and / or a polynucleotide encoding pendrin (e.g., Nucleic acid vectors containing the SLC26A4 promoter described herein (e.g., a promoter of a BAC, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOS: 1 and 20-27) operably linked to a polynucleotide encoding SEQ ID NOS: 10 or 11) may also be effective in reducing or alleviating endolymphatic hydrops in subjects with Meniere's disease. Such treatments may be used to treat hearing loss, tinnitus, or vestibular dysfunction (e.g., vertigo) in subjects with Meniere's disease, and may relieve a feeling of fullness or congestion in the ears.

[0165] In some embodiments, the compositions described herein are used to treat a subject having or at risk of developing a vestibular dysfunction (e.g., vertigo, dizziness, imbalance, oscillopsia, balance disorders, or bilateral vestibular disorders). In some embodiments, the vestibular dysfunction is a pendrin-associated vestibular dysfunction associated with DFNB4 or Pendred syndrome (e.g., imbalance or loss of balance associated with DFNB4 or Pendred syndrome). Pendrin-associated vestibular dysfunction can be characterized by the presence of 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35), and / or a polynucleotide encoding pendrin (e.g., a polynucleotide encoding SEQ ID NO: 10 or SEQ ID NO: 11). The subject can be treated by administering a nucleic acid vector containing an SLC26A4 promoter described herein (e.g., a promoter of the formula BAC, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOS: 1 and 20-27) operably linked to a nucleic acid vector. In some embodiments, the subject may have or be identified as having a mutation in SLC26A4 and may have severe, moderate, or mild vestibular dysfunction when treatment is initiated, or may be treated prior to the onset of symptoms (e.g., prophylactic treatment).

[0166] Vestibular dysfunction can also result from damage or loss of vestibular hair cells. Thus, the compositions and methods described herein can be used to treat subjects who have or are at risk of developing vestibular hair cell damage or loss (e.g., vestibular hair cell damage or loss associated with disease or infection, head trauma, ototoxic drugs (e.g., vestibulotoxic drugs), or aging), subjects who have or are at risk of developing vestibular dysfunction (e.g., dizziness, vertigo, imbalance, bilateral vestibular disorders, oscillopsia, or balance disorders), subjects who carry a genetic mutation associated with vestibular dysfunction, or subjects with a family history of inherited 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 condition in which an autoimmune response contributes to hair cell damage or cell 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 conditions, such as Lyme disease and syphilis, can also cause vestibular dysfunction (e.g., by inducing autoantibody production). Viral infections, such as rubella, cytomegalovirus (CMV), lymphocytic choriomeningitis virus (LCMV), HSV types 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 has vestibular dysfunction related to or resulting from the loss of hair cells (e.g., vestibular hair cells). In some embodiments, the compositions and methods described herein can be used to treat a subject suffering from or at risk of developing oscillopsia, hi some embodiments, the compositions and methods described herein can be used to treat a subject suffering from or at risk of developing bilateral vestibular dysfunction.In some embodiments, the compositions and methods described herein can be used to treat subjects suffering from or at risk of developing a balance disorder (e.g., imbalance). The compositions and methods described herein can also be administered as a prophylactic treatment to subjects at risk of developing a vestibular dysfunction, such as subjects with a family history of vestibular dysfunction (e.g., genetic vestibular dysfunction), subjects with a genetic mutation associated with vestibular dysfunction who have not yet shown 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 with idiopathic vestibular dysfunction.

[0167] The compositions and methods described herein can be used to induce or increase vestibular hair cell regeneration in a subject. Subjects who can benefit from compositions that promote or induce vestibular hair cell regeneration include those suffering from or at risk of developing vestibular dysfunction due to hair cell loss (e.g., vestibular hair cell loss associated with trauma (e.g., head trauma), disease or infection, ototoxic drugs, or aging), and subjects with abnormal vestibular hair cells (e.g., vestibular hair cells that 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 reduced numbers of vestibular hair cells due to genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase the maturation of vestibular hair cells, which can result in improved vestibular function. In some embodiments, the compositions and methods described herein promote or increase the maturation of regenerated vestibular hair cells.

[0168] 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 cell death (e.g., vestibular hair cell loss) in subjects who have been treated with an ototoxic drug, or who are currently undergoing or about to begin treatment with an ototoxic drug. Ototoxic drugs are toxic to cells of the inner ear and can cause vestibular dysfunction (e.g., vertigo, dizziness, imbalance, bilateral vestibular dysfunction, 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, antineoplastic agents (e.g., platinum-containing chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin, or other chemotherapeutic agents such as nitrogen mustard 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 nitrogen mustard, vincristine, gentamicin, streptomycin, and tobramycin, have been specifically identified as vestibulotoxic agents. In some embodiments, the methods and compositions described herein can be used to treat bilateral vestibular dysfunction 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 suffering from aminoglycoside-induced bilateral vestibular dysfunction or oscillopsia).

[0169] Vestibular dysfunction associated with vestibular hair cell damage or loss (e.g., vestibular hair cell damage or loss associated with disease or infection, head trauma, ototoxic drugs (e.g., vestibulotoxic drugs), or aging) can be mediated by the use of a polynucleotide encoding 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35), and / or a polynucleotide encoding Atoh1 (e.g., SEQ ID NO: 36 or SEQ ID NO: 38). Treatment can be achieved by administering a nucleic acid vector containing the SLC26A4 promoter described herein (e.g., a promoter of a BAC, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOS: 1 and 20-27) operably linked to a polynucleotide encoding pendrin (e.g., a polynucleotide encoding SEQ ID NOS: 10 or 11). Such nucleic acid vectors can also induce or increase vestibular hair cell regeneration or vestibular hair cell maturation in a subject in need thereof.

[0170] The methods described herein may include screening the subject for mutations in one or more genes known to be associated with hearing loss or vestibular dysfunction (e.g., SLC26A4) prior to treatment or administration with a composition described herein. Subjects can be screened for genetic mutations using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein may also include assessing the subject's hearing prior to treatment with or administration of a composition described herein. Hearing can 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 subject's hearing after treatment or administration with a composition described herein. In some embodiments, the methods described herein include assessing vestibular function in the subject prior to treatment or administration with a composition described herein. Vestibular function can be assessed using standard tests, such as eye movement tests (e.g., electronystagmography (ENG) or videonystagmography (VNG)), vestibulo-ocular reflex (VOR) tests (e.g., head impulse tests (Halmagyi-Curthoys tests), which can be performed at the bedside or using video head impulse tests (VHIT), or caloric reflex tests), stabilometry, rotary-chair testing, ECOG, vestibular evoked myogenic potential tests (VEMPs), and outpatient balance tests such as those described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010). These tests can also be used to assess vestibular function in a subject after treatment or administration with a composition described herein.

[0171] For treating a subject as described herein, an SLC26A4 promoter (e.g., a promoter of a BAC, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1 and 20-27) and / or an SLC26A4 enhancer (e.g., A polynucleotide encoding an expression product operably linked to a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35 can be used in combination with a polynucleotide encoding pendrin (e.g., the sequence of SEQ ID NO: 10 or SEQ ID NO: 11). The polynucleotide may be a polynucleotide encoding the amino acid sequence of SLC26A4 (e.g., a polynucleotide encoding the amino acid sequence of SLC26A4-expressing SLC26A4), a polynucleotide encoding Atoh1 (e.g., a polynucleotide encoding the amino acid sequence of SLC26A4-expressing SLC26A4), a polynucleotide encoding a wild-type version of a protein expressed in SLC26A4-expressing inner ear cells that is mutated in subjects with sensorineural hearing loss or vestibular dysfunction, a polynucleotide encoding another protein of interest (e.g., a reporter protein, e.g., a fluorescent protein, lacZ, or luciferase), or a polynucleotide that can be transcribed to produce an RNA molecule, e.g., an shRNA, an ASO, a component of a gene editing system (e.g., a nuclease, e.g., CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.The polynucleotide may 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 may encode wild-type pendrin, or if the subject's vestibular dysfunction is age-related or ototoxic drug-induced vestibular dysfunction associated with hair cell loss, the polynucleotide may 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.

[0172] 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 typically contains a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route and formulation of administration, are within the skill of those in the art. The unit dose need not be administered as a single injection, but may comprise a continuous infusion over a predetermined period of time. Administration may be performed using a syringe pump to control the rate of infusion to minimize damage to the inner ear (e.g., the cochlea and / or vestibular system). When the nucleic acid vector is an AAV vector (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, or PHP.S vector), the viral vector can be, for example, 1 μL to 200 μL (e.g., 1 μL to 200 μL). μL, 2μL, 3μL, 5μL, 6μL, 7μL, 8μL, 9μL, 10μL, 15μL, 20μL, 25μL, 30μL, 35μL, 40μL, 45μL, 50μL, 55μL, 60μL, 65μL, 70μL, 75μL , approximately 1 9 Vector genome (VG) / mL ~ approx. 1 x 10 16 VG / mL (e.g., 1 × 10 9VG / mL、2×10 9 VG / mL、3×10 9 VG / mL、4×10 9 VG / mL、5×10 9 VG / mL、6×10 9 VG / mL、7×10 9 VG / mL、8×10 9 VG / mL、9×10 9 VG / mL、1×10 10 VG / mL、2×10 10 VG / mL、3×10 10 VG / mL、4×10 10 VG / mL、5×10 10 VG / mL、6×10 10 VG / mL、7×10 10 VG / mL、8×10 10 VG / mL、9×10 10 VG / mL、1×10 11 VG / mL、2×10 11 VG / mL、3×10 11 VG / mL、4×10 11 VG / mL、5×10 11 VG / mL、6×10 11 VG / mL、7×10 11 VG / mL、8×10 11 VG / mL、9×10 11 VG / mL、1×10 12 VG / mL、2×10 12 VG / mL、3×10 12 VG / mL、4×10 12 VG / mL、5×10 12 VG / mL、6×10 12 VG / mL、7×10 12 VG / mL、8×10 12 VG / mL、9×10 12 VG / mL、1×10 13 VG / mL、2×10 13 VG / mL、3×10 13 VG / mL、4×10 13 VG / mL、5×10 13 VG / mL、6×10 13 VG / mL、7×10 13 VG / mL、8×10 13 VG / mL、9×1013 VG / mL, 1 × 10 14 VG / mL, 2 × 10 14 VG / mL, 3 × 10 14 VG / mL, 4 x 10 14 VG / mL, 5 × 10 14 VG / mL, 6 × 10 14 VG / mL, 7 × 10 14 VG / mL, 8 x 10 14 VG / mL, 9 x 10 14 VG / mL, 1 × 10 15 VG / mL, 2 × 10 15 VG / mL, 3 × 10 15 VG / mL, 4 x 10 15 VG / mL, 5 × 10 15 VG / mL, 6 × 10 15 VG / mL, 7 × 10 15 VG / mL, 8 x 10 15 VG / mL, 9 x 10 15 VG / mL, or 1 × 10 16 The AAV vector may be administered to a patient at a dose of approximately 1 x 10 7 VG / ear ~ approx. 2 x 10 15 VG / ear (e.g., 1 x 10 7 VG / ear, 2×10 7 VG / ear, 3×10 7 VG / ear, 4×10 7 VG / ear, 5×10 7 VG / ear, 6×10 7 VG / ear, 7×10 7 VG / ear, 8×10 7 VG / ear, 9×10 7 VG / ear, 1×10 8 VG / ear, 2×10 8 VG / ear, 3×10 8 VG / ear, 4×10 8 VG / ear, 5×10 8 VG / ear, 6×10 8 VG / ear, 7×10 8 VG / ear, 8×10 8 VG / ear, 9×10 8 VG / ear, 1×10 9 VG / ear, 2×10 9 VG / ear, 3×10 9 VG / ear, 4×109 VG / ear, 5×10 9 VG / ear, 6×10 9 VG / ear, 7×10 9 VG / ear, 8×10 9 VG / ear, 9×10 9 VG / ear, 1×10 10 VG / ear, 2×10 10 VG / ear, 3×10 10 VG / ear, 4×10 10 VG / ear, 5×10 10 VG / ear, 6×10 10 VG / ear, 7×10 10 VG / ear, 8×10 10 VG / ear, 9×10 10 VG / ear, 1×10 11 VG / ear, 2×10 11 VG / ear, 3×10 11 VG / ear, 4×10 11 VG / ear, 5×10 11 VG / ear, 6×10 11 VG / ear, 7×10 11 VG / ear, 8×10 11 VG / ear, 9×10 11 VG / ear, 1×10 12 VG / ear, 2×10 12 VG / ear, 3×10 12 VG / ear, 4×10 12 VG / ear, 5×10 12 VG / ear, 6×10 12 VG / ear, 7×10 12 VG / ear, 8×10 12 VG / ear, 9×10 12 VG / ear, 1×10 13 VG / ear, 2×10 13 VG / ear, 3×10 13 VG / ear, 4×10 13 VG / ear, 5×10 13 VG / ear, 6×10 13 VG / ear, 7×10 13 VG / ear, 8×10 13 VG / ear, 9×10 13 VG / ear, 1×10 14 VG / ear, 2×10 14 VG / ear, 3×10 14 VG / ear, 4×10 14VG / ear, 5×10 14 VG / ear, 6×10 14 VG / ear, 7×10 14 VG / ear, 8×10 14 VG / ear, 9×10 14 VG / ear, 1×10 15 VG / ear, or 2x10 15 The subject may be administered a dose of 100 mg / ear (VG / ear).

[0173] The compositions described herein are administered in an amount sufficient to improve or restore (e.g., rescue) hearing, inhibit or slow the progression of hearing loss (e.g., sensorineural hearing loss), alleviate tinnitus (e.g., in a subject with Meniere's disease), reduce vestibular dysfunction, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), treat bilateral vestibular disorders, treat oscillopia, inhibit or slow the progression of vestibular dysfunction, reduce aural fullness (e.g., in a subject with Meniere's disease), increase or promote vestibular hair cell regeneration, increase or induce hair cell maturation (e.g., maturation of regenerated vestibular hair cells), or increase or induce expression of an expression product in SLC26A4-expressing cells (e.g., interdental cells, spiral ridge cells, root cells, or vestibular supporting cells). Hearing may be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions) and may improve by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to hearing measurements obtained before treatment. In some embodiments, the composition is administered in an amount sufficient to improve the subject's ability to understand speech. The compositions described herein may also be administered in an amount sufficient to delay or prevent the onset of sensorineural hearing loss or hearing loss (e.g., in subjects who carry a mutation in SLC26A4 but who do not exhibit hearing impairment at the time of treatment or who exhibit mild to moderate hearing loss at the time of treatment).Vestibular function can be assessed using standard tests for balance and vertigo, such as eye movement tests (e.g., ENG or VNG), VOR tests (e.g., head impulse tests (Halmagyi-Curthoys tests, e.g., VHIT), or caloric reflex testing, stabilometry, and rotary-chair testing. Vestibular dysfunction may be assessed using MRI, ECOG, VEMP, and outpatient balance testing, and may improve by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to measurements obtained before treatment. The compositions described herein may also be administered in an amount sufficient to slow or prevent the onset or progression of vestibular dysfunction (e.g., in subjects with a 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 who do not exhibit vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), or in subjects who exhibit mild to moderate vestibular dysfunction). The nucleic acid vector administered to the subject or cell may contain an SLC26A4 promoter. Expression of the protein encoded by the transgene operably linked to the target and / or enhancer may be assessed using immunohistochemical staining, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detecting proteins or mRNA, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, Vestibular hair cell regeneration can be assessed indirectly based on testing of vestibular function and can be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to vestibular hair cell regeneration before administration of the compositions described herein or compared to an untreated subject.The compositions and methods described herein may also reduce toxicity associated with administration of a nucleic acid vector compared to toxicity observed following administration of a nucleic acid vector that does not include the SLC26A4 promoter and / or enhancer described herein (e.g., administration of a nucleic acid vector in which the same transgene is expressed using a ubiquitous promoter and / or without the SLC26A4 enhancer). These effects may occur, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more weeks after administration of a composition described herein. Patients may be evaluated 1, 2, 3, 4, 5, 6, or more months after administration of the composition, depending on the dose and route of administration used for treatment. Depending on the results of the evaluation, patients may receive additional treatment.

[0174] kit The compositions described herein can be provided in a kit for use in treating sensorineural hearing loss or vestibular dysfunction. The compositions can include one or more SLC26A4 enhancers (e.g., polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35) and / or SLC26A4 promoters (e.g., polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1 and 20-27) described herein. The kit may include a promoter of the formula BAC, such as a polynucleotide having at least one sequence identity to the promoter of the formula BAC), a nucleic acid vector containing such a polynucleotide, or a nucleic acid vector containing the 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, e.g., a protein that can be expressed in SLC26A4-expressing inner ear cells and treat hearing loss (e.g., pendrin) or a protein that can treat vestibular dysfunction (e.g., pendrin or Atoh1), or a transgene encoding an RNA molecule, such as an inhibitory RNA molecule). The nucleic acid vector may be packaged in an AAV viral capsid (e.g., AAV1, AAV2, AAV2quad(YF), AAV6, AAV8, AAV9, Anc80, Anc80L65, AAV-DJ, AAV-DJ / 9, 7m8, or PHP.B). The kit may further include a package insert instructing a user of the kit, such as a physician, to practice the methods described herein. The kit may optionally include a syringe or other device for administering the composition. [Example]

[0175] The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be merely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0176] Example 1. Transfection of AAV-DJ vectors containing various promoters and enhancers driving GFP expression into the lateral wall of the mouse cochlea The lateral walls of the cochleae of 6- to 8-week-old male C57BL / 6J mice (000664, The Jackson Laboratory) were excised for culture with AAV-DJ viral vectors containing a nuclear-targeted H2B-EGFP fusion transgene driven by various promoters (CMV and various SLC26A4 promoters described herein without the SLC26A4 enhancer), as well as AAV-DJ vectors containing an H2B-EGFP fusion transgene driven by either the PEND3.1.8 promoter (SEQ ID NO: 1) or the PEND3.1.6 promoter (SEQ ID NO: 23) and various mouse or human SLC26A4 enhancers. Figures 1A-1C show the general design of the transgene plasmids used to generate the AAV viral vectors, and Figure 7 shows the relative size and alignment of the various SLC26A4 promoters used in these experiments. After euthanasia, animals were sacrificed by CO2 euthanasia, and the temporal bones were collected. The lateral cochlear walls were dissected in ice-cold DMEM / F-12 solution (11039021, Gibco) and cultured in DMEM / F-12 supplemented with 10% FBS (F4135, Sigma) and 10 μg / ml ciprofloxacin (AC456880050, Fisher Scientific) in GlutaMax culture medium (10565018, Gibco) on glass-bottom culture dishes (10810-054, Matsunami Glass). AAV was added to the culture medium in 250 μl of medium. After incubation for 3 days, the cultured lateral cochlear walls were washed with 2 ml of fresh medium prepared as described above. The cultured lateral cochlear walls were then maintained in culture for an additional 2 days (a total of 5 days of culture after AAV addition).

[0177] At the end of the incubation period, samples were fixed with 4% formaldehyde in fresh 1X PBS at room temperature for 1 hour and rinsed three times with 1X PBS for 5 minutes each. The tissues were blocked with 10% normal donkey serum, 0.5% Triton X-100 in PBS, pH 7.4, for 1 hour at room temperature, followed by overnight incubation at 4°C with a primary antibody against pendrin (BiCell Scientific 20501) diluted 1:100 in 1X PBS with 0.5% Triton X-100. The next day, after three 5-minute washes with PBS, the tissues were incubated with a 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, tissues were washed with PBS (3x, 5 min) and then mounted in Slowfade Diamond Antifade Mounting Media (DAKO) (ThermoFisher Molecular probes, s36963).

[0178] After mounting, the lateral wall was imaged using a Zeiss LSM 880 confocal microscope in both the 488 (EGFP) and 568 (pendrin) channels. Laser power and gain were set to achieve the highest EGFP signal without saturating the detector. After establishing imaging settings, all groups were imaged at the same laser power and gain throughout the study (all images in Figures 3A-3I were imaged with the same settings; all images in Figures 4A-4B were imaged with the same settings; all images in Figure 5 were imaged with the same settings; and all images in Figure 10A were imaged with the same settings), allowing comparisons between groups.

[0179] To assess the performance of these promoters in driving expression, we quantified EGFP intensity in pendrin-expressing cells in the lateral wall. For quantification, a surface was fitted to the signal from the 568 channel to identify pendrin-containing cells, and a spot was fitted to the 488 channel to quantify EGFP-containing nuclei. A mask between these two surfaces was then used to identify cells that expressed both pendrin and EGFP using the Imaris software package (Oxford Instruments, Imaris 9.9.1). These features were used to measure the percentage of pendrin-expressing cells that also expressed EGFP, the total number of EGFP-expressing cells located inside or outside the outer spiral groove of the mouse lateral wall (the pendrin-expressing region), and the average EGFP intensity in all pendrin-expressing cells per sample. See Figure 6, panels A–D.

[0180] As can be seen in Figure 2A, most pendrin-expressing cells are located in the tight junctions of the spiral protuberance and stria vascularis. AAV expressing EGFP under the control of the ubiquitous CMV promoter conferred EGFP expression throughout the lateral wall, not limited to the pendrin-expressing region, demonstrating the ubiquitous AAV-DJ targeting in the lateral wall (Figure 2B). Replacing the CMV promoter with various SLC26A4 promoters disclosed herein resulted in EGFP expression being restricted primarily to cells that also express pendrin, with little or no EGFP expression in other cells of the lateral wall (Figures 3A-I).

[0181] Inclusion of certain SLC26A4 enhancer sequences described herein (mouse E6 (SEQ ID NO: 7), mouse E2 (SEQ ID NO: 6), or human E6.4 (SEQ ID NO: 5)) fused to the 5' end of the PEND3.1.8 promoter (SEQ ID NO: 1) in an AAV vector expressing EGFP resulted in increased EGFP expression without loss of specificity for pendrin-expressing cells (Figures 4A-4C). Interestingly, one of the other SLC26A4 enhancers (human E6.1; SEQ ID NO: 4) resulted in decreased EGFP expression while maintaining specificity for pendrin-expressing cells (Figure 4D). Two other enhancers (human E6.2 (SEQ ID NO: 8) and E6.3 (SEQ ID NO: 9)) resulted in loss of specificity for pendrin-expressing cells and resulted in EGFP expression throughout the lateral wall (Figures 4E-4F).

[0182] Inclusion of certain SLC26A4 enhancer sequences described herein (mouse E6 (SEQ ID NO: 7) or human E6.4 (SEQ ID NO: 5)) fused to the 5' end of the PEND3.1.6 promoter (SEQ ID NO: 23) in an AAV vector expressing EGFP resulted in increased expression of EGFP without loss of specificity for pendrin-expressing cells (Figures 5A-5B).

[0183] Quantification of EGFP expression in pendrin-positive and pendrin-negative regions of the lateral wall using the PEND3.1.8 promoter with or without the mouse E6 or E2 enhancer or one of the human E6.1 or E6.4 enhancers is shown in Figure 6. Inclusion of the mouse E6 or E2 enhancer or the human E6.4 enhancer increased the percentage of pendrin-expressing cells that also expressed EGFP compared with the PEND3.1.8 promoter alone (Figure 6, panel A). The presence of the human E.6.4 enhancer increased the number of EGFP-expressing cells in the pendrin-positive region of the lateral wall compared with the PEND3.1.8 promoter alone (Figure 6, panel B).

[0184] In a separate experiment, quantification of EGFP expression from AAV vectors containing the mouse E2 and E6 SLC26A4 enhancers fused to each other and to the 5' end of the PEND3.1.8 promoter generated from transgene plasmid P1708 (Figure 9) was compared with EGFP expression from AAV vectors containing only one or the other of the E2 or E6 SLC26A4 enhancers. As can be seen in Figure 10A, each of these AAV vectors contained expression specific to pendrin-positive cells, but expression was appreciably lower in the AAV vector with only the E2 enhancer. As shown in Figure 10B, inclusion of both enhancers did not significantly increase EGFP expression in pendrin-expressing cells as much as the AAV vector with only the mouse E6 enhancer. Compared to previous experiments using these same vectors (compare Figure 4 with Figure 10A), the apparently lower GFP intensity observed in this experiment using AAV vectors with only the mouse E2 enhancer or only the mouse E6 enhancer is due to the use of lower laser power to visualize EGFP in the latter experiments.

[0185] Example 2. Single-cell RNA-seq analysis of mouse lateral parietal cells transfected with AAV-DJ vectors containing various promoters and enhancers Lateral walls from the cochleae of 6- to 8-week-old male C57BL / 6J mice (000664, The Jackson Laboratory) were excised as described in Example 1 and simultaneously transfected with different AAV construct combinations, each containing a unique promoter or promoter-enhancer combination and a unique barcode. Controls included a construct lacking the ubiquitous Pgk promoter and a promoterless construct. The promoterless construct contains a stuffer sequence belonging to the ATP-binding cassette subfamily A member 4 (ABCA4) gene. The promoterless minP construct is a shortened version of the same stuffer sequence with a downstream minimal β-globin promoter sequence. Promoter-containing AAV constructs were generated from transgene plasmids with the general design shown in Figures 1A-1B. Seven days after AAV transfection, cultured lateral wall explants were processed for scRNA-seq experiments. 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.

[0186] To identify various cell types at the lateral wall, we selected a target capture of 10,000 cells per sample using the high-throughput droplet microfluidics GemCode platform from 10x Genomics with v3.1 chemistry. Each droplet contained a single cell in cell lysis buffer and gel beads hybridized with oligo(dT) primers encoding a unique cell barcode and unique molecular identifier (UMI). After capturing mRNA using the oligo(dT) primer-hybridized beads, the transcriptomes captured on the gel beads were pooled and reverse transcribed into cDNA. Reverse transcription and PCR amplification of the cDNA, as well as library preparation from the 3' end, were performed according to the manufacturer's published procedures.

[0187] In addition to the total RNA libraries described above (used to assess each cell's transcriptome), custom libraries were also generated for each sample to detect unique barcodes assigned to each promoter construct, providing promoter-construct-specific expression readouts. Libraries were sequenced on an Illumina NovaSeq 6000. Reads were demultiplexed, mapped to a custom GRCm38 mm10 reference genome, and filtered. Cell barcodes and UMIs were then 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.

[0188] Single-cell RNA-seq data were analyzed using Seurat v3 (Butler et al., Nat Biotechnol 36: 411-420, 2018). Cells with fewer than 100 genes were detected, and more than 20% of reads from mitochondrial genes and 40% of reads from ribosome content were filtered out. Expected doublets were removed using Scrublet (Wollock et al., Cell Syst. 8:281-291, 2019 e9). Variable genes were selected, and cells were clustered using the default Seurat function. Cell types were assigned to each cluster based on gene expression correlations and expression of known marker genes from total RNA libraries containing previously marked datasets from the cochlea. Adding promoter-construct reads per cell derived from the custom RNA library to Seurat objects with matching cell IDs allowed for pairing reads derived from viral constructs in each cell with their transcriptome. For each construct, the percentage of cells positive (above the noise threshold) was calculated for each cell type. The average expression of each construct in a cell type was calculated using CellRanger's method for calculating average expression (support.10xgenomics.com / single-cell-gene-expression / software / pipelines / latest / algorithms / overview). Points are plotted for cell types where more than 2% of cells express the construct.

[0189] Figure 8 shows the results of this analysis. The top four clusters of cell types listed in Figure 8 (spiral process / root cells / Böttcher cells; inner / outer sulcus / spiral ridge cells; interdental cells; and Reissner's membrane epithelial cells), identified as described above, are the cells that express the highest levels of native pendrin. As can be seen in Figure 8, use of the pendrin promoter described herein restricts expression to these top four cell clusters, as well as to the majority of supporting cells, compared to the ubiquitous Pgk promoter. Inclusion of the SLC26A4 enhancer, and in particular the human E6.4 or mouse E6 enhancer, increases both the proportion of cells in these four cell clusters that express and the overall expression level of the construct (as detected by barcoding).

[0190] Example 3. Administration of AAV-DJ vectors containing the Pend3.1.8 promoter and human E6.4 enhancer to Slc26a4 knockout mice Based on the ex vivo 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 described in Example 1 (Figure 4A, right panel), the same vector was tested to determine whether it would increase EGFP expression in target cell types in both the cochlea and vestibule in vivo. A plasmid containing these elements, P1517 (Figure 11), was packaged into AAV-DJ. As a control, a plasmid containing an expression cassette encoding the ubiquitous CMV promoter (without any enhancer) driving expression of nuclear-targeted EGFP packaged into AAV1 was used.

[0191] AAV-DJ vectors generated by the plasmid P1527 were locally administered into both sides of the posterior semicircular canal (IL) of newborn Slc26a4 knockout mice at age P3. These Slc26a4 KO mice were generated from mice on a C57BL / 6 background by CRISPR / Cas9-mediated deletion of exons 3–5 of the Slc26a4 gene on mouse chromosome 12. A CMV-driven control AAV1 vector was injected into both sides of the posterior semicircular canal (IL) of other KO mice at ages P3–P4. At age P22, control and test mice were sacrificed, and whole ears were fixed, decalcified, and paraffin-embedded. Sections were imaged for EGFP using a fluorescent microscope. To visualize cochlear structures, sections were further stained with Kcnj10 (Abnova H00003766-M01). In ears treated with AAV-DJ containing the E6.4 enhancer linked to the Pend3.1.8 promoter, EGFP fluorescence was detected in the nuclei of the interdental (ID) and spiral eminence (SP) cells of the cochlea (Figure 12, panels A–D). In contrast, ears treated with the control AAV1 containing the CMV promoter resulted in widespread, nonspecific expression throughout the cochlea (Figure 12, panels E–F).

[0192] Example 4. Administration of a composition containing a nucleic acid vector containing an SLC26A4 promoter and / or an SLC26A4 enhancer to a subject with sensorineural hearing loss According to the methods disclosed herein, a practitioner of skill in the art can treat a patient, such as a human patient, with hearing loss (e.g., pendrin-associated hearing loss, such as DFNB4 or Pendred syndrome) to improve or restore hearing. To this end, a practitioner of skill in the art can use at least one SLC26A4 promoter described herein (e.g., an SLC26A4 promoter of a formula BAC described hereinabove, such as any one of SEQ ID NOS: 1 and 20-27, e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1) 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). An AAV vector (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.eB) containing a 26A4 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 more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35).A composition containing an AAV vector containing a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of at least one SLC26A4 enhancer described herein (e.g., SLC26A4 promoter, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 2) 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) may be administered to a human patient. 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, or injection through the round window membrane) to treat sensorineural hearing loss.

[0193] After administering the composition to a patient, a physician skilled in the art can monitor the patient's improvement in response to treatment by a variety of methods. For example, the physician can monitor the patient's hearing by performing standard tests such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions after administering the composition. The finding that the patient shows improved hearing in one or more tests after administering the composition compared to the hearing test results before administering the composition indicates that the patient is responding favorably to treatment. Subsequent doses can be determined and administered as needed.

[0194] Example 5. Administration of a composition containing a nucleic acid vector containing an SLC26A4 promoter and / or an SLC26A4 enhancer to a subject with vestibular dysfunction According to the methods disclosed herein, a practitioner of skill in the art can treat a patient, such as a human patient, with a vestibular dysfunction (e.g., a vestibular dysfunction associated with hair cell loss, such as age-related vestibular dysfunction or ototoxic drug-induced vestibular dysfunction) to improve or restore vestibular function. To this end, a practitioner of skill in the art can use a polynucleotide sequence to encode a SLC26A4 promoter described herein (e.g., any one of SEQ ID NOS: 1 and 20-27, e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1, in a BAC of the formula described hereinabove. and 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 11109, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 1 or 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 more sequence identity) to any one of at least one SLC26A4 enhancer described herein (e.g., SEQ ID NO: 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, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 2) operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a wild-type form of Atoh1, such as a polynucleotide encoding SEQ ID NO: 36 or SEQ ID NO: 38).A composition containing an AAV vector (e.g., an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vector) containing a polynucleotide having sequence identity (e.g., a polynucleotide having sequence identity equal to or greater than 1000 nucleotides) can be administered to a human patient. The composition containing the AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or endolymph, injection through the round window membrane, or injection into the semicircular canal) to treat vestibular dysfunction.

[0195] After administering the composition to the patient, a practitioner skilled in the art can monitor the expression of the therapeutic protein encoded by the transgene and the patient's improvement in response to treatment by a variety of methods. For example, the practitioner can monitor the patient's vestibular function by performing standard tests, such as electronystagmography, videonystagmography, VOR testing (e.g., head impulse testing (Halmagyi-Curthoys test, e.g., VHIT), or caloric reflex testing), rotation testing, vestibular evoked myogenic potential testing, or computerized dynamic stabilometry. A finding that the patient shows improvement in vestibular function in one or more tests after administration of the composition compared to test results obtained before administration of the composition indicates that the patient is responding well to treatment. Subsequent doses can be determined and administered as needed.

[0196] Example 6. Administration of a composition containing a nucleic acid vector containing an SLC26A4 promoter and / or an SLC26A4 enhancer to a subject with Meniere's disease According to the methods disclosed herein, a practitioner of skill in the art can treat a patient, such as a human patient with Meniere's disease, to reduce vertigo, improve hearing, reduce tinnitus, or reduce aural fullness. To this end, a practitioner of skill in the art can use a gene encoding at least one SLC26A4 promoter described herein (e.g., an SLC26A4 promoter of a BAC formula described hereinabove, such as any one of SEQ ID NOS: 1 and 20-27, e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1) 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). An AAV vector (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.eB) containing a 26A4 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 more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35).A composition containing an AAV vector containing a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of at least one SLC26A4 enhancer described herein (e.g., SLC26A4 promoter, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 2) 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) may be administered to a human patient. Compositions containing AAV vectors can be administered to patients, for example, by local administration to the inner ear (e.g., injection into the perilymph or endolymph, injection through the round window membrane, or injection into the semicircular canals) to treat Meniere's disease.

[0197] After administering the composition to the patient, a physician skilled in the art can monitor the expression of the therapeutic protein encoded by the transgene and the patient's improvement in response to treatment by a variety of methods. For example, the physician can monitor the patient's vestibular function by performing standard tests such as electronystagmography, videonystagmography, VOR tests (e.g., head impulse tests (Halmagyi-Curthoys tests, e.g., VHIT), or caloric reflex tests), rotation tests, vestibular-evoked myogenic potential tests, or computerized dynamic stabilometry, and can monitor the patient's hearing by performing standard tests such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions after administering the composition. The physician can also rely on the patient's reports of vertigo, tinnitus, and aural fullness. A patient showing improvement in vestibular function or hearing in one or more of the tests, or reporting a reduction in vertigo, tinnitus, or a feeling of fullness in the ears after administration of the composition compared to test results obtained before administration of the composition, indicates that the patient is responding well to treatment. Subsequent doses can be determined and administered as needed.

[0198] Exemplary embodiments of the present invention are described in the following listed paragraphs. E1. A nucleic acid vector comprising an SLC26A4 promoter of the formula 5'-BAC-3', 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 more sequence identity) to SEQ ID NO:1; 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 more 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; 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 more sequence identity) to SEQ ID NO:3, or a portion thereof comprising at least the first 159 contiguous nucleotides of SEQ ID NO:3; The nucleic acid vector as described above, wherein the SLC26A4 promoter has 1,481 bases or less.

[0199] E2. The nucleic acid vector of E1, wherein A has the sequence of SEQ ID NO:1. E3. The nucleic acid vector of E1 or E2, wherein B is absent. E4. The nucleic acid vector according to E1 or E2, wherein B has the sequence of SEQ ID NO: 2 or a portion thereof comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2.

[0200] E5. The nucleic acid vector according to E4, wherein B has a partial sequence of SEQ ID NO: 2, comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2. E6. The nucleic acid vector of E5, wherein the portion of SEQ ID NO:2 is the first 307 contiguous nucleotides (set forth in SEQ ID NO:14) from the 3' end of SEQ ID NO:2.

[0201] E7. The nucleic acid vector of E4, wherein B has the sequence of SEQ ID NO:2. E8. The nucleic acid vector according to any one of E1 to E7, wherein C is absent. E9. The nucleic acid vector of any one of E1 to E7, 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.

[0202] E10. The nucleic acid vector of E9, wherein C has the sequence of a portion of SEQ ID NO:3, comprising at least the first 159 nucleotides of SEQ ID NO:3. E11. The nucleic acid vector of E10, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NOs:15, 16, 17, 18, and 19, respectively).

[0203] E12. The nucleic acid vector of E11, wherein the portion of SEQ ID NO:3 is the first 159 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:15). E13. The nucleic acid vector of E11, wherein the portion of SEQ ID NO:3 is the first 324 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:16).

[0204] E14. The nucleic acid vector of E11, wherein the portion of SEQ ID NO:3 is the first 341 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:17). E15. The nucleic acid vector of E11, wherein the portion of SEQ ID NO:3 is the first 716 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:18).

[0205] E16. The nucleic acid vector of E11, wherein the portion of SEQ ID NO:3 is the first 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:19). E17. The nucleic acid vector of E9, wherein C has the sequence of SEQ ID NO:3.

[0206] E18. The nucleic acid vector of any one of E1, E3, and E8, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0207] E19. The nucleic acid vector of E13, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E20. The nucleic acid vector of any one of E1, E2, E4, E7, and E8, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 20.

[0208] E21. The nucleic acid vector of E15, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 20. E22. The nucleic acid vector described in any one of E1 to E3, E9, and E12, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 21.

[0209] E23. The nucleic acid vector of E17, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 21. E24. The nucleic acid vector described in any one of E1, E2, E4 to E6, and E8, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 22.

[0210] E25. The nucleic acid vector of E19, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 22. E26. The nucleic acid vector described in any one of E1 to E3, E9 to E11, and E15, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 23.

[0211] E27. The nucleic acid vector of E21, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 23. E28. The nucleic acid vector described in any one of E1 to E3, E9 to E11, and E16, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 24.

[0212] E29. The nucleic acid vector of E23, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 24. E30. The nucleic acid vector described in any one of E1, E2, E4 to E6, E9 to E11, and E15, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 25.

[0213] E31. The nucleic acid vector of E25, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 25. E32. The nucleic acid vector described in any one of E1, E2, E4 to E6, E9 to E11, and E13, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 26.

[0214] E33. The nucleic acid vector of E27, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 26. E34. The nucleic acid vector described in any one of E1, E2, E4 to E6, and E9 to E12, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 27.

[0215] E35. The nucleic acid vector of E29, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 27. E36. The nucleic acid vector according to any one of E1 to E35, further comprising an SLC26A4 enhancer operably linked to the SLC26A4 promoter.

[0216] E37. The nucleic acid vector of E36, wherein 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 more sequence identity) to any one of SEQ ID NOs: 4 to 9, 34, and 35.

[0217] E38. The nucleic acid vector of E37, wherein 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 more sequence identity) to SEQ ID NO:4.

[0218] E39. The nucleic acid vector of E38, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:4. E40. The nucleic acid vector of E37, wherein 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 more sequence identity) to SEQ ID NO:5.

[0219] E41. The nucleic acid vector of E40, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:5. E42. The nucleic acid vector of E37, wherein 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 more sequence identity) to SEQ ID NO:6.

[0220] E43. The nucleic acid vector of E42, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:6. E44. The nucleic acid vector of E37, wherein 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 more sequence identity) to SEQ ID NO:7.

[0221] E45. The nucleic acid vector of E44, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:7. E46. The nucleic acid vector of E37, wherein 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 more sequence identity) to SEQ ID NO:8.

[0222] E47. The nucleic acid vector of E46, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:8. E48. The nucleic acid vector of E37, wherein 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 more sequence identity) to SEQ ID NO:9.

[0223] E49. The nucleic acid vector of E48, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:9. E50. The nucleic acid vector of E37, wherein 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 more sequence identity) to SEQ ID NO: 34.

[0224] E51. The nucleic acid vector of E50, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 34. E52. The nucleic acid vector of E37, wherein 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 more sequence identity) to SEQ ID NO: 35.

[0225] E53. The nucleic acid vector of E52, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 35. E54. The nucleic acid vector according to any one of E36 to E53, wherein the SLC26A4 enhancer is directly linked (eg, fused) to the SLC26A4 promoter.

[0226] E55. The SLC26A4 enhancer is a sequence of 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500). The nucleic acid vector of any one of E36 to E53, wherein the SLC26A4 promoter is linked to the SLC26A4 promoter via a nucleic acid linker of about 100 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.

[0227] E56. The nucleic acid vector according to E55, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter via a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 nucleotides, for example, about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0228] E57. The nucleic acid vector according to any one of E36 to E56, wherein the SLC26A4 enhancer is located 5' of the SLC26A4 promoter. E58. The nucleic acid vector according to any one of E36 to E56, wherein the SLC26A4 enhancer is located 3' of the SLC26A4 promoter.

[0229] E59. The nucleic acid vector of any one of E1 to E58, wherein the SLC26A4 promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0230] E60. The nucleic acid vector of E59, wherein the expression product is a heterologous expression product. E61. The nucleic acid vector according to E59 or E60, wherein the expression product is an expression product endogenously expressed in an SLC26A4-expressing cell.

[0231] E62. The nucleic acid vector of E61, wherein the expression product is an expression product that is endogenously expressed in SLC26A4-expressing inner ear cells. E63. The nucleic acid vector of E62, wherein the expression product is endogenously expressed in interdental cells, spiral ridge cells, cochlear root cells, and / or vestibular supporting cells (e.g., expressed in at least one of these cell types).

[0232] E64. The nucleic acid vector of any one of E61 to E63, wherein the expression product is pendrin (eg, a mammalian pendrin protein). E65. The nucleic acid vector of E64, wherein the pendrin (the mammalian pendrin protein) is the wild-type isoform endogenously expressed in the mammalian inner ear.

[0233] E66. The nucleic acid vector of E64 or E65, wherein the pendrin (the mammalian pendrin protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 10 or SEQ ID NO: 11.

[0234] E67. The nucleic acid vector of E66, wherein the pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO: 10 or SEQ ID NO: 11. E68. The nucleic acid vector according to any one of E59 to E63, wherein the expression product is Atoh1 (eg, mammalian Atoh1).

[0235] E69. The nucleic acid vector of E68, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the mammalian inner ear. E70. The nucleic acid vector of any one of E68 or E69, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 36 or SEQ ID NO: 38.

[0236] E71. The nucleic acid vector of E70, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO: 36 or SEQ ID NO: 38. E72. The nucleic acid vector of E59 or E60, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0237] E73. The nucleic acid vector of any one of E37 to E72, wherein the nucleic acid vector comprises two or more different SLC26A4 enhancers, each enhancer 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 more sequence identity) to any one of SEQ ID NOs: 4 to 9, 34, and 35.

[0238] E74. The nucleic acid vector of E73, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NOs: 4-9, 34, and 35.

[0239] E75. The nucleic acid vector of E73, wherein the nucleic acid vector comprises a first enhancer having at least 85% sequence identity to SEQ ID NO:6 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and a second enhancer having at least 85% sequence identity to SEQ ID NO:7 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0240] E76. The nucleic acid vector of 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. E77. The nucleic acid vector of E75 or E76, wherein the first and second enhancers are both located 5' of the SLC26A4 promoter.

[0241] E78. The nucleic acid vector of E77, wherein the first or second enhancer is directly fused to the other of the first or second enhancer, which is directly fused to the SLC26A4 promoter (i.e., the enhancer and promoter elements are directly linked to each other without any intervening nucleic acid).

[0242] E79. The nucleic acid vector according to E78, comprising, in 5' to 3' order, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, and the sequence of the SLC26A4 promoter (e.g., a polynucleotide sequence consisting of the polynucleotide sequence of SEQ ID NO: 6-SEQ ID NO: 7 of the SLC26A4 promoter).

[0243] E80. The nucleic acid vector according to any one of E75 to E79, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E81. The nucleic acid vector described in any one of E37 to E74, wherein the nucleic acid vector 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 more sequence identity) to any one of SEQ ID NOs: 4 to 9, 34, and 35.

[0244] E82. The nucleic acid vector of E81, wherein 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. E83. A polynucleotide comprising an SLC26A4 enhancer operably linked to a promoter, the polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35, wherein the enhancer is directly or indirectly linked to the promoter, and is operably linked to a promoter comprising 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 600, 1 to 650, 1 to 700, 1 to 750, 1 to 800, 1 to 900, 1 to 950, 1 to 1000, 1 to 1500, 1 to 1000, 1 to 1500, 1 to 2000, 1 to 3500, 1 to 4500, 1 to 5000, 1 to 1000, 1 to 1500, 1 to 2500, 1 to 3500, 1 to 4500, 1 to 5000, 1 to 1000, 1 to 1500, 1 to 2000, 1 to 3500, 1 to 4500, 1 to 5000, 1 to 1000, 1 to 150 the polynucleotides are linked via a nucleic acid linker of up to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500 nucleotides, for example, about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides.

[0245] E84. The polynucleotide according to E83, wherein the SLC26A4 enhancer is linked to the promoter via a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 nucleotides, for example, about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0246] E85. The polynucleotide of E83, wherein the SLC26A4 enhancer is directly linked (eg, fused) to the promoter. E86. The polynucleotide of any one of E83 to E85, wherein the SLC26A4 enhancer is located 5' of the promoter.

[0247] E87. The polynucleotide of any one of E83 to E85, wherein the SLC26A4 enhancer is located 3' of the promoter. E88. The polynucleotide of any one of E83 to E87, wherein 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 more sequence identity) to SEQ ID NO:4.

[0248] E89. The polynucleotide of E88, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:4. E90. The polynucleotide of any one of E83 to E87, wherein 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 more sequence identity) to SEQ ID NO:5.

[0249] E91. The polynucleotide of E90, wherein said SLC26A4 enhancer has the sequence of SEQ ID NO:5. E92. The polynucleotide of any one of E83 to E87, wherein 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 more sequence identity) to SEQ ID NO:8.

[0250] E93. The polynucleotide of E92, wherein said SLC26A4 enhancer has the sequence of SEQ ID NO:8. E94. The polynucleotide of any one of E83 to E87, wherein 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 more sequence identity) to SEQ ID NO: 9.

[0251] E95. The polynucleotide of E94, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:9. E96. The polynucleotide of any one of E83 to E87, wherein 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 more sequence identity) to SEQ ID NO: 34.

[0252] E97. The polynucleotide of E96, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 34. E98. The polynucleotide of any one of E83 to E87, wherein 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 more sequence identity) to SEQ ID NO: 35.

[0253] E99. The polynucleotide of E98, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 35. E100. The polynucleotide of any one of E83 to E999, wherein the promoter is a minimal promoter, a core promoter, or a constitutive promoter.

[0254] E101. The polynucleotide of 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 phosphoglycerol kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, an HSV promoter, or an SV40 promoter.

[0255] E102. The polynucleotide of E101, wherein the promoter is a minimal β-globin promoter, a CMV mini 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.

[0256] E103. The polynucleotide of E100, wherein the promoter is a minimal promoter. E104. The polynucleotide of any one of E83 to E99, wherein the promoter is a mammalian SLC26A4 promoter.

[0257] E105. The polynucleotide of E104, wherein the SLC26A4 promoter is a human or mouse SLC26A4 promoter. E106. The SLC26A4 promoter has the formula 5'-BAC-3'; 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 more sequence identity) to SEQ ID NO:1; 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 more 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; 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 more sequence identity) to SEQ ID NO:3, or a portion thereof comprising at least the first 159 contiguous nucleotides of SEQ ID NO:3; The polynucleotide of E104 or E105, wherein the SLC26A4 promoter is 1,481 bases or less.

[0258] E107. The polynucleotide of E106, wherein A has the sequence of SEQ ID NO:1. E108. The polynucleotide of E106 or E107, wherein B is absent. E109. The polynucleotide according to E106 or E107, wherein B has the sequence of SEQ ID NO: 2 or a part thereof comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2.

[0259] E110. The polynucleotide according to E109, wherein B has a sequence of a portion of SEQ ID NO: 2, comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2. E111. The polynucleotide of E110, wherein the portion of SEQ ID NO:2 is the first 307 contiguous nucleotides (set forth in SEQ ID NO:14) from the 3' end of SEQ ID NO:2.

[0260] E112. The polynucleotide of E109, wherein B has the sequence of SEQ ID NO:2. E113. The polynucleotide according to any one of E106 to E112, wherein C is absent.

[0261] E114. The polynucleotide of any one of E106 to E112, 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.

[0262] E115. The polynucleotide of E114, wherein C has the sequence of a portion of SEQ ID NO:3, comprising at least the first 159 nucleotides of SEQ ID NO:3. E116. The polynucleotide of E115, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NOs:15, 16, 17, 18, and 19, respectively).

[0263] E117. The polynucleotide of E116, wherein the portion of SEQ ID NO:3 is the first 159 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:15). E118. The polynucleotide of E116, wherein the portion of SEQ ID NO:3 is the first 324 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:16).

[0264] E119. The polynucleotide of E116, wherein the portion of SEQ ID NO:3 is the first 341 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:17). E120. The polynucleotide of E116, wherein the portion of SEQ ID NO:3 is the first 716 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:18).

[0265] E121. The polynucleotide of E116, wherein the portion of SEQ ID NO:3 is the first 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:19). E122. The polynucleotide of E114, wherein C has the sequence of SEQ ID NO:3.

[0266] E123. The polynucleotide of any one of E106, E108, and E113, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0267] E124. The polynucleotide of E123, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E125. The polynucleotide of any one of E106, E107, E109, E112, and E113, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 20.

[0268] E126. The polynucleotide of E125, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 20. E127. The polynucleotide described in any one of E106 to E108, E114, and E122, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 21.

[0269] E128. The polynucleotide of E127, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 21. E129. The polynucleotide described in any one of E106, E107, E109 to E111, and E113, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 22.

[0270] E130. The polynucleotide of E129, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 22. E131. The polynucleotide described in any one of E106 to E108, E114 to E116, and E119, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 23.

[0271] E132. The polynucleotide of E131, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 23. E133. The polynucleotide described in any one of E106 to E108, E114 to E116, and E121, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 24.

[0272] E134. The polynucleotide of E133, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 24. E135. The polynucleotide of any one of E106, E107, E109 to E111, E114 to E116, and E120, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 25.

[0273] E136. The polynucleotide of E135, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 25. E137. The polynucleotide of any one of E106, E107, E109 to E111, E114 to E116, and E118, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 26.

[0274] E138. The polynucleotide of E137, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 26. E139. The polynucleotide described in any one of E106, E107, E109 to E111, and E114 to E117, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 27.

[0275] E140. The polynucleotide of E139, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 27. E141. The polynucleotide of E104 or E105, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 28.

[0276] E142. The polynucleotide of E141, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 28. E143. The polynucleotide of E104 or E105, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 29 to 33.

[0277] E144. The polynucleotide according to E143, wherein the SLC26A4 promoter has a sequence set forth in any one of SEQ ID NOs: 29 to 33. E145. The polynucleotide of E104 or E105, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 41.

[0278] E146. The polynucleotide of E145, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 41. E147. The polynucleotide of any one of E83 to E146, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0279] E148. The polynucleotide of E147, wherein the expression product is a heterologous expression product. E149. The polynucleotide of E147 or E148, wherein the expression product is an expression product endogenously expressed in an SLC26A4-expressing cell.

[0280] E150. The polynucleotide of E149, wherein the expression product is an expression product endogenously expressed in SLC26A4-expressing inner ear cells. E151. The polynucleotide of E150, wherein the expression product is endogenously expressed in (e.g., expressed in at least one of) interdental cells, spiral ridge cells, cochlear root cells, and / or vestibular supporting cells.

[0281] E152. The polynucleotide of any one of E149 to E151, wherein the expression product is pendrin (eg, a mammalian pendrin protein). E153. The polynucleotide of E152, wherein said pendrin (the mammalian pendrin protein) is a wild-type isoform endogenously expressed in the inner ear of mammals.

[0282] E154. The polynucleotide of any one of E152 or E153, wherein the pendrin (the mammalian pendrin protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 10 or SEQ ID NO: 11.

[0283] E155. The polynucleotide of E154, wherein the pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO: 10 or SEQ ID NO: 11. E156. The polynucleotide of any one of E147 to E151, wherein the expression product is Atoh1 (eg, mammalian Atoh1).

[0284] E157. The polynucleotide of E156, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the mammalian inner ear.

[0285] E158. The polynucleotide of any one of E156 or E157, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 36 or SEQ ID NO: 38.

[0286] E159. The polynucleotide of E158, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO: 36 or SEQ ID NO: 38. E160. The polynucleotide of E147 or E148, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0287] E161. The polynucleotide of any one of E83 to E160, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, each enhancer 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0288] E162. The polynucleotide of E161, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0289] E163. A polynucleotide described in any one of E83 to 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0290] E164. The polynucleotide of E163, wherein each copy of the two or more copies of the SLC26A4 enhancer has the sequence of one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0291] E165. A polynucleotide comprising the SLC26A4 promoter in the format 5'-BAC-3', operably linked to a polynucleotide that can be transcribed to produce an expression product, 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 more sequence identity) to SEQ ID NO:1; 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 more 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; 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 more sequence identity) to SEQ ID NO:3, or a portion thereof comprising at least the first 159 contiguous nucleotides of SEQ ID NO:3; A polynucleotide in which the SLC26A4 promoter is 1,481 bases or less.

[0292] E166. The polynucleotide of E165, wherein A has the sequence of SEQ ID NO:1. E167. The polynucleotide of E165 or E166, wherein B is absent. E168. The polynucleotide according to E165 or E166, wherein B has the sequence of SEQ ID NO: 2 or a part thereof comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2.

[0293] E169. The polynucleotide according to E168, wherein B has a sequence of a portion of SEQ ID NO: 2, comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2. E170. The polynucleotide of E169, wherein the portion of SEQ ID NO:2 is the first 307 contiguous nucleotides (set forth in SEQ ID NO:14) from the 3' end of SEQ ID NO:2.

[0294] E171. The polynucleotide of E168, wherein B has the sequence of SEQ ID NO:2. E172. The polynucleotide of any one of E165 to E171, wherein C is absent.

[0295] E173. The polynucleotide of any one of E165 to E171, 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.

[0296] E174. The polynucleotide of E173, wherein C has a sequence of a portion of SEQ ID NO:3, comprising at least the first 159 nucleotides of SEQ ID NO:3. E175. The polynucleotide of E174, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NOs:15, 16, 17, 18, and 19, respectively).

[0297] E176. The polynucleotide of E175, wherein the portion of SEQ ID NO:3 is the first 159 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:15). E177. The polynucleotide of E175, wherein the portion of SEQ ID NO:3 is the first 324 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:16).

[0298] E178. The polynucleotide of E175, wherein the portion of SEQ ID NO:3 is the first 341 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:17). E179. The polynucleotide of E175, wherein the portion of SEQ ID NO:3 is the first 716 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:18).

[0299] E180. The polynucleotide of E175, wherein the portion of SEQ ID NO:3 is the first 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:19). E181. The polynucleotide of E173, wherein C has the sequence of SEQ ID NO:3.

[0300] E182. The polynucleotide of any one of E165, E167, and E172, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0301] E183. The polynucleotide of E182, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E184. The polynucleotide of any one of E165, E166, E168, E171, and E172, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 20.

[0302] E185. The polynucleotide of E184, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 20. E186. The polynucleotide described in any one of E165 to E167, E173, and E181, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 21.

[0303] E187. The polynucleotide of E186, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 21. E188. The polynucleotide described in any one of E165, E166, E168 to E170, and E172, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 22.

[0304] E189. The polynucleotide of E188, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 22. E190. The polynucleotide described in any one of E165 to E167, E173 to E175, and E178, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 23.

[0305] E191. The polynucleotide of E190, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 23. E192. The polynucleotide described in any one of E165 to E167, E173 to E175, and E180, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 24.

[0306] E193. The polynucleotide of E192, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 24. E194. The polynucleotide of any one of E165, E166, E168 to E170, E173 to E175, and E179, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 25.

[0307] E195. The polynucleotide of E194, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 25. E196. The polynucleotide described in any one of E165, E166, E168 to E170, E173 to E175, and E177, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 26.

[0308] E197. The polynucleotide of E196, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 26. E198. The polynucleotide described in any one of E165, E166, E168 to E170, and E173 to E176, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 27.

[0309] E199. The polynucleotide of E198, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 27. E200. The polynucleotide of any one of E165 to E199, further comprising an SLC26A4 enhancer operably linked to the SLC26A4 promoter.

[0310] E201. The polynucleotide of E200, wherein 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 more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35.

[0311] E202. The polynucleotide of E201, wherein 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 more sequence identity) to SEQ ID NO:4.

[0312] E203. The polynucleotide of E202, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:4. E204. The polynucleotide of E201, wherein 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 more sequence identity) to SEQ ID NO:5.

[0313] E205. The polynucleotide of E204, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:5. E206. The polynucleotide of E201, wherein 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 more sequence identity) to SEQ ID NO:6.

[0314] E207. The polynucleotide of E206, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:6. E208. The polynucleotide of E201, wherein 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 more sequence identity) to SEQ ID NO:7.

[0315] E209. The polynucleotide of E208, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:7. E210. The polynucleotide of E201, wherein 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 more sequence identity) to SEQ ID NO:8.

[0316] E211. The polynucleotide of E210, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:8. E212. The polynucleotide of E201, wherein 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 more sequence identity) to SEQ ID NO:9.

[0317] E213. The polynucleotide of E212, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:9. E214. The polynucleotide of E201, wherein 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 more sequence identity) to SEQ ID NO: 34.

[0318] E215. The polynucleotide of E214, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 34. E216. The polynucleotide of E201, wherein 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 more sequence identity) to SEQ ID NO: 35.

[0319] E217. The polynucleotide of E216, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 35. E218. The polynucleotide of any one of E200 to E217, wherein the SLC26A4 enhancer is directly linked to (eg, fused to) the SLC26A4 promoter.

[0320] E219. The SLC26A4 enhancer is selected from the group consisting of 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500). The polynucleotide of any one of E200 to E217, wherein the polynucleotide is linked to the SLC26A4 promoter via a nucleic acid linker of about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides.

[0321] E220. The polynucleotide according to E219, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter via a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 nucleotides, for example, about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0322] E221. The polynucleotide of any one of E200 to E220, wherein the SLC26A4 enhancer is located 5' of the SLC26A4 promoter. E222. The polynucleotide of any one of E200 to E220, wherein the SLC26A4 enhancer is located 3' of the SLC26A4 promoter.

[0323] E223. The polynucleotide according to any one of E165 to E222, wherein the expression product is a heterologous expression product. E224. The polynucleotide according to any one of E165 to E222, wherein the expression product is an expression product endogenously expressed in an SLC26A4-expressing cell.

[0324] E225. The polynucleotide of E224, wherein the expression product is an expression product endogenously expressed in SLC26A4-expressing inner ear cells. E226. The polynucleotide of E225, wherein the expression product is endogenously expressed in (e.g., expressed in at least one of) interdental cells, spiral ridge cells, cochlear root cells, and / or vestibular supporting cells.

[0325] E227. The polynucleotide of any one of E224 to E226, wherein the expression product is pendrin (eg, a mammalian pendrin protein). E228. The polynucleotide of E227, wherein said pendrin (the mammalian pendrin protein) is the wild-type isoform endogenously expressed in the mammalian inner ear.

[0326] E229. The polynucleotide of any one of E227 or E228, wherein the pendrin (the mammalian pendrin protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 10 or SEQ ID NO: 11.

[0327] E230. The polynucleotide of E229, wherein the pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO: 10 or SEQ ID NO: 11. E231. The polynucleotide according to any one of E165 to E226, wherein the expression product is Atoh1 (eg, mammalian Atoh1).

[0328] E232. The polynucleotide of E231, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the mammalian inner ear.

[0329] E233. The polynucleotide of any one of E231 or E232, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 36 or SEQ ID NO: 38.

[0330] E234. The polynucleotide of E233, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO: 36 or SEQ ID NO: 38. E235. The polynucleotide described in any one of E165 to E223, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as Cas9, TALEN, or ZFN, or a gRNA), or a microRNA.

[0331] E236. The polynucleotide of any one of E200 to E235, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, each enhancer 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 more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35.

[0332] E237. The polynucleotide of E236, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NOs: 4-9, 34, and 35.

[0333] E238. The polynucleotide of E236, 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 more sequence identity) to 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 more sequence identity) to SEQ ID NO:7.

[0334] E239. The polynucleotide of 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. E240. The polynucleotide of E238 or E239, wherein the first and second enhancers are both located 5' to the SLC26A4 promoter.

[0335] E241. The polynucleotide of E240, wherein the first or second enhancer is directly fused to the other of the first or second enhancer, which is directly fused to the SLC26A4 promoter (i.e., the enhancer and promoter elements are directly linked to each other without any intervening nucleic acid).

[0336] E242. The polynucleotide according to E241, comprising, in 5' to 3' order, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, and the sequence of the SLC26A4 promoter (for example, a polynucleotide sequence consisting of the polynucleotide sequence of SEQ ID NO: 6-SEQ ID NO: 7 of the SLC26A4 promoter).

[0337] E243. The polynucleotide according to any one of E238 to E242, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 1. E244. The polynucleotide of any one of E200 to E237, 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 more sequence identity) to any one of SEQ ID NOs: 4 to 9, 34, and 35.

[0338] E245. The polynucleotide of E244, wherein 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.

[0339] E246. Polynucleotides (a) SLC26A4 promoter of formula 5'-BAC-3', 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 more sequence identity) to SEQ ID NO:1; 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 more 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; 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 more 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 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35, wherein the enhancer is directly or indirectly connected to a promoter ... an SLC26A4 enhancer linked via a nucleic acid linker of 0, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 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; The polynucleotide comprising:

[0340] E247. The polynucleotide of E246, wherein A has the sequence of SEQ ID NO:1. E248. The polynucleotide of E246 or E247, wherein B is absent. E249. The polynucleotide according to E246 or E247, wherein B has the sequence of SEQ ID NO: 2 or a part thereof comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2.

[0341] E250. The polynucleotide according to E249, wherein B has a sequence of a portion of SEQ ID NO: 2, comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2. E251. The polynucleotide of E250, wherein the portion of SEQ ID NO:2 is the first 307 contiguous nucleotides (set forth in SEQ ID NO:14) from the 3' end of SEQ ID NO:2.

[0342] E252. The polynucleotide of E249, wherein B has the sequence of SEQ ID NO:2. E253. The polynucleotide according to any one of E246 to E252, wherein C is absent.

[0343] E254. The polynucleotide of any one of E246 to 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.

[0344] E255. The polynucleotide of E254, wherein C has the sequence of a portion of SEQ ID NO:3, comprising at least the first 159 nucleotides of SEQ ID NO:3. E256. The polynucleotide of E255, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NOs:15, 16, 17, 18, and 19, respectively).

[0345] E257. The polynucleotide of E256, wherein the portion of SEQ ID NO:3 is the first 159 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:15). E258. The polynucleotide of E256, wherein the portion of SEQ ID NO:3 is the first 324 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:16).

[0346] E259. The polynucleotide of E256, wherein the portion of SEQ ID NO:3 is the first 341 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:17). E260. The polynucleotide of E256, wherein the portion of SEQ ID NO:3 is the first 716 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:18).

[0347] E261. The polynucleotide of E256, wherein the portion of SEQ ID NO:3 is the first 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:19). E262. The polynucleotide of E254, wherein C has the sequence of SEQ ID NO:3.

[0348] E263. The polynucleotide of any one of E246, E248, and E253, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0349] E264. The polynucleotide of E263, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E265. The polynucleotide of any one of E246, E247, E249, E252, and E253, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 20.

[0350] E266. The polynucleotide of E265, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 20. E267. The polynucleotide described in any one of E246 to E248, E254, and E262, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 21.

[0351] E268. The polynucleotide of E267, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 21. E269. The polynucleotide of any one of E246, E247, E249 to E251, and E253, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 22.

[0352] E270. The polynucleotide of E269, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 22. E271. The polynucleotide described in any one of E246 to E248, E254 to E256, and E259, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 23.

[0353] E272. The polynucleotide of E271, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 23. E273. The polynucleotide described in any one of E246 to E248, E254 to E256, and E261, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 24.

[0354] E274. The polynucleotide of E273, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 24. E275. The polynucleotide of any one of E246, E247, E249 to E251, E254 to E256, and E253, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 25.

[0355] E276. The polynucleotide of E275, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 25. E277. The polynucleotide of any one of E246, E247, E249 to E251, E254 to E256, and E258, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 26.

[0356] E278. The polynucleotide of E277, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 26. E279. The polynucleotide described in any one of E246, E247, E249 to E251, and E254 to E257, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 27.

[0357] E280. The polynucleotide of E279, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 27. E281. The polynucleotide described in any one of E246 to E280, wherein 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 more sequence identity) to SEQ ID NO:4.

[0358] E282. The polynucleotide of E281, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:4. E283. The polynucleotide described in any one of E246 to E280, wherein 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 more sequence identity) to SEQ ID NO:5.

[0359] E284. The polynucleotide of E283, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:5. E285. The polynucleotide described in any one of E246 to E280, wherein 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 more sequence identity) to SEQ ID NO: 6.

[0360] E286. The polynucleotide of E285, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:6. E287. The polynucleotide described in any one of E246 to E280, wherein 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 more sequence identity) to SEQ ID NO: 7.

[0361] E288. The polynucleotide of E287, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:7. E289. The polynucleotide described in any one of E246 to E280, wherein 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 more sequence identity) to SEQ ID NO:8.

[0362] E290. The polynucleotide of E289, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:8. E291. The polynucleotide described in any one of E246 to E280, wherein 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 more sequence identity) to SEQ ID NO:9.

[0363] E292. The polynucleotide of E291, wherein said SLC26A4 enhancer has the sequence of SEQ ID NO:9. E293. The polynucleotide of any one of E246 to E280, wherein 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 more sequence identity) to SEQ ID NO: 34.

[0364] E294. The polynucleotide of E293, wherein said SLC26A4 enhancer has the sequence of SEQ ID NO: 34. E295. The polynucleotide described in any one of E246 to E280, wherein 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 more sequence identity) to SEQ ID NO: 35.

[0365] E296. The polynucleotide of E295, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 35. E297. The polynucleotide of any one of E246 to E296, wherein the SLC26A4 enhancer is directly linked to (eg, fused to) the SLC26A4 promoter.

[0366] E298. The SLC26A4 enhancer is selected from the group consisting of 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500). The polynucleotide of any one of E246 to E296, wherein the polynucleotide is linked to the SLC26A4 promoter via a nucleic acid linker of about 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides.

[0367] E299. The polynucleotide according to E298, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter via a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 nucleotides, for example, about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0368] E300. The polynucleotide of any one of E246 to E299, wherein the SLC26A4 enhancer is located 5' of the SLC26A4 promoter. E301. The polynucleotide of any one of E246 to E299, wherein the SLC26A4 enhancer is located 3' of the SLC26A4 promoter.

[0369] E302. The polynucleotide of any one of E246 to E301, wherein the SLC26A4 promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0370] E303. The polynucleotide of E302, wherein the expression product is a heterologous expression product. E304. The polynucleotide according to E302, wherein the expression product is an expression product endogenously expressed in an SLC26A4-expressing cell.

[0371] E305. The polynucleotide of E304, wherein the expression product is an expression product endogenously expressed in SLC26A4-expressing inner ear cells. E306. The polynucleotide of E305, wherein the expression product is endogenously expressed in (e.g., expressed in at least one of) interdental cells, spiral ridge cells, cochlear root cells, and / or vestibular supporting cells.

[0372] E307. The polynucleotide of any one of E304 to E306, wherein the expression product is pendrin (eg, a mammalian pendrin protein). E308. The polynucleotide of E307, wherein said pendrin (the mammalian pendrin protein) is a wild-type isoform endogenously expressed in the mammalian inner ear.

[0373] E309. The polynucleotide of any one of E307 or E308, wherein the pendrin (the mammalian pendrin protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 10 or SEQ ID NO: 11.

[0374] E310. The polynucleotide of E309, wherein said pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO: 10 or SEQ ID NO: 11. E311. The polynucleotide according to any one of E302 to E306, wherein the expression product is Atoh1 (eg, mammalian Atoh1).

[0375] E312. The polynucleotide of E311, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the mammalian inner ear.

[0376] E313. The polynucleotide of any one of E311 or E312, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 36 or SEQ ID NO: 38.

[0377] E314. The polynucleotide of E313, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO: 36 or SEQ ID NO: 38. E315. The polynucleotide of E302 or E303, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as Cas9, TALEN, or ZFN, or a gRNA), or a microRNA.

[0378] E316. The polynucleotide of any one of E246 to E315, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, each enhancer 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 more sequence identity) to any one of SEQ ID NOs: 4-9, 34, and 35.

[0379] E317. The polynucleotide of E316, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NOs: 4-9, 34, and 35.

[0380] E318. The polynucleotide of 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 more sequence identity) to 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 more sequence identity) to SEQ ID NO: 7.

[0381] E319. The polynucleotide of 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. E320. The polynucleotide of E318 or E319, wherein the first and second enhancers are both located 5' to the SLC26A4 promoter.

[0382] E321. The polynucleotide of E320, wherein the first or second enhancer is directly fused to the other of the first or second enhancer, which is directly fused to the SLC26A4 promoter (i.e., the enhancer and promoter elements are directly linked to each other without any intervening nucleic acid).

[0383] E322. The polynucleotide according to E321, comprising, in 5' to 3' order, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, and the sequence of the SLC26A4 promoter (for example, a polynucleotide sequence consisting of the polynucleotide sequence of SEQ ID NO: 6-SEQ ID NO: 7 of the SLC26A4 promoter).

[0384] E323. The polynucleotide according to any one of E318 to E322, wherein the SLC26A4 promoter has the nucleotide sequence of SEQ ID NO: 1. E324. A polynucleotide described in any one of E246 to E317, 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 more sequence identity) to any one of SEQ ID NOs: 4 to 9, 34, and 35.

[0385] E325. The polynucleotide of E324, wherein 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.

[0386] E326. A nucleic acid vector comprising the polynucleotide according to any one of E83 to E325. E327. A nucleic acid vector comprising 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0387] E328. The nucleic acid vector of E327, wherein 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 more sequence identity) to SEQ ID NO:4.

[0388] E329. The nucleic acid vector of E328, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:4. E330. The nucleic acid vector of E327, wherein 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 more sequence identity) to SEQ ID NO:5.

[0389] E331. The nucleic acid vector of E330, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:5. E332. The nucleic acid vector of E327, wherein 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 more sequence identity) to SEQ ID NO:8.

[0390] E333. The nucleic acid vector of E52, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:8. E334. The nucleic acid vector of E327, wherein 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 more sequence identity) to SEQ ID NO:9.

[0391] E335. The nucleic acid vector of E334, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO:9. E336. The nucleic acid vector of E327, wherein 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 more sequence identity) to SEQ ID NO: 34.

[0392] E337. The nucleic acid vector of E336, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 34. E338. The nucleic acid vector of E327, wherein 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 more sequence identity) to SEQ ID NO: 35.

[0393] E339. The nucleic acid vector of E338, wherein the SLC26A4 enhancer has the sequence of SEQ ID NO: 35. E340. The nucleic acid vector according to any one of E327 to E339, wherein the SLC26A4 enhancer is operably linked to a promoter.

[0394] E341. The SLC26A4 enhancer is selected from the group consisting of 1 to 500 nucleotides (e.g., 1 to 50, 1 to 100, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500 , 250-500 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).

[0395] E342. The nucleic acid vector according to E341, wherein the SLC26A4 enhancer is linked to the promoter via a nucleic acid linker of 1 to 100 nucleotides (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 nucleotides, for example, about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0396] E343. The nucleic acid vector of E340, wherein the SLC26A4 enhancer is directly linked to (eg, fused to) the promoter. E344. The nucleic acid vector according to any one of E340 to E343, wherein the SLC26A4 enhancer is located 5' of the promoter.

[0397] E345. The nucleic acid vector according to any one of E340 to E343, wherein the SLC26A4 enhancer is located 3' of the promoter. E346. The nucleic acid vector according to any one of E340 to E345, wherein the promoter is a minimal promoter, a core promoter, or a constitutive promoter.

[0398] 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 phosphoglycerol kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, an HSV promoter, or an SV40 promoter.

[0399] E348. The nucleic acid vector of E347, wherein the promoter is a minimal β-globin promoter, a CMV mini 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.

[0400] E349. The nucleic acid vector of E346, wherein the promoter is a minimal promoter. E350. The nucleic acid vector according to any one of E340 to E345, wherein the promoter is a mammalian SLC26A4 promoter.

[0401] E351. The nucleic acid vector of E350, wherein the SLC26A4 promoter is a human or mouse SLC26A4 promoter. E352. The SLC26A4 promoter has the formula 5'-BAC-3', wherein: 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 more sequence identity) to SEQ ID NO:1; 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 more 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; 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 more sequence identity) to SEQ ID NO:3, or a portion thereof comprising at least the first 159 contiguous nucleotides of SEQ ID NO:3; The nucleic acid vector of E1350 or E351, wherein the SLC26A4 promoter is 1481 bases or less.

[0402] E353. The nucleic acid vector of E352, wherein A has the sequence of SEQ ID NO:1. E354. The nucleic acid vector of E352 or E353, wherein B is absent. E355. The nucleic acid vector according to E352 or E353, wherein B has the sequence of SEQ ID NO: 2 or a part thereof comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2.

[0403] E356. The nucleic acid vector according to E355, wherein B has a partial sequence of SEQ ID NO: 2, comprising 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO: 2. E357. The nucleic acid vector of E356, wherein the portion of SEQ ID NO:2 is the first 307 contiguous nucleotides (set forth in SEQ ID NO:14) from the 3' end of SEQ ID NO:2.

[0404] E358. The nucleic acid vector of E355, wherein B has the sequence of SEQ ID NO:2. E359. The nucleic acid vector according to any one of E352 to E358, wherein C is absent.

[0405] E360. The nucleic acid vector according to any one of E352 to 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.

[0406] E361. The nucleic acid vector of E360, wherein C has the sequence of a portion of SEQ ID NO:3, comprising at least the first 159 nucleotides of SEQ ID NO:3. E362. The nucleic acid vector of E361, wherein the portion of SEQ ID NO:3 is the first 159, 324, 341, 716, or 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NOs:15, 16, 17, 18, and 19, respectively).

[0407] E363. The nucleic acid vector of E362, wherein the portion of SEQ ID NO:3 is the first 159 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:15). E364. The nucleic acid vector of E362, wherein the portion of SEQ ID NO:3 is the first 324 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:16).

[0408] E365. The nucleic acid vector of E362, wherein the portion of SEQ ID NO:3 is the first 341 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:17). E366. The nucleic acid vector of E362, wherein the portion of SEQ ID NO:3 is the first 716 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:18).

[0409] E367. The nucleic acid vector of E362, wherein the portion of SEQ ID NO:3 is the first 723 contiguous nucleotides of SEQ ID NO:3 (set forth in SEQ ID NO:19). E368. The nucleic acid vector of E360, wherein C has the sequence of SEQ ID NO:3.

[0410] E369. The nucleic acid vector of any one of E352, E354, and E359, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0411] E370. The nucleic acid vector of E369, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E371. The nucleic acid vector of any one of E352, E353, E355, E358, and E359, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 20.

[0412] E372. The nucleic acid vector of E371, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 20. E373. The nucleic acid vector described in any one of E352 to E354, E360, and E368, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 21.

[0413] E374. The nucleic acid vector of E373, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 21. E375. The nucleic acid vector of any one of E352, E353, E355 to E357, and E359, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 22.

[0414] E376. The nucleic acid vector of E375, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 22. E377. The nucleic acid vector described in any one of E352 to E354, E360 to E362, and E365, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 23.

[0415] E378. The nucleic acid vector of E377, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 23. E379. The nucleic acid vector described in any one of E352 to E354, E360 to E362, and E367, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 24.

[0416] E380. The nucleic acid vector of E379, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 24. E381. The nucleic acid vector described in any one of E352, E353, E355 to E357, E360 to E362, and E364, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 25.

[0417] E382. The nucleic acid vector of E381, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 25. E383. The nucleic acid vector described in any one of E352, E353, E355 to E357, E360 to E362, and E364, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 26.

[0418] E384. The nucleic acid vector of E383, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 26. E385. The nucleic acid vector described in any one of E352, E353, E355 to E357, and E360 to E363, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 27.

[0419] E386. The nucleic acid vector of E385, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 27. E387. The nucleic acid vector of E350 or E351, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 28.

[0420] E388. The nucleic acid vector of E387, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 28. E389. The nucleic acid vector of E350 or E351, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 29-33.

[0421] E390. The nucleic acid vector according to E389, wherein the SLC26A4 promoter has any one of the sequences set forth in SEQ ID NOs: 29 to 33. E391. The nucleic acid vector of E350 or E351, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 41.

[0422] E392. The nucleic acid vector of E391, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 41. E393. The nucleic acid vector according to any one of E340 to E392, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0423] E394. The nucleic acid vector of E393, wherein the expression product is a heterologous expression product. E395. The nucleic acid vector according to E393, wherein the expression product is an expression product that is endogenously expressed in an SLC26A4-expressing cell.

[0424] E396. The nucleic acid vector of E395, wherein the expression product is an expression product that is endogenously expressed in SLC26A4-expressing inner ear cells. E397. The nucleic acid vector of E396, wherein the expression product is endogenously expressed in interdental cells, spiral ridge cells, cochlear root cells, and / or vestibular supporting cells (e.g., expressed in at least one of these cell types).

[0425] E398. The nucleic acid vector according to any one of E395 to E397, wherein the expression product is pendrin (eg, a mammalian pendrin protein). E399. The nucleic acid vector of E398, wherein said pendrin (the mammalian pendrin protein) is the wild-type isoform endogenously expressed in the mammalian inner ear.

[0426] E400. The nucleic acid vector of any one of E398 or E399, wherein the pendrin (the mammalian pendrin protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 10 or SEQ ID NO: 11.

[0427] E401. The nucleic acid vector of E400, wherein said pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO: 10 or SEQ ID NO: 11. E402. The nucleic acid vector according to any one of E393 to E397, wherein the expression product is Atoh1 (eg, mammalian Atoh1).

[0428] E403. The nucleic acid vector according to E402, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the inner ear of mammals. E404. The nucleic acid vector of any one of E402 or E403, wherein the Atoh1 (the mammalian Atoh1 protein) has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 36 or SEQ ID NO: 38.

[0429] E405. The nucleic acid vector of E404, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO: 36 or SEQ ID NO: 38. E406. The nucleic acid vector of E393 or E394, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0430] E407. The nucleic acid vector described in any one of E327 to E406, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, each enhancer 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0431] E408. The nucleic acid vector of E407, wherein each different SLC26A4 enhancer is independently selected from enhancers having the sequence of one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0432] E409. A nucleic acid vector described in any one of E327 to 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 more sequence identity) to any one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0433] E410. The nucleic acid vector of E409, wherein each copy of the two or more copies of the SLC26A4 enhancer has the sequence of one of SEQ ID NOs: 4, 5, 8, 9, 34, and 35.

[0434] E411. The nucleic acid vector according to any one of E1 to E82 and E326 to E410, wherein the nucleic acid vector is a viral vector, a plasmid, a cosmid, or an artificial chromosome.

[0435] E412. The nucleic acid vector according to any one of E1 to E82 and E326 to E411, wherein the nucleic acid vector is a viral vector. E413. The nucleic acid vector of E412, wherein the viral vector is an adeno-associated viral vector (AAV), an adenovirus viral vector, or a lentivirus viral vector.

[0436] E414. The nucleic acid vector according to E413, wherein the viral vector is an AAV vector. E415. The nucleic acid vector of E414, wherein the AAV vector has a capsid of AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S.

[0437] E416. A composition comprising the nucleic acid vector according to any one of E1 to E81 and E326 to E415, and a pharmaceutically acceptable carrier, diluent, or excipient. E417. A cell comprising the polynucleotide according to any one of E83 to E325, or the nucleic acid vector according to any one of E1 to E82 and E326 to E415.

[0438] E418. The cell according to E417, wherein the cell is an SLC26A4-expressing cell. E419. The cell according to E418, wherein the cell is an SLC26A4-expressing inner ear cell. E420. The cell according to any one of E417 to E419, wherein the cell is a mammalian cell.

[0439] E421. The cell according to E420, wherein the mammalian cell is a human cell. E422. The cell according to any one of E417 to E431, wherein the cell is an interdental cell, a spiral ridge cell, a cochlear root cell, or a vestibular supporting cell.

[0440] E423. A method for expressing an expression product in a cell, the method comprising contacting the cell with a nucleic acid vector described in any one of E1 to E82 and E326 to E415, or a composition described in E416.

[0441] E424. The method according to E423, wherein the cells are inner ear cells. E425. The method of E173 or E174, wherein the cell is an SLC26A4-expressing cell.

[0442] E426. The method according to any one of E423 to E425, wherein the cells are SLC26A4-expressing inner ear cells. E427. The method of E426, wherein the SLC26A4-expressing inner ear cells are interdental cells, spiral eminence cells, cochlear root cells, or vestibular supporting cells.

[0443] E428. The method according to any one of E423 to E427, wherein the cell is a mammalian cell. E429. The method according to E428, wherein the mammalian cells are human cells.

[0444] E430. The method according to any one of E423 to E429, wherein the contacting is performed inside a subject (eg, in vivo). E431. A method of treating a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss), comprising administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector described in any one of E1 to E82 and E326 to E415 or a composition described in E416.

[0445] E432. The method according to E431, wherein the hearing loss is pendrin-associated hearing loss. E433. The method according to E432, wherein the expression product is pendrin. E434. The method of E432 or E433, wherein the pendrin-associated hearing loss is Pendred syndrome or DFNB4-associated hearing loss.

[0446] E435. A method for treating hearing loss associated with Meniere's disease in a subject in need thereof, comprising administering to the inner ear of the subject an effective amount of a nucleic acid vector described in any one of E1 to E82 and E326 to E415 or a composition described in E416.

[0447] E436. A method for treating hearing loss associated with Meniere's disease in a subject in need thereof, comprising administering to the inner ear of the subject an effective amount of a nucleic acid vector described in any one of E1 to E82 and E326 to E415 or a composition described in E416.

[0448] E437. The method according to E435 or E436, wherein the expression product is pendrin. E438. A method for treating hearing loss associated with Meniere's disease in a subject in need of treatment for vestibular dysfunction associated with Meniere's disease, comprising administering to the inner ear of the subject an effective amount of a nucleic acid vector described in any one of E1 to E82 and E326 to E415 or a composition described in E416.

[0449] E439. The method according to E438, wherein the expression product is pendrin or Atoh1. E440. The method according to E438 or E439, wherein the vestibular dysfunction is vertigo.

[0450] E441. A method for treating a subject having or at risk of developing a vestibular dysfunction, comprising administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector described in any one of E1 to E182 and E326 to E415 or a composition described in E416.

[0451] E442. The method according to E441, wherein the vestibular dysfunction is pendrin-associated vestibular dysfunction. E443. The method according to E442, wherein the expression product is pendrin.

[0452] E444. The method according to E442 or E443, wherein the pendrin-associated vestibular dysfunction is vestibular dysfunction associated with Pendred syndrome or DFNB4. E445. The method according to E441, wherein the expression product is pendrin or Atoh1.

[0453] E446. A method for inducing or increasing differentiation of vestibular supporting cells into vestibular hair cells, the method comprising the step of contacting the vestibular supporting cells with the nucleic acid vector described in any one of E1 to E82 and E326 to E415 or the composition described in E416.

[0454] E447. The method according to E446, wherein the expression product is Atoh1. E448. The method according to E446 or E447, wherein the contacting is performed in vivo (eg, inside a subject).

[0455] E449. The method of E448, wherein the subject has or is at risk of developing vestibular dysfunction. E450. A method for inducing or increasing vestibular hair cell regeneration in a subject in need thereof, comprising administering to the inner ear of said subject an effective amount of a nucleic acid vector described in any one of E1 to E82 and E326 to E415 or a composition described in E416.

[0456] E451. The method according to E450, wherein said expression product is pendrin or Atoh1. E452. The method of E450 or E451, wherein the subject has or is at risk of developing vestibular dysfunction.

[0457] E453. A method for improving the function of SLC26A4-expressing cells, comprising the step of contacting the SLC26A4-expressing cells with the nucleic acid vector described in any one of E1 to E182 and E326 to E415 or the composition described in E416.

[0458] E454. The method according to E453, wherein the contacting is performed in vivo (eg, inside a subject). E455. The method of E454, wherein the subject has or is at risk of developing hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction.

[0459] E456. The method according to any one of E441 to E445, wherein the vestibular dysfunction is vertigo, dizziness, imbalance (e.g., balance disorder or equilibrium disorder), oscillopsia, or bilateral vestibular dysfunction.

[0460] E457. The method according to any one of E441 to E456, wherein the vestibular dysfunction is associated with damage or loss of vestibular hair cells. E458. The method of E457, wherein the damage or loss of vestibular hair cells is associated with aging (the vestibular dysfunction is age-related vestibular dysfunction), exposure to an ototoxic (e.g., vestibulotoxic) drug (the vestibular dysfunction is ototoxic drug-induced vestibular dysfunction), disease or infection (the vestibular dysfunction is disease- or infection-associated vestibular dysfunction), or head trauma (the vestibular dysfunction is head trauma-associated vestibular dysfunction).

[0461] E459. The method of E458, wherein the ototoxic drug is an aminoglycoside (aminoglycoside antibiotics, e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin, and netilmicin), viomycin, antineoplastic drug (e.g., platinum-containing chemotherapeutic agents such as cisplatin, carboplatin, or oxaliplatin, or other chemotherapeutic agents such as nitrogen mustard or vincristine), a loop diuretic (e.g., ethacrynic acid or furosemide), a salicylate, or quinine.

[0462] E460. The method of any one of E431 to E437, further comprising assessing the hearing of the subject prior to administration of the nucleic acid vector or the composition. E461. The method of any one of E431 to E437 and E460, further comprising assessing the hearing of the subject after administration of the nucleic acid vector or the composition.

[0463] E462. The method of any one of E438 to E459, further comprising assessing the vestibular function of the subject prior to administration of the nucleic acid vector or the composition. E463. The method according to any one of E438 to E459 and E462, further comprising assessing the vestibular function of the subject after administration of the nucleic acid vector or the composition.

[0464] E464. The method according to any one of E423 to E463, wherein the nucleic acid vector or the composition is administered locally. E465. The method of E464, wherein the nucleic acid vector or the composition is administered to the inner ear.

[0465] E466. The method of E464, wherein the nucleic acid vector or the composition is administered to the middle ear. E467. The method according to E464, wherein the nucleic acid vector or the composition is administered transtympanically or intratympanically.

[0466] E468. The method of E464, wherein the nucleic acid vector or the composition is administered into the perilymph. E469. The method of E464, wherein the nucleic acid vector or the composition is administered into the endolymph.

[0467] E470. The method of E464, wherein the nucleic acid vector or the composition is administered to or through the oval window. E471. The method of E464, wherein the nucleic acid vector or the composition is administered to or through the round window.

[0468] E472. The method according to E464, wherein the nucleic acid vector or the composition is administered to a semicircular canal. E473. The method of any one of E423 to E472, wherein the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, delay the onset of hearing loss, slow the progression of hearing loss, improve hearing, increase or induce expression of an expression product in SLC26A4-expressing cells, 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 aural fullness, increase vestibular hair cell regeneration, induce or increase differentiation of vestibular supporting cells into vestibular hair cells, increase or induce hair cell maturation (e.g., maturation of regenerated hair cells), or improve vestibular supporting cell function.

[0469] E474. The method according to any one of E423 to E473, wherein the subject is a human subject. E475. A cell comprising the polynucleotide according to any one of E83 to E325, the nucleic acid vector according to any one of E1 to E82 and E326 to E415, or the composition according to E416.

[0470] Other embodiments Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unnecessarily limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. Other embodiments are within the scope of the claims.

Claims

1. 1. A nucleic acid vector comprising an SLC26A4 promoter of the formula 5′-BAC-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 from the 3' end of SEQ ID NO:2; 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; The diffusion vector, wherein the SLC26A4 promoter is 1,481 bases or less.

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

1.

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

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

2.

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

2.

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

2.

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

2.

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

9. 8. The nucleic acid vector of claim 1, 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. 10. The nucleic acid vector of claim 9, wherein C has the sequence of a portion of SEQ ID NO:3, comprising at least the first 159 nucleotides of SEQ ID NO:

3.

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

3.

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

3.

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

14. The nucleic acid vector of claim 13, wherein the SLC26A4 enhancer has at least 85% sequence identity with any one of SEQ ID NOs: 4-7, 34, and 35.

15. The nucleic acid vector of claim 14, wherein the SLC26A4 enhancer has the sequence of any one of SEQ ID NOs: 4 to 7, 34, and 35.

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

17. The nucleic acid vector according to any one of claims 13 to 15, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter via 1 to 500 nucleic acid linkers.

18. The nucleic acid vector of claim 17, wherein the SLC26A4 enhancer is linked to the SLC26A4 promoter via 1 to 100 nucleic acid linkers.

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

20. The nucleic acid vector of any one of claims 13 to 19, wherein the vector comprises two or more different SLC26A4 enhancers, each SLC26A4 enhancer independently selected from enhancers having at least 85% sequence identity with any one of SEQ ID NOs: 4 to 7, 34, and 35.

21. 21. The nucleic acid vector of claim 20, 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.

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

7.

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

25. The nucleic acid vector of claim 24, comprising, in 5'→3' order, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

1.

26. The nucleic acid vector of any one of claims 13 to 21, wherein the vector comprises two or more copies of an SLC26A4 enhancer having at least 85% sequence identity to any one of SEQ ID NOs: 4 to 7, 34, and 35.

27. 27. The nucleic acid vector of claim 26, wherein each copy of the two or more copies of the SLC26A4 enhancer has the sequence of any one of SEQ ID NOs: 4 to 7, 34, and 35.

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

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

30. 29. The nucleic acid vector of claim 28, wherein the expression product is an expression product that is endogenously expressed in SLC26A4-expressing inner ear cells.

31. 31. The nucleic acid vector of claim 30, wherein the SLC26A4-expressing inner ear cell is an interdental cell, a spiral ridge cell, a cochlear root cell, or a vestibular supporting cell.

32. 32. The nucleic acid vector of any one of claims 28, 30, and 31, wherein the expression product is a mammalian pendrin protein.

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

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

11.

35. 32. The nucleic acid vector of any one of claims 28, 30, and 31, wherein the expression product is a 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, optionally 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.

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

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

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

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

4.

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

5.

42. A polynucleotide comprising: a. SLC26A4 promoter of the formula 5'-BAC-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 from the 3' end of SEQ ID NO:2; 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. The polynucleotide comprising 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 linked to the promoter directly or via a nucleic acid linker of 1 to 500 nucleotides.

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

1.

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

45. B has the sequence of SEQ ID NO:2 or a portion thereof including 1 to 917 consecutive nucleotides from the 3' end of SEQ ID NO:

2.

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

2.

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

2.

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

2.

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

50. 49. The polynucleotide of any one of claims 42 to 48, 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.

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

3.

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

3.

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

3.

54. 50. The polynucleotide of claim 49, wherein B is absent and C is absent.

55. The polynucleotide of any one of claims 42 to 54, wherein the SLC26A4 promoter is 1,481 bases or less.

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

4.

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

5.

58. 56. The polynucleotide of any one of claims 42 to 55, wherein the polynucleotide comprises two or more different SLC26A4 enhancers, each SLC26A4 enhancer independently selected from enhancers having at least 85% sequence identity to any one of SEQ ID NOs: 4 to 7, 34, and 35.

59. 59. The polynucleotide of 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. 59. The polynucleotide of claim 58, 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. 61. The nucleic acid vector of 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. 62. The nucleic acid vector of claim 61, wherein the first or second enhancer is directly fused to the other of the first or second enhancer, which is directly fused to the SLC26A4 promoter.

63. 63. The nucleic acid vector of claim 62, comprising, in 5'→3' order, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

1.

64. 64. The polynucleotide of any one of claims 36 to 63, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

65. 65. The polynucleotide of claim 64, wherein the expression product is a heterologous expression product.

66. 65. The polynucleotide of claim 64, wherein the expression product is an expression product endogenously expressed in SLC26A4-expressing inner ear cells.

67. 67. The polynucleotide of claim 66, wherein the SLC26A4-expressing inner ear cell is an interdental cell, a spiral eminence cell, a cochlear root cell, or a vestibular supporting cell.

68. 68. The polynucleotide of any one of claims 64, 66, and 67, wherein the expression product is a mammalian pendrin protein.

69. 69. The polynucleotide of claim 68, wherein the mammalian pendrin protein is the wild-type isoform endogenously expressed in the mammalian ear.

70. 70. The polynucleotide of claim 69, wherein the mammalian pendrin protein has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:

11.

71. 68. The polynucleotide of any one of claims 64 to 67, wherein the expression product is a mammalian Atoh1 protein.

72. A nucleic acid vector comprising the polynucleotide of any one of claims 36 to 71.

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

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

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

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

77. 71. The method of claim 70, wherein the contacting occurs internally in the subject.

78. A method for treating a subject having or at risk of developing pendrin-associated hearing loss, comprising administering to the subject a therapeutically effective amount of a nucleic acid vector described in claims 1 to 35 and 72, wherein the expression product is pendrin.

79. 73. The method of claim 72, wherein the pendrin-associated hearing loss is Pendred syndrome or DFNB4.

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

81. A method for treating a subject having or at risk of developing a pendrin-associated vestibular dysfunction, comprising administering to the subject a therapeutically effective amount of a nucleic acid vector described in claims 1 to 35 and 72, wherein the expression product is pendrin.

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

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

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