SLC26A4 regulatory elements and uses thereof

Abstract

The present disclosure provides an SL26A4 enhancer and an SLC26A4 promoter, as well as vectors containing them, that can increase gene expression in SLC26A4-expressing 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 disorder.

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 created on July 31, 2023, is named 51124-098WO3_Sequence_Listing_7_31_23 and is 33,576 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 the 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 sequence encoding a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) or at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) operably linked to a promoter. 9% or more sequence identity), wherein the distance between the enhancer and promoter in the polynucleotide is less than 3 kilobases (3 kb) (e.g., about 3.0 kb, 2.75 kb, 2.5 kb, 2.25 kb, 2.0 kb, 1.75 kb, 1.55 kb, 1.25 kb, 1.0 kb, 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases, 100 bases, 50 bases, or less). In some embodiments, the distance between the enhancer and promoter in the polynucleotide is less than 2 kb. In some embodiments, the distance between the enhancer and promoter in the polynucleotide is less than 1 kb. In some embodiments, the distance between the enhancer and promoter in the polynucleotide is less than 0.5 kb. In some embodiments, the distance between the enhancer and the promoter in the polynucleotide is less than 100 bases.

[0005] In another aspect, the present invention provides a nucleic acid vector comprising a polynucleotide according to the first aspect. In another aspect, the invention provides a nucleic acid vector comprising a polynucleotide comprising an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) or at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0006] In some embodiments of any of the above aspects, the enhancer is operably linked to a promoter. In some embodiments, the promoter is a minimal promoter, a core promoter, or a constitutive promoter. In some embodiments, the promoter is a CAG promoter, a CBA promoter, a smCBA promoter, a CASI promoter, a dihydrofolate reductase (DHFR) promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, 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. In some embodiments, the promoter is a mammalian SLC26A4 promoter. In some embodiments, the SLC26A4 promoter is a human or mouse SLC26A4 promoter. In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 1. In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 12-16. In some embodiments, the SLC26A4 promoter has the sequence of any one of SEQ ID NOs: 12-16. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO:12.In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 13. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 14. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 15. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 16. In some embodiments, the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 17.

[0007] In another aspect, the invention provides a nucleic acid vector containing a polynucleotide comprising 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 SEQ ID NO: 1. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 1.

[0008] In another aspect, the invention provides a polynucleotide comprising 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 SEQ ID NO: 1 operably linked to a polynucleotide that can be transcribed to produce an expression product. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 1.

[0009] In another aspect, the invention provides a nucleic acid vector containing a polynucleotide comprising 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 SEQ ID NO: 17. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 17.

[0010] In another aspect, the invention provides a polynucleotide comprising 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 SEQ ID NO: 17 operably linked to a polynucleotide that can be transcribed to produce an expression product. In some embodiments, the SLC26A4 promoter has the sequence of SEQ ID NO: 17.

[0011] In some embodiments of any of the aforementioned aspects, the SLC26A4 promoter is operably linked to an enhancer. In some embodiments, the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0012] In some embodiments of any of the aforementioned aspects, the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2. In some embodiments of any of the aforementioned aspects, the enhancer has the sequence of SEQ ID NO: 2.

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

[0014] In some embodiments of any of the aforementioned aspects, the enhancer is located 5' of the promoter of the polynucleotide. In some embodiments of any of the aforementioned aspects, the enhancer is located 3' of the promoter of the polynucleotide. In some embodiments of any of the aforementioned aspects, the enhancer is fused directly to the promoter. In some embodiments of any of the aforementioned aspects, the enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleic acids).

[0015] In some embodiments of any of the aforementioned aspects, the promoter is operably linked to a polynucleotide that can be transcribed to generate an expression product. In some embodiments of any of the aforementioned aspects, the expression product is a heterologous expression product. In some embodiments of any of the aforementioned aspects, the expression product is an expression product endogenously expressed in SLC26A4-expressing cells. In some embodiments, the expression product is an expression product endogenously expressed in SLC26A4-expressing inner ear cells. In some embodiments, the expression product is an expression product endogenously expressed in interdental cells, spiral eminence cells, cochlear root cells, and / or vestibular supporting cells (e.g., expressed in at least one of these cell types). In some embodiments of any of the aforementioned aspects, the expression product is pendrin (e.g., mammalian pendrin). In some embodiments, pendrin (e.g., mammalian pendrin) is the wild-type isoform endogenously expressed in the mammalian inner ear. In some embodiments, pendrin has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, pendrin has the sequence of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments of any of the foregoing aspects, the expression product is Atoh1 (e.g., mammalian Atoh1). In some embodiments, Atoh1 (e.g., mammalian Atoh1) is the wild-type isoform endogenously expressed in the mammalian inner ear. In some embodiments, Atoh1 has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:8 or SEQ ID NO:10. In some embodiments, Atoh1 has the sequence of SEQ ID NO:8 or SEQ ID NO:10.In some embodiments of any of the foregoing aspects, the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0016] In some embodiments of any of the aforementioned aspects, the polynucleotide comprises an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2 and an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 3. In some embodiments, the polynucleotide comprises an enhancer having the sequence of SEQ ID NO: 2 and an enhancer having the sequence of SEQ ID NO: 3. In some embodiments of any of the aforementioned aspects, the polynucleotide comprises, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and a promoter having the sequence of SEQ ID NO: 17. In some more specific embodiments of any of the aforementioned aspects, the polynucleotide contains the sequence of SEQ ID NO: 18.

[0017] In some embodiments of any of the aforementioned aspects, the polynucleotide contains two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and / or two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) (e.g., two or more copies of one or both enhancers). In some embodiments, the polynucleotide contains two or more copies of an enhancer having the sequence of SEQ ID NO:2 and / or SEQ ID NO:3 (eg, two or more copies of one or both enhancers).

[0018] 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 virus (AAV), an adenovirus, or a lentivirus. 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.

[0019] 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.

[0020] 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.

[0021] 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).

[0022] 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 preceding aspects and embodiments. In some embodiments, the hearing loss is pendrin-associated hearing loss. In some embodiments, the expression product is pendrin. In some embodiments, the pendrin-associated hearing loss is hearing loss associated with Pendred syndrome or DFNB4.

[0023] 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 subject's inner ear an effective amount of a nucleic acid vector or composition of any of the preceding aspects and embodiments, hi some embodiments, the expression product is pendrin.

[0024] 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 preceding aspects and embodiments, hi some embodiments, the expression product is pendrin.

[0025] 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, the expression product is pendrin or Atoh1. In some embodiments, the vestibular dysfunction is vertigo.

[0026] 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 foregoing aspects and embodiments. In some embodiments, the vestibular dysfunction is a pendrin-associated vestibular dysfunction. In some embodiments, the expression product is pendrin. In some embodiments, the pendrin-associated vestibular dysfunction is a vestibular dysfunction associated with Pendred syndrome or DFNB4. In some embodiments, the expression product is pendrin or Atoh1. In some embodiments, the vestibular dysfunction is vertigo, dizziness, imbalance (e.g., balance disorder or equilibrium disorder), oscillopia, or bilateral vestibular dysfunction. In some embodiments, 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 an ototoxic (e.g., vestibulotoxic) drug (i.e., the vestibular dysfunction is ototoxic drug-induced vestibular dysfunction), disease or infection (i.e., the vestibular dysfunction is disease- or infection-associated vestibular dysfunction), or head trauma (i.e., the vestibular dysfunction is head trauma-associated 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.

[0027] 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 of any of the preceding aspects and embodiments. In some embodiments, the expression product is 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.

[0028] 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 of any of the preceding aspects and embodiments. In some embodiments, the expression product is 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] In some embodiments of any of the aforementioned aspects, the nucleic acid vector or composition is administered in an amount sufficient to prevent or 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).

[0034] 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.

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

[0036] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., a nucleic acid vector containing an SLC26A4 enhancer and / or an SLC26A4 promoter) to a subject by any effective route. Exemplary administration routes are described herein below.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] [Table 1]

[0041] 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).

[0042] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein refer to an amount sufficient to 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.

[0043] 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).

[0044] As used herein, the term "express" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., 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.

[0045] 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 human cell, e.g., a cell such as a human cochlear supporting cell). Exogenous material includes materials provided from an external source to an organism or culture extracted therefrom.

[0046] 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.

[0047] As used herein, the terms "increasing" and "decreasing" refer to modulating a metric's function, expression, or activity 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 indicator is measured after administration, at a time when the administration has had the described effect, for example, at least 1 week, 1 month, 3 months, or 6 months after the start of the treatment regimen.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] As used herein, the term "polynucleotide" refers to a polymer of nucleosides. Typically, polynucleotides are composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. The term encompasses molecules containing nucleosides or nucleoside analogs 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 application refers to a polynucleotide, it is understood that both DNA, RNA, and in each case, both single- and double-stranded forms (and the complement of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or sequence information (i.e., a series of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in a 5' to 3' orientation unless otherwise indicated.

[0052] 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 reference 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 reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for 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 the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full 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.

[0053] 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., The term also refers 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 99.9% identity or greater) to 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).

[0054] 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 can control gene expression in SLC26A4-expressing cells. SLC26A4 enhancers for use in the compositions and methods described herein have at least 85% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or greater). The SLC26A4 enhancers described herein can be operably linked to a promoter that is operably linked to a polynucleotide encoding an expression product to increase the expression level of the expression product in SLC26A4-expressing cells and / or increase the number of SLC26A4-expressing cells in which the expression product is expressed.

[0055] 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 or enhancer promoter provided in Table 3). SLC26A4 promoters 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 SEQ ID NO: 1, at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17, 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), 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: 12-16.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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).

[0063] As used herein, the terms "subject" and "patient" refer to an animal (e.g., a mammal such as a human). A subject treated in accordance with 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 of skill in the art will understand that a subject treated in accordance with the present disclosure may have undergone standard testing or may have been identified as at risk without testing due to the presence of one or more risk factors associated with the disease or condition.

[0064] 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).

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 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 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.

[0069] 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]

[0070] [Figure 1] 1 is a plasmid map of plasmid P1236. [Figure 2] 1 is a plasmid map of plasmid P1240. [Figure 3A] A series of images showing the inner ear tropism of AAV1 containing a green fluorescent protein (GFP) transgene driven by a ubiquitous promoter (CMV). Sections of the mouse inner ear cochlea and vestibular apparatus showing expression of the enhanced green fluorescent protein (EGFP) transgene. [Figure 3B] A series of images showing the inner ear tropism of AAV1 containing a green fluorescent protein (GFP) transgene driven by a ubiquitous promoter (CMV). AAV1 tropism, as determined by EGFP expression in the cochlea, includes, but is not limited to, spiral ganglion neurons (SGNs), Reissner's membrane (RM), fibrocytes (F), spiral eminence cells (SP), and root cells (RC). [Figure 3C] A series of images showing the inner ear tropism of AAV1 containing a green fluorescent protein (GFP) transgene driven by a ubiquitous promoter (CMV). AAV1 tropism in the vestibular system includes, but is not limited to, hair cells (HCs), supporting cells (SCs), mesenchymal cells (MCs), and cells that make up the roof of the otolith organs. [Figure 4] A series of images showing the lack of EGFP transgene expression in inner ear organs when driven by the mouse SLC26A4 promoter of SEQ ID NO: 1 without an enhancer sequence. Sections of the mouse cochlea and vestibular organs showed no EGFP expression (A and C, respectively). No EGFP expression was observed in sections of the mouse cochlea (shown as a square in A and at higher resolution in B) or vestibular organs (shown as a rectangle in C and at higher resolution in D). Scale bar: 100 μm. [Figure 5-1]A series of images showing restriction of EGFP transgene expression to specific cell types in the inner ear when driven by the mouse E2 enhancer element of SEQ ID NO:2 directly fused to the mouse SLC26A4 promoter of SEQ ID NO:1. Sections (top and bottom rows) of mouse cochlea and vestibular apparatus from two different animals showing EGFP expression. A low-magnification view of the section from animal #1 is shown in A. A high-magnification view of the left-most rectangle is shown in B. The top rectangle is shown in C. The right-most rectangle is shown in D. A low-magnification view of the section from animal #2 is shown in E. A high-magnification view of the top left rectangle is shown in F. The top right rectangle is shown in G. The bottom rectangle is shown in H. EGFP expression was restricted to interdental cells (ID) (B), spiral eminence cells (SP) (D, F, and G), and root cells (RC) (G) in the cochlea of both animals. EGFP expression was restricted to supporting cells (SC) within the vestibular otolithic apparatus (H). Scale bar: 100 μm. [Figure 5-2] Same as above. [Figure 6] A series of images showing restriction of EGFP transgene expression to specific cell types in the inner ear when driven by the mouse E6 enhancer element of SEQ ID NO: 3 directly fused to the mouse SLC26A4 promoter of SEQ ID NO: 1. Sections of mouse inner ear and vestibular organ showing EGFP expression (A) and their corresponding higher magnification images (top rectangle shown in B, bottom rectangle shown in C). EGFP expression is observed in the spiral prominence (SP) and root cells (RC) of the cochlea (B). Weak EGFP expression was observed in supporting cells (SC) of the vestibular organ (C). Scale bar: 100 μm. [Figure 7] 1 is a plasmid map of plasmid P1669. [Figure 8] 1 is a plasmid map of plasmid P1670. [Figure 9]Figure 1 shows a series of images assessing pendrin and EGFP expression. (A) Images of whole-mount views of mouse lateral wall explants stained with an antibody specific for pendrin. Different layers of the lateral wall are shown in the images. (B) Images of whole-mount views of mouse lateral wall transduced with an AAV vector expressing EGFP under the control of the ubiquitous CMV promoter and stained with an antibody specific for pendrin. Images showing pendrin staining alone, GFP alone, and the combination of pendrin and GFP are shown. [Figure 10] This is a series of images showing whole-mount views of mouse lateral wall explants transduced with an AAV1 viral vector expressing EGFP under the control of the mouse minimal SLC26A4 promoter and both the mouse E2 and E6 enhancers (A), the mouse core SLC26A4 promoter and both the mouse E2 and E6 enhancers (B), or the mouse core SLC26A4 promoter and only the mouse E2 enhancer (C), counterstained with an antibody specific for pendrin. The top row of each figure shows both pendrin and GFP. The second row shows only pendrin staining in that micrograph. The third row shows only GFP. The fourth row shows only GFP, with gain adjusted to better visualize GFP in C. [Figure 11-1]

[0033] Figure 7 is a series of different magnifications of the same field showing EGFP expression in the cochlea after in vivo administration of an AAV1 vector (plasmid P1669, see Figure 7) containing an EGFP expression cassette encoding the E2 enhancer (SEQ ID NO: 2) directly fused to the 5' end of the E6 enhancer (SEQ ID NO: 3) directly fused to the 5' end of the mouse SLC26A4 minimal promoter (SEQ ID NO: 17) to pendrin knockout mice. Panel A is a photomicrograph at the lowest magnification. The area indicated by the box labeled "B" in panel A is shown at higher magnification in panel B. The boxes labeled "C" and "D" in panel B are shown at higher magnification in panels C and D, respectively. [Figure 11-2] Same as above. [Figure 12]7 is a photomicrograph showing EGFP expression in the cochlea after in vivo administration of an AAV1 vector (plasmid P1669, FIG. 7) containing an EGFP expression cassette encoding the E2 enhancer (SEQ ID NO: 2) directly fused to the 5′ end of the E6 enhancer (SEQ ID NO: 3) directly fused to the 5′ end of the mouse SLC26A4 minimal promoter (SEQ ID NO: 17) into wild-type non-human primates. Arrows point to cells expressing nuclear EGFP. DETAILED DESCRIPTION OF THE INVENTION

[0071] 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 enhancers that can be operably linked to a promoter to induce transgene expression in SLC26A4-expressing cells (e.g., SLC26A4-expressing inner ear cells). SLC26A4 enhancers can also increase gene expression levels and cell numbers of SLC26A4-expressing cells in which gene expression can be detected. In some embodiments, SLC26A4 enhancers can reduce or minimize off-target expression in non-SLC26A4-expressing cells (e.g., when operably linked to a constitutive promoter). Thus, an SLC26A4 enhancer can be operably linked to a promoter, which in turn is 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 SLC26A4 promoters that can be used to induce expression of an operably linked expression product specifically in SLC26A4-expressing cells. One or more of the SLC26A4 enhancers described herein can be operably linked to the SLC26A4 promoters described herein.The invention also features nucleic acid vectors containing one or more SLC26A4 enhancers 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 3) operably linked to a polynucleotide encoding an expression product, and nucleic acid vectors containing an SLC26A4 promoter (e.g., an SLC26A4 promoter having at least 85% sequence identity to SEQ ID NO: 1) operably linked to a polynucleotide encoding an expression product, as well as methods of using these vectors to treat hearing loss (e.g., pendrin-associated hearing loss), vestibular dysfunction (e.g., imbalance or loss of balance associated with pendrin-associated vestibular dysfunction or loss of vestibular hair cells), or Meniere's disease.

[0072] 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.

[0073] 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.

[0074] The present invention is based, in part, on the discovery of SLC26A4 enhancers and promoters that can be used to induce gene expression in SLC26A4-expressing cells while minimizing off-target expression in non-SLC26A4-expressing cells. The SLC26A4 enhancer can be operably linked to a promoter, such as the SLC26A4 promoter, which in turn can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding pendrin). The SLC26A4 enhancers and promoters 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). The 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 in SLC26A4-expressing cells (e.g., a protein, such as pendrin or another protein, endogenously expressed in SLC26A4-expressing cells, or an RNA molecule, such as an inhibitory RNA molecule) to treat a subject with or at risk of developing hearing loss (e.g., sensorineural hearing loss) or hearing loss (e.g., pendrin-associated hearing loss), a subject with or at risk of developing vestibular dysfunction (e.g., pendrin-associated vestibular dysfunction or vestibular dysfunction associated with loss of vestibular hair cells), or a subject with Meniere's disease. The discovery of SLC26A4 enhancers and promoters that can improve cell-type-specific expression can improve the safety and efficacy of gene therapy by reducing toxicity resulting from off-target expression.

[0075] The compositions and methods described herein may include a SLC26A4 enhancer listed in Table 2 (e.g., SEQ ID NO:2 or SEQ ID NO:3) or a variant thereof, e.g., a polynucleotide sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, an SLC26A4 enhancer for use in the compositions and methods described herein has the sequence of SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, compositions described herein contain two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) SLC26A4 enhancers, which may have the same sequence (e.g., multiple copies of the same SLC26A4 enhancer) or different sequences (e.g., one or more copies of each of the SLC26A4 enhancers listed in Table 2). In some embodiments, compositions described herein contain one of each of the SLC26A4 enhancers listed in Table 2 (e.g., a single copy of SEQ ID NO:2 and a single copy of SEQ ID NO:3). In some embodiments, compositions described herein contain multiple copies of SEQ ID NO:2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies) or multiple copies of SEQ ID NO:3 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies). In some embodiments, the compositions described herein contain a single copy of SEQ ID NO:2 and multiple copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies) of SEQ ID NO:3, or a single copy of SEQ ID NO:3 and multiple copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies) of SEQ ID NO:2. In some embodiments, the compositions described herein contain multiple copies of both SEQ ID NO:2 and SEQ ID NO:3 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of each enhancer).In embodiments in which the composition contains two or more enhancers (e.g., one or more copies of each of SEQ ID NO:2 and SEQ ID NO:3), the enhancers may be included in any order, may be positioned immediately adjacent to each other (e.g., linked without any intervening sequence between the enhancer sequences, e.g., the 3' end of a first enhancer is positioned immediately before the 5' end of a second enhancer), or may be joined by a nucleic acid linker (e.g., a nucleic acid linker is positioned between each enhancer sequence included in the composition, or between at least two of the enhancer sequences in the composition).

[0076] Exemplary SLC26A4 enhancer sequences are listed in Table 2.

[0077] [Table 2]

[0078] The SLC26A4 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 promoter of SEQ ID NO: 1 or SEQ ID NO: 17), which 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 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). According to the methods described herein, a composition containing one or more of the above-mentioned polynucleotides (e.g., 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 SEQ ID NO: 2 or SEQ ID NO: 3) operably linked to a promoter operably linked to a polynucleotide encoding an expression product can be administered to a subject. The 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).

[0079] The compositions and methods described herein may also include an SLC26A4 promoter. In some embodiments, the SLC26A4 promoter is operably linked to one or more of the aforementioned enhancer sequences (e.g., SEQ ID NO: 2, SEQ ID NO: 3, or both). The SLC26A4 promoter may be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding pendrin, a polynucleotide encoding a protein or RNA molecule endogenously expressed in SLC26A4-expressing cells, 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. Exemplary SLC26A4 promoters are provided in Table 3 below. In some embodiments, an SLC26A4 promoter for use in the compositions and methods described herein has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 1. In some embodiments, an SLC26A4 promoter for use in the compositions and methods described herein has the sequence of SEQ ID NO: 1. In some embodiments, an SLC26A4 promoter for use in the compositions and methods described herein has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 17. In some embodiments, the SLC26A4 promoter for use in the compositions and methods described herein has the sequence of SEQ ID NO:17.

[0080] [Table 3-1]

[0081]

Table 3-2

[0082]

Table 3-3

[0083]

Table 3-4

[0084]

Table 3-5

[0085] In some embodiments, a polynucleotide encoding wild-type pendrin, or a variant thereof, e.g., a polynucleotide sequence encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) pendrin (e.g., SEQ ID NO: 4 or SEQ ID NO: 5), is selected from the group consisting of the SLC26A4 promoter described herein (e.g., a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 17, or a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 17). For example, a promoter having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or SEQ ID NO:3) or a promoter (e.g., an SLC26A4 promoter such as an SLC26A4 promoter or enhancer-promoter provided in Table 3, a minimal promoter, a core promoter, or a constitutive promoter) operably linked to one or more SLC26A4 enhancers described herein (e.g., one or more copies of an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or SEQ ID NO:3). In some embodiments, a polynucleotide encoding wild-type pendrin, or a variant thereof, is operably linked to a polynucleotide containing, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and a promoter having the sequence of SEQ ID NO: 17.In some more specific embodiments, the polynucleotide encoding wild-type pendrin, or a variant thereof, is operably linked to the sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide sequence encoding the 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: 4 or SEQ ID NO: 5, 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, the polynucleotide sequence encoding pendrin is any polynucleotide sequence that, due to redundancy in the genetic code, encodes SEQ ID NO: 4 or SEQ ID NO: 5. The polynucleotide sequence encoding pendrin may be partially or completely codon-optimized for expression (e.g., in human SLC26A4-expressing inner ear cells). Pendrin proteins may also be encoded by polynucleotides containing single nucleotide polymorphisms (SNPs) known to be non-pathogenic in human subjects (e.g., SNPs that do not result in hearing loss). Human pendrin may be encoded by a polynucleotide having the sequence of SEQ ID NO: 6. Mouse pendrin may be encoded by a polynucleotide having the sequence of SEQ ID NO: 7. The pendrin protein may be a homolog of the human pendrin protein or a human pendrin 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 in Table 4 below.A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer described herein operably linked to a promoter (e.g., an SLC26A4 promoter, e.g., an SLC26A4 promoter or an enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 3) operably linked to a polynucleotide encoding pendrin, or a nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 promoter described herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 17) operably linked to a polynucleotide encoding 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).

[0086] [Table 4-1]

[0087] [Table 4-2]

[0088] [Table 4-3]

[0089] [Table 4-4]

[0090] Expression of exogenous polynucleotides in mammalian cells The compositions and methods described herein can be used to treat a variety of diseases, including: the development of a leukemia, autism, or autism spectrum disorders, by administering a nucleic acid vector containing at least one SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) operably linked to a promoter (e.g., an SLC26A4 promoter, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 3) operably linked to a polynucleotide encoding an expression product (e.g., a protein or RNA molecule of interest, e.g., an inhibitory RNA), or SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 1, or at least 85% 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. Expression of an exogenous polynucleotide (e.g., a gene endogenously expressed in SLC26A4-expressing cells, such as a polynucleotide encoding pendrin) can be induced or increased 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) by administering a nucleic acid vector containing an exogenous polynucleotide (e.g., a polynucleotide having a sequence identity (7%, 98%, 99%, or more) to an SLC26A4-expressing cell, such as interdental cells, root cells, spiral ridge cells, and vestibular supporting cells). In some embodiments, the nucleic acid vector contains, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO:2, an enhancer having the sequence of SEQ ID NO:3, and a promoter having the sequence of SEQ ID NO:17. In some embodiments, the nucleic acid vector contains the sequence of SEQ ID NO:18.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.

[0091] 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: 2 or SEQ ID NO: 3) 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 3), or (For example, a nucleic acid vector containing a polynucleotide 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) or a polynucleotide having at least 85% sequence identity to SEQ ID NO: 17 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity)) can be used to express the polynucleotide in one or more SLC26A4-expressing cells (e.g., SLC26A4-expressing inner ear cells). In some embodiments, the nucleic acid vector contains, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and a promoter having the sequence of SEQ ID NO: 17. In some embodiments, the nucleic acid vector contains the sequence of SEQ ID NO: 18.Exemplary polynucleotides that can be expressed using the nucleic acid vectors described herein include polynucleotides encoding proteins expressed in healthy SLC26A4-expressing cells, such as 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).

[0092] In some embodiments, a polynucleotide encoding wild-type Atoh1, or a variant thereof, e.g., a polynucleotide sequence encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) Atoh1 (e.g., SEQ ID NO: 8 or SEQ ID NO: 10), is used in combination with the SLC26A4 promoter described herein (e.g., a promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 17, or a polynucleotide sequence encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 17). For example, a promoter having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or SEQ ID NO:3) or a promoter (e.g., an SLC26A4 promoter such as an SLC26A4 promoter or enhancer-promoter provided in Table 3, a minimal promoter, a core promoter, or a constitutive promoter) operably linked to one or more SLC26A4 enhancers described herein (e.g., one or more copies of an enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 and / or SEQ ID NO:3). In some embodiments, a polynucleotide encoding wild-type Atoh1, or a variant thereof, is operably linked to a polynucleotide containing, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and a promoter having the sequence of SEQ ID NO: 17.In some embodiments, a polynucleotide encoding wild-type Atoh1, or a variant thereof, is operably linked to a polynucleotide having the sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide sequence encoding the Atoh1 protein encodes an amino acid sequence containing one or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more conservative amino acid substitutions) relative to SEQ ID NO: 4, provided that the encoded Atoh1 analog retains the therapeutic function of wild-type Atoh1 (e.g., the ability to promote hair cell proliferation). Not more than 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: 8 or SEQ ID NO: 10 due to redundancy in the genetic code. The polynucleotide sequence encoding Atoh1 can be partially or completely codon-optimized for expression (e.g., in human SLC26A4-expressing inner ear cells, e.g., 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:9. Mouse Atoh1 can be encoded by a polynucleotide having the sequence of SEQ ID NO:11. 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.A nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 enhancer described herein operably linked to a promoter (e.g., an SLC26A4 promoter, e.g., an SLC26A4 promoter or enhancer-promoter, minimal promoter, core promoter, or constitutive promoter provided in Table 3) operably linked to a polynucleotide encoding Atoh1, or a nucleic acid vector (e.g., an AAV vector) containing an SLC26A4 promoter described herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 17) operably linked to a polynucleotide encoding Atoh1, can be administered to a subject to treat, alleviate, or prevent vestibular dysfunction associated with damage or loss of vestibular hair cells (e.g., imbalance or loss of balance, dizziness, or vertigo) (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).

[0093] [Table 5-1]

[0094] [Table 5-2]

[0095] 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 through 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 concatamer formation in the nucleus of the mammalian cell). A gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein. To introduce an exogenous gene into mammalian cells, the gene can be incorporated into a vector. Vectors can be introduced into cells by various methods, including transformation, transfection, transduction, direct uptake, particle bombardment, and encapsulation of the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in further detail, for example, in Green, et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014); and Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0096] 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.

[0097] 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., the SLC26A4 promoter or enhancer-promoter provided in Table 3), 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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 cells of interest. Mammalian cells, such as human cells, exposed to an external electric field in this manner are then susceptible to the 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.

[0102] Further techniques useful for transfection of 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.

[0103] 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 can be associated with polynucleotides to confer a positive charge favoring interaction with cell membranes include activated dendrimers (e.g., as described in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Another useful tool for inducing the uptake of exogenous polynucleotide by target cells 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 and allow 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.

[0108] 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 the site-specific cleavage of endogenous polynucleotide sequences, thereby preparing the genome of the cell for covalent integration of a target polynucleotide, 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], Methylation changes in early embryonic genes in cancer [abstract], Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0109] 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 include at least one SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 2 or SEQ ID NO: 3), and / or an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 2 or SEQ ID NO: 3), operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein of interest or a polynucleotide that can be transcribed to produce an RNA molecule such as an inhibitory RNA). For example, a promoter 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) or a promoter having at least 85% sequence identity to SEQ ID NO:17 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), and additional sequences 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 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.

[0110] 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.

[0111] 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. rAAV vectors useful in the compositions and methods described herein include: (1) a promoter (e.g., the SLC26A4 promoter, 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) to SEQ ID NO: 17; or (2) 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) to SEQ ID NO: 17; A recombinant polynucleotide construct includes (1) a promoter having 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a promoter of a gene encoding a gene encoding a gene of interest (e.g., a promoter having 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a promoter of ... of interest (e.g., a promoter having 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a promoter of a gene of interest (e.g., a promoter having 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a promoter of a gene of interest (e.g., a promoter having 8%, 89%, In some embodiments, the rAAV vector further comprises at least one SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3). In some embodiments, rAAV vectors useful in the compositions and methods described herein contain, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO:2, an enhancer having the sequence of SEQ ID NO:3, and a promoter having the sequence of SEQ ID NO:17. In some embodiments, the rAAV vector contains the sequence of SEQ ID NO:18.In a typical application, the 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, such as Atoh1, that can promote the differentiation of vestibular supporting cells into vestibular hair cells. Such rAAV vectors may also contain a marker or reporter gene. Useful rAAV vectors lack one or more AAV WT genes, in whole or in part, but retain functional flanking ITR sequences. AAV ITRs can be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs can 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.

[0112] 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.

[0113] 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.

[0114] 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).

[0115] 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).

[0116] Pharmaceutical Composition The SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2 or SEQ ID NO: 3) and / or the SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1), or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 1) A polynucleotide having at least 85% sequence identity to SEQ ID NO:17 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) can be operably linked 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) 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) or vestibular dysfunction (e.g., vestibular dysfunction associated with damage or loss of vestibular hair cells, or pendrin-associated vestibular dysfunction). Pharmaceutical compositions containing vectors, such as viral vectors, containing the SLC26A4 enhancer and / or SLC26A4 promoter described herein operably linked to a polynucleotide encoding an expression product can be prepared using methods known in the art. For example, such compositions can be prepared in a desired form, such as a lyophilized formulation or aqueous solution, using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013), incorporated herein by reference).

[0117]

[0039] 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 SEQ ID NO: 2 or SEQ ID NO: 3) and / or an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 1) described herein operably linked to a polynucleotide encoding an expression product. Mixtures of nucleic acid vectors containing polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) can be prepared in water, suitably mixed with one or more excipients, carriers, or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. These preparations may contain a preservative to prevent the growth of microorganisms under ordinary conditions of storage and use. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In any case, the formulation may be sterile and may have fluidity to the extent that easy syringability exists. The formulation may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, 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 dispersions, and by the use of surfactants.Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0118] 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 vehicles will be known to those skilled in the art in light of this disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous fluid or injected at the intended injection site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition may be formulated to contain a synthetic perilymph solution. An exemplary synthetic perilymph solution contains 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl, 1-10 mM glucose, and 2-50 mM HEPES, with a pH of about 6-9 and an osmolality of about 300 mOsm / kg. The individual responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations can meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0119] 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 of administration in any given case will vary depending on the particular composition being administered, the patient, the pharmaceutical formulation method, the method of administration (e.g., time and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. The 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).

[0120] 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 a SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) operably linked to a polynucleotide encoding pendrin (e.g., a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5), and / or a SLC26A4 promoter (e.g., a polynucleotide encoding SEQ ID NO:5). Treatment can be by administering a nucleic acid vector containing a polynucleotide 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) to SEQ ID NO: 17, or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17. In some embodiments, the nucleic acid vector contains, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and a promoter having the sequence of SEQ ID NO: 17 operably linked to a polynucleotide encoding pendrin. In some embodiments, the nucleic acid vector contains a polynucleotide having the sequence of SEQ ID NO: 18 operably linked to a polynucleotide encoding pendrin. The subject may have or be identified as having a mutation in SLC26A4 and may have severe, moderate, or mild hearing loss when treatment is initiated, or may be treated before the onset of symptoms (e.g., prophylactic treatment).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.

[0121] In some embodiments, the compositions described herein are used to treat a subject with Meniere's disease. Both subjects with a mutation in SLC26A4 and subjects with Meniere's disease have endolymphatic hydrops, and therefore, compositions that can be used to treat a subject with a mutation in SLC26A4 include, for example, an SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) operably linked to a polynucleotide encoding pendrin (e.g., a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5), and / or A nucleic acid vector containing an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17 (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) may also be effective in reducing or ameliorating 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.

[0122] 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 treated by administering to a subject a SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3) operably linked to a polynucleotide encoding pendrin (e.g., a polynucleotide encoding SEQ ID NO:4 or SEQ ID NO:5), and / or an SLC26A4 promoter (e.g., a polynucleotide encoding SEQ ID NO:1) or a nucleic acid 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 SEQ ID NO: 17. In some embodiments, the subject may have, or may have been identified as having, a mutation in SLC26A4 and may have severe, moderate, or mild vestibular dysfunction when treatment is initiated, or may be treated prior to the onset of symptoms (e.g., prophylactic treatment).

[0123] 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.

[0124] 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.

[0125] 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).

[0126] Vestibular dysfunction associated with vestibular hair cell damage or loss (e.g., damage or loss of vestibular hair cells associated with disease or infection, head trauma, ototoxic drugs (e.g., vestibulotoxic drugs), or aging-related vestibular hair cell damage or loss) can be treated by administering to a subject a SLC26A4 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110, 111, 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, 16 The treatment can be by administering a nucleic acid 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 SEQ ID NO: 1, and / or a nucleic acid vector containing an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17). In some embodiments, the nucleic acid vector contains, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and a promoter having the sequence of SEQ ID NO: 17 operably linked to a polynucleotide encoding Atoh1 or pendrin. In some embodiments, the nucleic acid vector contains a polynucleotide having the sequence of SEQ ID NO: 18 operably linked to a polynucleotide encoding Atoh1 or pendrin. Such nucleic acid vectors can also induce or increase vestibular hair cell regeneration or vestibular hair cell maturation in a subject in need thereof.

[0127] 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 a 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.

[0128] For the treatment of a subject described herein, a polynucleotide having an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 1, or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 17, a polynucleotide encoding an expression product operably linked to a SLC26A4 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or SEQ ID NO:3). can be a polynucleotide encoding pendrin (e.g., a polynucleotide encoding the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5), a polynucleotide encoding Atoh1 (e.g., a polynucleotide encoding the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:10), 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.In some embodiments, the expression product is operably linked to a polynucleotide sequence comprising, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and a promoter having the sequence of SEQ ID NO: 17. In some embodiments, the expression product is operably linked to a polynucleotide having the sequence of SEQ ID NO: 18. The polynucleotide can be selected based on the cause of the subject's hearing loss or vestibular dysfunction (e.g., if the subject's hearing loss is associated with a mutation in SLC26A4, the polynucleotide can encode wild-type pendrin, or if the subject's vestibular dysfunction is age-related or ototoxic drug-induced vestibular dysfunction associated with hair cell loss, the polynucleotide can encode Atoh1), the severity of the subject's hearing loss, the health of the subject's inner ear cells, the subject's age, the subject's family history of hearing loss, or other factors.

[0129] 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 9 VG / mL, 2 × 10 9 VG / mL, 3 x 10 9 VG / mL, 4 x 10 9 VG / mL, 5 × 10 9 VG / mL, 6 × 10 9 VG / mL, 7 × 10 9 VG / mL, 8 x 10 9 VG / mL, 9 x 10 9 VG / mL, 1 × 10 10 VG / mL, 2 × 10 10 VG / mL, 3 x 10 10VG / 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、9×10 13 VG / mL、1×10 14 VG / mL、2×10 14 VG / mL、3×10 14 VG / mL、4×10 14 VG / mL、5×10 14 VG / mL、6×10 14 VG / mL、7×10 14 VG / mL、8×10 14 VG / mL、9×10 14 VG / mL、1×10 15 VG / mL、2×1015 VG / mL, 3 x 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, 4×10 9 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 10VG / 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 14 VG / 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).

[0130] 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 can 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 taken 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.

[0131] kit The compositions described herein can be provided in a kit for use in treating sensorineural hearing loss or vestibular dysfunction. The compositions may contain 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 SEQ ID NO:2 or SEQ ID NO:3) and / or SLC26A4 promoters (e.g., polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:17, as described herein. The SLC26A4 enhancer and / or promoter may comprise a polynucleotide having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to 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 is expressed in SLC26A4-expressing inner ear cells and can 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]

[0132] 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.

[0133] Example 1. Demonstration of AAV1-CMV.H2B.EGFP tropism in whole mouse inner ear tissue To test the hypothesis that AAV1 transduces a wide range of cell types throughout the inner ear, including those that do not express pendrin, AAV1 virus was injected into adult C57BL / 6 mice via the posterior semicircular canal (intracochlear (IL)) at 7.19 × 10 10 The AAV1 virus was delivered at a dose of 3.59 x 10 vg / ear. 13 The virus was packaged into a plasmid containing an expression cassette encoding a cytomegalovirus (CMV) promoter driving the expression of nuclear-targeted enhanced green fluorescent protein (EGFP fused to the H2B fragment of the histone 2b gene) at a titer of 10 ...

[0134] Example 2. Expression of an EGFP transgene is significantly reduced in mouse inner ear tissue under the control of the mouse SLC26A4 core promoter In vivo experiments were performed to assess whether AAV1 EGFP transgene expression could be restricted to SLC26A4-expressing cells in the cochlea and vestibule by replacing the ubiquitous CMV promoter with the mouse SLC26A4 core promoter. To test this, a plasmid containing an expression cassette encoding the mouse SLC26A4 core promoter (SEQ ID NO: 1) without any enhancer sequence driving nuclear-targeted EGFP expression was transfected into 6.28 × 10 cells. 13 The resulting AAV virus was packaged into AAV1 at a titer of 6.28 × 10 vg / mL and injected into adult C57BL / 6 mice via the posterior semicircular canal (IL). 10 The virus was administered topically at a dose of 1000 mg / ear. Two weeks after virus administration, whole ears were fixed, decalcified, paraffin-embedded, and sections were imaged for EGFP using a fluorescence microscope. EGFP fluorescence was not detected in any cell type in the cochlea (Figures 4A-4D) or vestibule (data not shown). The fluorescence observed in the wider fields of view in Figures 4A and 4C is unrelated to EGFP expression and is the result of naturally fluorescent structures within the mouse ear observed when the display light intensity was significantly increased, as this was done to detect EGFP-specific staining.

[0135] Example 3. Addition of an enhancer to the mouse SLC26A4 core promoter resulted in increased expression of an EGFP transgene in target cell types within the cochlea and vestibule To test whether the combination of the mouse SLC26A4 core promoter and enhancer element(s) increases EGFP expression in target cell types within both the cochlea and vestibule, plasmids containing expression cassettes encoding the E2 enhancer (SEQ ID NO:2) directly fused to the 5' end of the mouse SLC26A4 core promoter (SEQ ID NO:1) (plasmid P1240, Figure 2) or the E6 enhancer (SEQ ID NO:3) directly fused to the 5' end of the mouse SLC26A4 core promoter (SEQ ID NO:1) (plasmid P1236, Figure 1) driving nuclear-targeted EGFP expression were each transfected into 7.12 x 10 cells. 13 and 6.68 x 10 13Each of these AAV1 vectors was separately packaged into AAV1 at a titer of 7.12 × 10 10 and 6.68 x 10 10 AAV1 containing the E2 enhancer was administered topically via the posterior semicircular canal (IL) to different adult C57BL / 6 mice at a dose of 1000 mg / ear. Two weeks after viral administration, whole ears were fixed, decalcified, paraffin-embedded, and sections were imaged for EGFP using a fluorescence microscope. In ears treated with AAV1 containing the E2 enhancer, EGFP fluorescence was detected in the nuclei of the interdental cells (ID), spiral eminence cells (SP), and root cells (RC) of the cochlea (Figures 5A-5G), as well as the supporting cells of the vestibular otolith (Figures 5E and 5H). In ears treated with AAV1 containing the E6 enhancer, EGFP fluorescence was detected in the nuclei of the spiral eminence cells (SP) and root cells (RC) of the cochlea (Figures 6A-6B). Weak EGFP fluorescence signals were detected in the supporting cells (SC) of the vestibular otolith (Figures 6A and 6C).

[0136] Example 4. The presence of multiple enhancers in AAV1 vectors increases SLC26A4 promoter-driven GFP expression in mouse cochlear lateral wall explants without affecting cell specificity Lateral walls were excised from the cochleae of 6-8 week-old male C57BL / 6J mice (000664, The Jackson Laboratory) and cultured with AAV1 viral vectors derived from transgene plasmids containing nuclear-targeted H2B-EGFP fusion transgenes driven by various promoters (CMV and various enhancer-less SLC26A4 promoters described herein), as well as AAV1 vectors containing H2B-EGFP fusion transgenes driven by the mouse core SLC26A4 promoter (SEQ ID NO: 1) and both the mouse E2 enhancer (SEQ ID NO: 2) and the mouse E6 enhancer (SEQ ID NO: 3) (P1670, Figure 8), or the mouse minimal SLC26A4 promoter (SEQ ID NO: 17) and both the mouse E2 enhancer and the mouse E6 enhancer (P1669, Figure 7). 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 walls were washed with 2 ml of fresh medium prepared as described above. The cultured lateral walls were then maintained in culture for an additional 2 days (a total of 5 days of culture after AAV addition).

[0137] 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).

[0138] After mounting, the lateral walls were 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 the imaging settings, all groups within a study were imaged at the same laser power and gain to allow comparisons between groups.

[0139] As can be seen in Figure 9A, most SLC26A4-expressing cells are located in the tight junctions of the spiral prominence 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 SLC26A4-expressing region, demonstrating the ubiquitous AAV1 targeting in the lateral wall (Figure 9B). Replacing the CMV promoter with a mouse core or minimal promoter and combining it with the E2 and / or E6 enhancers disclosed herein resulted in EGFP expression being restricted primarily to cells that also expressed pendrin, with little or no EGFP expression in other cells of the lateral wall (Figures 10A-C).

[0140] By including both the mouse E6 and E2 SLC26A4 enhancer sequences fused to the 5' end of either the minimal or core mouse promoter in an AAV vector expressing EGFP, EGFP was expressed without losing specificity for SLC26A4-expressing cells (Fig. 10A and 10B). Interestingly, when both the mouse E2 and mouse E6 enhancers were fused to either the mouse minimal promoter (Fig. 10A) or the mouse core promoter (Fig. 10B), both resulted in stronger specific expression of EGFP than when only the mouse E2 enhancer was fused to the mouse core promoter (Fig. 10C).

[0141] Example 5. In vivo administration of AAV1 vectors containing multiple enhancers increases SLC26A4 promoter-driven GFP expression in the mouse cochlea To test whether combining both the E2 and E6 enhancers would further increase EGFP expression in vivo as shown in Example 3, plasmids containing an EGFP expression cassette encoding the E2 enhancer (SEQ ID NO: 2) directly fused to the 5' end of the E6 enhancer (SEQ ID NO: 3) directly fused to the 5' end of either the mouse SLC26A4 core promoter (SEQ ID NO: 1) (plasmid P1670, Figure 8) or the mouse SLC26A4 minimal promoter (SEQ ID NO: 17) (plasmid P1669, Figure 7) were transfected with 4.41 x 10 13(Lot 1), 2.70 x 10 13 (Lot 2), and 4.60 x 10 13 The virus was packaged into AAV1 at a titer of 1000 vg / mL.

[0142] Lot 2 of the AAV1 vector containing the minimal SLC26A4 promoter containing an expression cassette encoding the E2 enhancer (SEQ ID NO: 2) and E6 enhancer (SEQ ID NO: 3) was injected bilaterally into P1-P3-aged pendrin knockout mice via the posterior semicircular canal (IL), at a dose of 1.5x10 10 The pendrin KO mice were locally administered at a dose of 1000 mg / ear. These pendrin KO mice were generated from mice with a C57BL / 6 background by CRISPR / Cas9-mediated deletion of exons 3–5 of the SLC26A4 gene on mouse chromosome 12. At age P21, whole ears were fixed, decalcified, paraffin-embedded, and 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 AAV1 containing the E2 and E6 enhancers linked to a minimal promoter, EGFP fluorescence was detected in the nuclei of the interdental cells (ID), spiral eminence cells (SP), and root cells (RC) of the cochlea (Figure 11).

[0143] Example 6. In vivo administration of AAV1 vectors containing multiple SLC26A4 enhancers and SLC26A4 promoters induces GFP expression in SLC26A4-expressing cells in the cochlea of non-human primates A plasmid (plasmid P1669) containing an EGFP expression cassette encoding the E2 enhancer (SEQ ID NO: 2) directly fused to the 5' end of the E6 enhancer (SEQ ID NO: 3) directly fused to the 5' end of the mouse SLC26A4 minimal promoter (SEQ ID NO: 17) was packaged into AAV. The virus was administered topically to 2-4 year-old cynomolgus monkeys (Macaca fascicularis). Administration was performed bilaterally by first creating a fenestration in the PSCC to allow fluid outflow, followed by delivery of 60 μL / ear of virus through the round window membrane. Two weeks after birth, animals were perfused, and whole ears were harvested, decalcified, paraffin-embedded, and sections were imaged for EGFP using a fluorescence microscope. In ears treated with AAV1 containing the E2 and E6 enhancers linked to a minimal promoter, EGFP fluorescence was detected in the nuclei of the spiral eminence cells (SP), root cells (RC), and outer sulcus cells of the cochlea (Figure 12).

[0144] Example 7. Administration of a composition containing a nucleic acid vector containing an SLC26A4 enhancer and an SLC26A4 promoter 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-related hearing loss such as DFNB4 or Pendred syndrome) to improve or restore hearing. To this end, a practitioner of skill in the art will recognize an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:1, or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:17) operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a wild-type version of pendrin, e.g., SEQ ID NO:4 or SEQ ID NO:5). A vector containing at least one linked SLC26A4 enhancer described herein (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 SEQ ID NO:2 and / or SEQ ID NO:3). A composition containing an AV vector (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vector) can be administered to a human patient. The 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.

[0145] After administering the composition to the 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.

[0146] Example 8. Administration of a composition containing a nucleic acid vector containing an SLC26A4 enhancer and an SLC26A4 promoter 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, having 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 will recognize an SLC26A4 promoter operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a wild-type form of Atoh1, e.g., a polynucleotide encoding SEQ ID NO:8 or SEQ ID NO:10) (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:17). and at least one SLC26A4 enhancer described herein (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 SEQ ID NO:2 and / or SEQ ID NO:3) operably linked to A composition containing an AAV vector (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vector) 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.

[0147] 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.

[0148] Example 9. Administration of a composition containing a nucleic acid vector containing an SLC26A4 enhancer and an SLC26A4 promoter 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 a feeling of fullness in the ears. To this end, a practitioner of skill in the art will recognize an SLC26A4 promoter (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:1, or a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:17) operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a wild-type version of pendrin, e.g., SEQ ID NO:4 or SEQ ID NO:5). A vector containing at least one linked SLC26A4 enhancer described herein (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 SEQ ID NO:2 and / or SEQ ID NO:3). The composition containing AV vector (for example, 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) can be administered to human patients.The composition containing AAV vector can be administered to patients by, for example, local administration to the inner ear (for example, injection into the perilymph or endolymph, injection through the round window membrane or injection into the semicircular canal) to treat Meniere's disease.

[0149] 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 a feeling of fullness in the ears. 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.

[0150] Exemplary embodiments of the present invention are described in the following listed paragraphs. E1. A nucleic acid sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or at least 85% 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, operably linked to a promoter. a polynucleotide comprising an enhancer having a sequence similar to that of a promoter, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 3 kilobases (3 kb) (e.g., about 3.0 kb, 2.75 kb, 2.5 kb, 2.25 kb, 2.0 kb, 1.75 kb, 1.55 kb, 1.25 kb, 1.0 kb, 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases, 100 bases, 50 bases, or less).

[0151] E2. The polynucleotide of E1, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 2 kb. E3. The polynucleotide of E2, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 1 kb.

[0152] E4. The polynucleotide of E3, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 0.5 kb. E5. The polynucleotide according to E4, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 100 bases.

[0153] E6. The polynucleotide according to any one of E1 to E5, wherein the enhancer is located 5' of the promoter in the polynucleotide. E7. The polynucleotide according to any one of E1 to E5, wherein the enhancer is located 3' of the promoter in the polynucleotide.

[0154] E8. The polynucleotide of any one of E1 to E7, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0155] E9. The polynucleotide of E8, wherein the enhancer has the sequence of SEQ ID NO:2. E10. The polynucleotide of any one of E1 to E7, wherein the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0156] E11. The polynucleotide of E10, wherein the enhancer has the sequence of SEQ ID NO:3. E12. The polynucleotide of any one of E1 to E11, wherein the enhancer is fused directly to the promoter.

[0157] E13. The polynucleotide of any one of E1 to E11, wherein the enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleic acids).

[0158] E14. The polynucleotide according to any one of E1 to E13, wherein the promoter is a minimal promoter, a core promoter, or a constitutive promoter. E15. The polynucleotide of E14, wherein the promoter is a CAG promoter, a CBA promoter, a smCBA promoter, a CASI promoter, a dihydrofolate reductase (DHFR) promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, an HSV promoter, or an SV40 promoter.

[0159] E16. The polynucleotide of E15, 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.

[0160] E17. The polynucleotide according to any one of E1 to E13, wherein the promoter is a minimal promoter. E18. The polynucleotide according to any one of E1 to E13, wherein the promoter is a mammalian SLC26A4 promoter.

[0161] E19. The polynucleotide of E18, wherein the SLC26A4 promoter is a human or mouse SLC26A4 promoter. E20. The polynucleotide of E19, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0162] E21. The polynucleotide of E20, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E22. The polynucleotide of E19, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 12 to 16.

[0163] E23. The polynucleotide according to E22, wherein the SLC26A4 promoter has any one of the sequences set forth in SEQ ID NOs: 12 to 16. E24. The polynucleotide of E19, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 17.

[0164] E25. The polynucleotide of E24, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 17. E26. The polynucleotide of any one of E1 to E25, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0165] E27. The polynucleotide of E26, wherein the expression product is a heterologous expression product. E28. The polynucleotide according to E26, wherein the expression product is an expression product endogenously expressed in an SLC26A4-expressing cell.

[0166] E29. The polynucleotide of E28, wherein the expression product is an expression product endogenously expressed in SLC26A4-expressing inner ear cells. E30. The polynucleotide of E29, 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).

[0167] E31. The polynucleotide of any one of E28 to E30, wherein the expression product is pendrin (eg, a mammalian pendrin protein). E32. The polynucleotide of E31, wherein said pendrin (the mammalian pendrin protein) is a wild-type isoform endogenously expressed in the inner ear of mammals.

[0168] E33. The polynucleotide of any one of E31 or E32, 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:4 or SEQ ID NO:5.

[0169] E34. The polynucleotide of E33, wherein the pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. E35. The polynucleotide according to any one of E27 to E30, wherein the expression product is Atoh1 (eg, mammalian Atoh1).

[0170] E36. The polynucleotide of E35, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the inner ear of mammals. E37. The polynucleotide of any one of E35 or E36, 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:8 or SEQ ID NO:10.

[0171] E38. The polynucleotide of E37, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO:8 or SEQ ID NO:10. E39. The polynucleotide of E26 or E27, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0172] E40. The polynucleotide of any one of E1 to E39, comprising an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0173] E41. The polynucleotide of E40, comprising an enhancer having the sequence of SEQ ID NO:2 and an enhancer having the sequence of SEQ ID NO:3. E42. The polynucleotide of E41, comprising, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 17 (e.g., an enhancer having the sequence of SEQ ID NO: 2 directly fused to an enhancer having the sequence of SEQ ID NO: 3 directly fused to an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 17).

[0174] E43. The polynucleotide of E42, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 17. E44. The polynucleotide of E42 or E43, comprising the sequence of SEQ ID NO:18.

[0175] E45. The polynucleotide of any one of E1-E41, comprising two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and / or two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) (e.g., two or more copies of one or both enhancers).

[0176] E46. The polynucleotide of E45, comprising two or more copies of an enhancer having the sequence of SEQ ID NO:2 and / or SEQ ID NO:3 (eg, two or more copies of one or both enhancers).

[0177] E47. A nucleic acid vector comprising the polynucleotide according to any one of E1 to E46. E48. A nucleic acid vector comprising a polynucleotide comprising an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) or at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0178] E49. The nucleic acid vector of E48, wherein the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2.

[0179] E50. The nucleic acid vector of E49, wherein the enhancer has the sequence of SEQ ID NO:2. E51. The nucleic acid vector of E48, wherein the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0180] E52. The nucleic acid vector of E51, wherein the enhancer has the sequence of SEQ ID NO:3. E53. The nucleic acid vector according to any one of E48 to E52, wherein the enhancer is operably linked to a promoter.

[0181] E54. The nucleic acid vector of E53, wherein the enhancer is located 5' of the promoter. E55. The nucleic acid vector of E53, wherein the enhancer is located 3' of the promoter.

[0182] E56. The nucleic acid vector according to any one of E53 to E55, wherein the enhancer is fused directly to the promoter. E57. The nucleic acid vector of any one of E53 to E55, wherein the enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleic acids).

[0183] E58. The nucleic acid vector according to any one of E53 to E57, wherein the promoter is a minimal promoter, a core promoter, or a constitutive promoter. E59. The nucleic acid vector of E58, wherein the promoter is a CAG promoter, a CBA promoter, a smCBA promoter, a CASI promoter, a dihydrofolate reductase (DHFR) promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EF1α promoter, a β-globin promoter, a CMV promoter, an HSV promoter, or an SV40 promoter.

[0184] E60. The nucleic acid vector of E59, 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.

[0185] E61. The nucleic acid vector of E58, wherein the promoter is a minimal promoter. E62. The nucleic acid vector according to any one of E53 to E57, wherein the promoter is a mammalian SLC26A4 promoter.

[0186] E63. The nucleic acid vector of E62, wherein the SLC26A4 promoter is a human or mouse SLC26A4 promoter. E64. The nucleic acid vector of E63, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1.

[0187] E65. The nucleic acid vector of E64, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E66. The nucleic acid vector described in E63, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 12 to 16.

[0188] E67. The nucleic acid vector according to E66, wherein the SLC26A4 promoter has any one of the sequences set forth in SEQ ID NOs: 12 to 16. E68. The nucleic acid vector of E63, wherein the SLC26A4 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 17.

[0189] E69. The nucleic acid vector of E68, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 17. E70. The nucleic acid vector of any one of E53 to E69, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0190] E71. The nucleic acid vector of E70, wherein the expression product is a heterologous expression product. E72. The nucleic acid vector according to E70, wherein the expression product is an expression product that is endogenously expressed in an SLC26A4-expressing cell.

[0191] E73. The nucleic acid vector of E72, wherein the expression product is an expression product endogenously expressed in SLC26A4-expressing inner ear cells. E74. The nucleic acid vector of E73, 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).

[0192] E75. The nucleic acid vector of any one of E72 to E74, wherein the expression product is pendrin (eg, a mammalian pendrin protein). E76. The nucleic acid vector of E75, wherein the pendrin (the mammalian pendrin protein) is a wild-type isoform endogenously expressed in the inner ear of a mammal.

[0193] E77. The nucleic acid vector of E75 or E76, 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:4 or SEQ ID NO:5.

[0194] E78. The nucleic acid vector of E77, wherein the pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO:4 or SEQ ID NO:5. E79. The nucleic acid vector according to any one of E71 to E74, wherein the expression product is Atoh1 (eg, a mammalian Atoh1 protein).

[0195] E80. The nucleic acid vector of E79, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the mammalian inner ear. E81. The nucleic acid vector of any one of E79 or E80, 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:8 or SEQ ID NO:10.

[0196] E82. The nucleic acid vector of E81, wherein the Atoh1 (the mammalian Atoh1 protein) has the sequence of SEQ ID NO:8 or SEQ ID NO:10. E83. The nucleic acid vector of E70 or E71, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0197] E84. The nucleic acid vector of any one of E48 to E83, wherein the polynucleotide comprises an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0198] E85. The nucleic acid vector of E84, wherein the polynucleotide comprises an enhancer having the sequence of SEQ ID NO:2 and an enhancer having the sequence of SEQ ID NO:3. E86. The nucleic acid vector of E85, wherein the polynucleotide comprises, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 17 (e.g., an enhancer having the sequence of SEQ ID NO: 2 directly fused to an enhancer having the sequence of SEQ ID NO: 3 directly fused to an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 17).

[0199] E87. The nucleic acid vector of E86, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 17. E88. The nucleic acid vector of E86 or E87, wherein the polynucleotide comprises the sequence of SEQ ID NO:18.

[0200] E89. The nucleic acid vector of any one of E48 to E85, wherein the polynucleotide comprises two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and / or two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) (e.g., two or more copies of one or both enhancers).

[0201] E90. The nucleic acid vector of E89, wherein the polynucleotide comprises two or more copies of an enhancer (e.g., two or more copies of one or both enhancers) having the sequence of SEQ ID NO:2 and / or SEQ ID NO:3.

[0202] E91. A nucleic acid vector comprising a polynucleotide comprising 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 SEQ ID NO:1.

[0203] E92. The nucleic acid vector of E91, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E93. A nucleic acid vector comprising a polynucleotide comprising 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 SEQ ID NO: 17.

[0204] E94. The nucleic acid vector of E93, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 17. E95. The nucleic acid vector of any one of E91 to E94, wherein the promoter is operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0205] E96. The nucleic acid vector of E95, wherein the expression product is a heterologous expression product. E97. The nucleic acid vector according to E95, wherein the expression product is an expression product that is endogenously expressed in an SLC26A4-expressing cell.

[0206] E98. The nucleic acid vector of E97, wherein the expression product is an expression product that is endogenously expressed in SLC26A4-expressing inner ear cells. E99. The nucleic acid vector of E98, 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).

[0207] E100. The nucleic acid vector of any one of E97 to E99, wherein the expression product is pendrin (eg, a mammalian pendrin protein). E101. The nucleic acid vector of E100, wherein the pendrin (the mammalian pendrin protein) is the wild-type isoform endogenously expressed in the inner ear of mammals.

[0208] E102. The nucleic acid vector of E100 or E101, 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:4 or SEQ ID NO:5.

[0209] E103. The nucleic acid vector of E102, wherein the pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. E104. The nucleic acid vector according to any one of E95 to E99, wherein the expression product is Atoh1 (eg, a mammalian Atoh1 protein).

[0210] E105. The nucleic acid vector of E104, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the inner ear of mammals. E106. The nucleic acid vector of E104 or E105, 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:8 or SEQ ID NO:10.

[0211] E107. The nucleic acid vector of E106, wherein the Atoh1 (the encoded mammalian Atoh1) has the sequence of SEQ ID NO:8 or SEQ ID NO:10. E108. The nucleic acid vector of E95 or E96, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0212] E109. The nucleic acid vector according to any one of E91 to E108, wherein the promoter is operably linked to an enhancer. E110. The nucleic acid vector of E109, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) or at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0213] E111. The nucleic acid vector of E110, wherein the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:2.

[0214] E112. The nucleic acid vector of E111, wherein the enhancer has the sequence of SEQ ID NO:2. E113. The nucleic acid vector of E110, wherein the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:3.

[0215] E114. The nucleic acid vector of E113, wherein the enhancer has the sequence of SEQ ID NO:3. E115. The nucleic acid vector according to any one of E109 to E114, wherein the enhancer is located 5' of the promoter.

[0216] E116. The nucleic acid vector according to any one of E109 to E114, wherein the enhancer is located 3' of the promoter. E117. The nucleic acid vector according to any one of E109 to E116, wherein the enhancer is fused directly to the promoter.

[0217] E118. The nucleic acid vector according to any one of E109 to E116, wherein the enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleic acids).

[0218] E119. The nucleic acid vector of any one of E109 to E118, wherein the polynucleotide comprises an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0219] E120. The nucleic acid vector of E119, wherein the polynucleotide comprises an enhancer having the sequence of SEQ ID NO:2 and an enhancer having the sequence of SEQ ID NO:3. E121. The nucleic acid vector of E120, wherein the polynucleotide comprises, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 17 (e.g., an enhancer having the sequence of SEQ ID NO: 2 directly fused to an enhancer having the sequence of SEQ ID NO: 3 directly fused to an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 17).

[0220] E122. The nucleic acid vector of E121, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 17. E123. The nucleic acid vector of E121 or E122, wherein the polynucleotide comprises the sequence of SEQ ID NO: 18.

[0221] E124. The nucleic acid vector of any one of E109 to E120, wherein the polynucleotide comprises two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and / or two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) (e.g., two or more copies of one or both enhancers).

[0222] E125. The nucleic acid vector of E1124, wherein the polynucleotide comprises two or more copies of an enhancer (e.g., two or more copies of one or both enhancers) having the sequence of SEQ ID NO:2 and / or SEQ ID NO:3.

[0223] E126. A polynucleotide comprising 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 SEQ ID NO:1 operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0224] E127. The polynucleotide of E126, wherein the SLC26A4 promoter has the sequence of SEQ ID NO:1. E128. A polynucleotide comprising 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 SEQ ID NO: 17 operably linked to a polynucleotide that can be transcribed to produce an expression product.

[0225] E129. The polynucleotide of E128, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 17. E130. The polynucleotide according to any one of E126 to E129, wherein the expression product is a heterologous expression product.

[0226] E131. The polynucleotide according to any one of E126 to E129, wherein the expression product is an expression product endogenously expressed in an SLC26A4-expressing cell. E132. The polynucleotide of E131, wherein the expression product is an expression product endogenously expressed in SLC26A4-expressing inner ear cells.

[0227] E133. The polynucleotide of E132, 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).

[0228] E134. The polynucleotide of any one of E126 to E129 and E131 to E133, wherein the expression product is pendrin (eg, a mammalian pendrin protein).

[0229] E135. The polynucleotide of E134, wherein said pendrin (the mammalian pendrin protein) is the wild-type isoform endogenously expressed in the mammalian inner ear.

[0230] E136. The polynucleotide of E134 or E135, 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:4 or SEQ ID NO:5.

[0231] E137. The polynucleotide of E136, wherein the pendrin (the mammalian pendrin protein) has the sequence of SEQ ID NO:4 or SEQ ID NO:5. E138. The polynucleotide according to any one of E126 to E133, wherein the expression product is Atoh1 (eg, a mammalian Atoh1 protein).

[0232] E139. The polynucleotide of E138, wherein the Atoh1 (the mammalian Atoh1 protein) is a wild-type isoform endogenously expressed in the mammalian inner ear.

[0233] E140. The polynucleotide of E138 or E139, 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:8 or SEQ ID NO:10.

[0234] E141. The polynucleotide of E140, wherein the Atoh1 (the encoded mammalian Atoh1) has the sequence of SEQ ID NO:8 or SEQ ID NO:10. E142. The polynucleotide of any one of E126 to E133, wherein the expression product is a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA.

[0235] E143. The polynucleotide of any one of E126 to E142, wherein the promoter is operably linked to an enhancer. E144. The polynucleotide of E143, wherein the enhancer has at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) or at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0236] E145. The polynucleotide of E144, wherein the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:2.

[0237] E146. The polynucleotide of E145, wherein the enhancer has the sequence of SEQ ID NO:2. E147. The polynucleotide of E144, wherein the enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:3.

[0238] E148. The polynucleotide of E147, wherein the enhancer has the sequence of SEQ ID NO:3. E149. The polynucleotide according to any one of E143 to E148, wherein the enhancer is located 5' of the promoter.

[0239] E150. The polynucleotide according to any one of E143 to E148, wherein the enhancer is located 3' of the promoter. E151. The polynucleotide of any one of E143 to E150, wherein the enhancer is fused directly to the promoter.

[0240] E152. The polynucleotide of any one of E143 to E150, wherein the enhancer is linked to the promoter by a nucleic acid linker of 1 to 100 nucleic acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleic acids).

[0241] E153. The polynucleotide of any one of E143 to E152, comprising an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0242] E154. The polynucleotide of E153, comprising an enhancer having the sequence of SEQ ID NO:2 and an enhancer having the sequence of SEQ ID NO:3. E155. The polynucleotide of E154, comprising, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 17 (e.g., an enhancer having the sequence of SEQ ID NO: 2 directly fused to an enhancer having the sequence of SEQ ID NO: 3 directly fused to an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO: 17).

[0243] E156. The polynucleotide of E155, wherein the SLC26A4 promoter has the sequence of SEQ ID NO: 17. E157. The polynucleotide of E155 or E156, comprising the sequence of SEQ ID NO: 18.

[0244] E158. The polynucleotide of any one of E143 to E154, comprising two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) and / or two or more copies of an enhancer having at least 85% sequence identity to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) (e.g., two or more copies of one or both enhancers).

[0245] E159. The polynucleotide of E158, comprising two or more copies of an enhancer having the sequence of SEQ ID NO:2 and / or SEQ ID NO:3 (eg, two or more copies of one or both enhancers).

[0246] E160. A nucleic acid vector comprising the polynucleotide according to any one of E126 to E159. E161. The nucleic acid vector according to any one of E47 to E125 and E160, wherein the nucleic acid vector is a viral vector, a plasmid, a cosmid, or an artificial chromosome.

[0247] E162. The nucleic acid vector according to any one of E47 to E125, E160 and E161, wherein the nucleic acid vector is a viral vector. E163. The nucleic acid vector of E162, wherein the viral vector is an adeno-associated viral (AAV) viral vector, an adenovirus viral vector, or a lentivirus viral vector.

[0248] E164. The nucleic acid vector according to E163, wherein the viral vector is an AAV vector. E165. The nucleic acid vector of E164, 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.

[0249] E166. A composition comprising the nucleic acid vector according to any one of E47 to E125 and E160 to E165, and a pharmaceutically acceptable carrier, diluent, or excipient. E167. A cell comprising the polynucleotide according to any one of E1 to E46 and E126 to E159 or the nucleic acid vector according to any one of E47 to E125 and E160 to E165.

[0250] E168. The cell according to E167, wherein the cell is an SLC26A4-expressing cell. E169. The cell of E168, wherein the cell is an SLC26A4-expressing inner ear cell.

[0251] E170. The cell according to any one of E167 to E169, wherein the cell is a mammalian cell. E171. The cell of E170, wherein the mammalian cell is a human cell.

[0252] E172. The cell according to any one of E167 to E171, which is an interdental cell, a spiral ridge cell, a cochlear root cell, or a vestibular supporting cell. E173. A method for expressing an expression product in a cell, the method comprising contacting the cell with a nucleic acid vector according to any one of E47 to E125 and E160 to E165 or a composition according to E166.

[0253] E174. The method of E173, wherein the cell is an inner ear cell. E175. The method according to E173 or E174, wherein the cell is an SLC26A4-expressing cell.

[0254] E176. The method according to any one of E173 to E175, wherein the cells are SLC26A4-expressing inner ear cells. E177. The method of E176, wherein the SLC26A4-expressing inner ear cells are interdental cells, spiral eminence cells, cochlear root cells, or vestibular supporting cells.

[0255] E178. The method according to any one of E173 to E177, wherein the cell is a mammalian cell. E179. The method of E178, wherein the mammalian cells are human cells.

[0256] E180. The method of any one of E173 to E179, wherein the contacting is performed inside a subject (eg, in vivo). E181. 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 E47 to E125 and E160 to E165 or a composition described in E166.

[0257] E182. The method of E181, wherein the hearing loss is pendrin-associated hearing loss. E183. The method according to E182, wherein the expression product is pendrin. E184. The method of E182 or E183, wherein the pendrin-associated hearing loss is Pendred syndrome or DFNB4-associated hearing loss.

[0258] E185. 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 E47 to E125 and E160 to E165 or a composition described in E166.

[0259] E186. A method for 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 described in any one of E47 to E125 and E160 to E165 or a composition described in E166.

[0260] E187. The method of E185 or E186, wherein the expression product is pendrin. E188. A method for treating vestibular dysfunction 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 E47 to E125 and E160 to E165 or a composition described in E166.

[0261] E189. The method according to E188, wherein the expression product is pendrin or Atoh1. E190. The method according to E188 or E189, wherein said vestibular dysfunction is vertigo.

[0262] E191. 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 E47 to E125 and E160 to E165 or a composition described in E166.

[0263] E192. The method of E191, wherein said vestibular dysfunction is pendrin-associated vestibular dysfunction. E193. The method according to E192, wherein the expression product is pendrin.

[0264] E194. The method of E192 or E193, wherein the pendrin-associated vestibular dysfunction is vestibular dysfunction associated with Pendred syndrome or DFNB4. E195. The method of E191, wherein the expression product is pendrin or Atoh1.

[0265] E196. A method for inducing or increasing differentiation of vestibular supporting cells into vestibular hair cells, comprising the step of contacting the vestibular supporting cells with the nucleic acid vector described in any one of E47 to E125 and E160 to E165 or the composition described in E166, wherein the expression product is Atoh1.

[0266] E197. The method of E196, wherein the contacting is performed in vivo (eg, inside a subject). E198. The method of E197, wherein the subject has or is at risk of developing vestibular dysfunction.

[0267] E199. A method for inducing or increasing vestibular hair cell regeneration 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 E47 to E125 and E160 to E165 or a composition described in E166, wherein the expression product is Atoh1.

[0268] E200. The method of E199, wherein the subject has or is at risk of developing a vestibular dysfunction. E201. 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 E47 to E125 and E160 to E165 or the composition described in E166.

[0269] E202. The method of E201, wherein the contacting is performed in vivo (eg, inside a subject). E203. The method of E202, wherein the subject has or is at risk of developing hearing loss (e.g., sensorineural hearing loss) or vestibular dysfunction.

[0270] E204. The method of any one of E191 to E203, wherein the vestibular dysfunction is vertigo, dizziness, imbalance (e.g., loss of balance or equilibrium), oscillopsia, or bilateral vestibular dysfunction.

[0271] E205. The method of any one of E201-E204, wherein the vestibular dysfunction is associated with damage or loss of vestibular hair cells. E206. The method of E205, 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).

[0272] E207. The method of E206, wherein the ototoxic drug is an aminoglycoside (an aminoglycoside antibiotic, e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, amikacin, dibekacin, and netilmicin), viomycin, antineoplastic drug (e.g., a platinum-containing chemotherapeutic agent such as cisplatin, carboplatin, or oxaliplatin, or other chemotherapeutic agent such as nitrogen mustard or vincristine), a loop diuretic (e.g., ethacrynic acid or furosemide), a salicylate, or quinine.

[0273] E208. The method of any one of E181 to E187, further comprising assessing the subject's hearing prior to administration of the nucleic acid vector or the composition. E209. The method of any one of E181 to E187 and E208, further comprising assessing the hearing of the subject after administration of the nucleic acid vector or the composition.

[0274] E210. The method of any one of E188 to E207, further comprising assessing the vestibular function of the subject prior to administration of the nucleic acid vector or the composition. E211. The method of any one of E188 to E207 and E210, further comprising assessing the vestibular function of the subject after administration of the nucleic acid vector or the composition.

[0275] E212. The method according to any one of E173 to E211, wherein the nucleic acid vector or the composition is administered locally. E213. The method of E212, wherein the nucleic acid vector or the composition is administered to the inner ear.

[0276] E214. The method of E212, wherein the nucleic acid vector or the composition is administered to the middle ear. E215. The method of E212, wherein the nucleic acid vector or the composition is administered transtympanically or intratympanically.

[0277] E216. The method of E212, wherein the nucleic acid vector or the composition is administered into the perilymph. E217. The method of E212, wherein the nucleic acid vector or the composition is administered into the endolymph.

[0278] E218. The method of E212, wherein the nucleic acid vector or the composition is administered to or through the oval window. E219. The method of E212, wherein the nucleic acid vector or the composition is administered to or through the round window.

[0279] E220. The method of E212, wherein the nucleic acid vector or the composition is administered to a semicircular canal. E221. The method of any one of E173 to E220, 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.

[0280] E222. The method of any one of E173 to E221, wherein the subject is a human subject. E223. A kit comprising a polynucleotide according to any one of E1 to E46 or E126 to E159, a nucleic acid vector according to any one of E47 to E125 or E160 to E165, or a composition according to E166.

[0281] 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. A polynucleotide comprising an enhancer operably linked to a promoter, the enhancer having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 3 kilobases (3 kb).

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

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

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

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

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

3.

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

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

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

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

1.

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

1.

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

17.

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

17.

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

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

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

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

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

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

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

5.

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

3.

22. 22. A polynucleotide according to claim 21, comprising, in 5' to 3' order, an enhancer having the sequence of SEQ ID NO: 2, an enhancer having the sequence of SEQ ID NO: 3, and an SLC26A4 promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO:

17.

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

17.

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

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

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

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

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

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

30. 30. 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 according to any one of claims 24 to 28 or a composition according to claim 29.

31. 31. The method of claim 30, wherein the contacting occurs internally in a subject.

32. 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 the nucleic acid vector of claim 27.

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

34. 28. 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 the nucleic acid vector of claim 27.

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

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

37. 29. 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 the nucleic acid vector of claim 27 or 28.

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