GJB2 Regulatory Elements and Uses Thereof

Abstract

The present disclosure provides gap junction protein beta 2 (GJB2) promoters and enhancers, and vectors containing the same, which can be used to express expression products in GJB2-expressing cells, including cochlear supporting cells. The GJB2 promoters and enhancers described herein can be operably linked to a polynucleotide, such as a transgene, encoding a wild-type form of GJB2 for the treatment of a subject having or at risk of developing hearing loss (e.g., GJB2-associated hearing loss).

Description

[Background technology]

[0001] Hearing loss is a major public health problem that is estimated to affect nearly 15% of school-age children and one in three people by age 65. The most common type of hearing loss is sensorineural hearing loss, which is a type of hearing loss caused by defects in cells of the inner ear, such as cochlear hair cells and cochlear supporting cells, or in the neural pathways projecting from the inner ear to the brain. Sensorineural hearing loss is often acquired and has a variety of causes, including acoustic trauma, disease or infection, head trauma, ototoxic drugs, and aging. There are also genetic causes of sensorineural hearing loss, such as mutations in genes involved in the development and function of the inner ear. Mutations in more than 90 such genes have been identified, including mutations inherited in autosomal recessive, autosomal dominant, and X-linked patterns.

[0002] In recent years, efforts to treat hearing loss have increasingly focused on gene therapy as a possible solution.However, there are still few approaches to target specific cell populations in the cochlea.The gene therapy approach to hearing loss that induces the expression of exogenous genes in all cells of the inner ear may have off-target effects or may result in toxicity.Therefore, there is a need for new approaches to target specific cell populations in the cochlea for the treatment of hearing loss. Summary of the Invention

[0003] The present invention provides compositions and methods for promoting expression of a gene of interest in a particular cell type, such as a gene endogenously expressed in GJB2-expressing cells, a gene that can induce differentiation of cochlear supporting cells into cochlear hair cells, or a gene expressed in cochlear supporting cells that is mutated in a subject with hearing loss. The compositions and methods described herein relate to polynucleotides that can induce expression of a transgene in GJB2-expressing cells (e.g., GJB2-expressing inner ear cells). The polynucleotides described herein can be operably linked to a polynucleotide encoding an expression product, such as, for example, a protein or inhibitory RNA, and administered to a subject, such as a human subject, to treat or prevent hearing loss (e.g., sensorineural hearing loss, such as GJB2-associated hearing loss).

[0004] In a first aspect, the present invention provides a polynucleotide comprising SEQ ID NO:1, or at least one of SEQ ID NOs:3-12 (e.g., one or more of them), a functional portion or derivative thereof, operably linked to a second region 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 a functional portion or derivative thereof, comprising at least one of SEQ ID NOs:20-24 (e.g., one of many thereof). or a derivative thereof, the polynucleotide comprises a GJB2 promoter containing a first region having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), the distance between the first region and the second region in the polynucleotide (e.g., the distance between the 3' end of the first region and the 5' end of the second region) is 1 kb or less, and optionally, a linker containing 1 to 100 nucleotides between the first region and the second region is provided. In some embodiments, the distance between the first region and the second region in the polynucleotide is 0.5 kb or less. In some embodiments, the distance between the first region and the second region in the polynucleotide is 0.25 kb or less.

[0005] In another aspect, the invention provides a polynucleotide comprising SEQ ID NO:1, or at least one of SEQ ID NOs:3-12 (e.g., any one of many thereof) operably linked to a second region 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 a functional portion or derivative thereof comprising at least one of SEQ ID NOs:20-24 (e.g., any one of many thereof). In one embodiment, the present invention provides a polynucleotide comprising a GJB2 promoter containing a first region having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion or derivative thereof comprising one or more of the following:

[0006] In another aspect, the invention provides a polynucleotide comprising SEQ ID NO:1, or at least one of SEQ ID NOs:3-12 (e.g., a sequence identical to SEQ ID NO:1, SEQ ID NO:3-12, or a sequence identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO: The present invention provides a polynucleotide comprising a GJB2 promoter containing a first region having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion or derivative thereof comprising one or more of the following: In some embodiments, the heterologous polynucleotide encodes Gjb6 (e.g., encodes SEQ ID NO: 47). In some embodiments, the heterologous polynucleotide encodes brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NTF3). In some embodiments, the heterologous polynucleotide is a polynucleotide that encodes a protein or inhibitory RNA that can induce differentiation of cochlear supporting cells into cochlear hair cells or induce or increase proliferation of cochlear supporting cells (e.g., a polynucleotide listed in Table 5), or a transgene that corresponds to a wild-type form of a gene that is expressed in cochlear supporting cells and that is mutated in a subject with hearing loss (e.g., a transgene that corresponds to a wild-type form of a gene listed in Table 6).In some embodiments, the heterologous polynucleotide encodes (e.g., can be transcribed to produce) 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.

[0007] In another aspect, the present invention provides a polynucleotide comprising a GJB2 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to any one of SEQ ID NOs: 13-19. In some embodiments, the GJB2 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: 13. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 13. In some embodiments, the GJB2 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: 14. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 14. In some embodiments, the GJB2 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: 15. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 15. In some embodiments, the GJB2 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: 16. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 16. In some embodiments, the GJB2 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 GJB2 promoter has the sequence of SEQ ID NO: 17. In some embodiments, the GJB2 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: 18. In some embodiments, the GJB2 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: 19. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 19.

[0008] In another aspect, the present invention provides a polynucleotide comprising a GJB2 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to any one of SEQ ID NOs: 25-28. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 25. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 25. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:26. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:26. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:27. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:27. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 28. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 28.

[0009] In another aspect, the invention provides a polynucleotide containing a GJB2 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: 37. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:37.

[0010] In another aspect, the present invention provides a polynucleotide comprising a sequence similar to SEQ ID NO:2, or one of SEQ ID NOs:20-24, operably linked to a second region 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 functional portion or derivative thereof comprising at least one of SEQ ID NOs:3-12 (e.g., one or more of them). In one embodiment, the polynucleotide comprises a GJB2 promoter comprising a first region having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion or derivative thereof comprising at least one of the following (e.g., one of many of the following):

[0011] In another aspect, the invention provides a nucleic acid vector containing a polynucleotide according to any of the preceding aspects and embodiments.

[0012] In another aspect, the invention provides a nucleic acid vector containing a polynucleotide comprising a GJB2 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: 36. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:36.

[0013] In another aspect, the present invention provides a nucleic acid vector containing a polynucleotide comprising a GJB2 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to any one of SEQ ID NOs: 3-7. In some embodiments, the GJB2 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: 3. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 3. In some embodiments, the GJB2 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:4. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:4. In some embodiments, the GJB2 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:5. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:5. In some embodiments, the GJB2 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:6. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:6. In some embodiments, the GJB2 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:7.In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:7.

[0014] In another aspect, the present invention provides a nucleic acid vector containing a polynucleotide comprising a GJB2 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to any one of SEQ ID NOs: 2 and 20-24. In some embodiments, the GJB2 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: 2. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 2. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:20. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:20. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:21. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:21. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:22. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:22. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:23.In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 23. In some embodiments, the GJB2 promoter has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 24. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 24.

[0015] In another aspect, the present invention provides a nucleic acid vector comprising SEQ ID NO:1, or at least one of SEQ ID NOs:3-12, operably linked to a second region 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 a functional portion or derivative thereof comprising at least one of SEQ ID NOs:20-24 (e.g., one or more of them). The present invention provides a nucleic acid vector comprising a polynucleotide comprising a GJB2 promoter containing a first region having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion or derivative thereof, including one or more of the above (e.g., one or more of the above), and optionally comprising a linker containing 1 to 100 nucleotides between the first and second regions.

[0016] In some embodiments of any of the foregoing aspects, the first region 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 first region has the sequence of SEQ ID NO:1.

[0017] In some embodiments of any of the foregoing aspects, the first region 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: 3-12. In some embodiments, the first region 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, the first region has the sequence of SEQ ID NO: 3. In some embodiments, the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:4. In some embodiments, the first region has the sequence of SEQ ID NO:4. In some embodiments, the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:5. In some embodiments, the first region has the sequence of SEQ ID NO:5. In some embodiments, the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:6. In some embodiments, the first region has the sequence of SEQ ID NO:6. In some embodiments, the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:7. In some embodiments, the first region has the sequence of SEQ ID NO:7.In some embodiments, the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:8. In some embodiments, the first region has the sequence of SEQ ID NO:8. In some embodiments, the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:9. In some embodiments, the first region has the sequence of SEQ ID NO:9. In some embodiments, the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO: 10. In some embodiments, the first region has the sequence of SEQ ID NO: 10. In some embodiments, the first region 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: 11. In some embodiments, the first region has the sequence of SEQ ID NO: 11. In some embodiments, the first region 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: 12. In some embodiments, the first region has the sequence of SEQ ID NO: 12.

[0018] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:4 and the sequence of SEQ ID NO:12. In some embodiments, the first region 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:13. In some embodiments, the first region has the sequence of SEQ ID NO:13.

[0019] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:5 and the sequence of SEQ ID NO:12. In some embodiments, the first region 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:14. In some embodiments, the first region has the sequence of SEQ ID NO:14.

[0020] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:6 and the sequence of SEQ ID NO:12. In some embodiments, the first region 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:15. In some embodiments, the first region has the sequence of SEQ ID NO:15.

[0021] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:7 and the sequence of SEQ ID NO:12. In some embodiments, the first region 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:16. In some embodiments, the first region has the sequence of SEQ ID NO:16.

[0022] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:9 and the sequence of SEQ ID NO:12. In some embodiments, the first region 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 first region has the sequence of SEQ ID NO:17.

[0023] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:10 and the sequence of SEQ ID NO:12. In some embodiments, the first region 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:18. In some embodiments, the first region has the sequence of SEQ ID NO:18.

[0024] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:11 and the sequence of SEQ ID NO:12. In some embodiments, the first region 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:19. In some embodiments, the first region has the sequence of SEQ ID NO:19.

[0025] In some embodiments of any of the aforementioned aspects, the sequence of SEQ ID NO:12 precedes the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0026] In some embodiments of any of the foregoing aspects, the second region 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, the second region has the sequence of SEQ ID NO: 2.

[0027] In some embodiments of any of the foregoing aspects, the second region 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:20-24. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:20. In some embodiments, the second region has the sequence of SEQ ID NO:20. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:21. In some embodiments, the second region has the sequence of SEQ ID NO:21. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:22. In some embodiments, the second region has the sequence of SEQ ID NO:22. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:23. In some embodiments, the second region has the sequence of SEQ ID NO:23. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to SEQ ID NO:24. In some embodiments, the second region has the sequence of SEQ ID NO:24.

[0028] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:23 and the sequence of SEQ ID NO:20. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:25. In some embodiments, the second region has the sequence of SEQ ID NO:25.

[0029] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:24 and the sequence of SEQ ID NO:20. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:26. In some embodiments, the second region has the sequence of SEQ ID NO:26.

[0030] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:23 and the sequence of SEQ ID NO:22. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:27. In some embodiments, the second region has the sequence of SEQ ID NO:27.

[0031] In some embodiments of any of the foregoing aspects, the functional portion of SEQ ID NO:2 comprises the sequence of SEQ ID NO:24 and the sequence of SEQ ID NO:22. In some embodiments, the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:28. In some embodiments, the second region has the sequence of SEQ ID NO:28.

[0032] In some embodiments of any of the aforementioned aspects, the sequence of SEQ ID NO:20 precedes the sequences of SEQ ID NO:23 and SEQ ID NO:24. In some embodiments of any of the aforementioned aspects, the sequence of SEQ ID NO:22 precedes the sequences of SEQ ID NO:23 and SEQ ID NO:24.

[0033] In some embodiments of any of the aforementioned aspects, the first region is directly linked to the second region without a linker (e.g., the 3' end of the first region is immediately preceding the 5' end of the second region). In some embodiments of any of the aforementioned aspects, the GJB2 promoter has at least 85% sequence identity to SEQ ID NO:29 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity). In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:29. In some embodiments of any of the aforementioned aspects, the GJB2 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:30. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:30. In some embodiments of any of the aforementioned aspects, the GJB2 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:31. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO:31. In some embodiments of any of the aforementioned aspects, the GJB2 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: 32. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 32. In some embodiments of any of the aforementioned aspects, the GJB2 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: 33.In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 33. In some embodiments of any of the aforementioned aspects, the GJB2 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: 34. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 34. In some embodiments of any of the aforementioned aspects, the GJB2 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: 35. In some embodiments, the GJB2 promoter has the sequence of SEQ ID NO: 35.

[0034] In another aspect, the present invention provides a polynucleotide comprising a GJB2 enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63 (e.g., any one of SEQ ID NOs: 52-59) operably linked to a promoter, wherein the distance between the enhancer and the promoter in the polynucleotide is less than 3 kilobases (3 kb). In some embodiments, the distance between the enhancer and the promoter in the polynucleotide is less than 2 kb. In some embodiments, the distance between the enhancer and the promoter in the polynucleotide is less than 1 kb. In some embodiments, the distance between the enhancer and the promoter in the polynucleotide is less than 0.5 kb. In some embodiments, the polynucleotide comprising the GJB2 enhancer is contained in a nucleic acid vector. In some embodiments, the promoter is an inner ear cell type specific promoter (e.g., a cochlear supporting cell specific promoter). In some embodiments, the inner ear cell type specific promoter is a promoter listed in Table 9 (e.g., a promoter that can induce expression in GJB2-expressing inner ear cells). In some embodiments, the promoter is a GJB2 promoter.In some embodiments, the GJB2 promoter is a promoter as described herein above (e.g., a polynucleotide comprising a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof, and optionally a linker linking the first and second regions). In some embodiments, the GJB2 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: 1 and 8-11. In some embodiments, the GJB2 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 the promoters listed in Table 8 (SEQ ID NOs: 66-68). In some embodiments, the GJB2 promoter has the sequence of a promoter listed in Table 8.

[0035] In another aspect, the present invention provides a nucleic acid vector containing a polynucleotide comprising a GJB2 enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63 (e.g., any one of SEQ ID NOs: 52-59). In some embodiments, the enhancer is operably linked to a promoter. In some embodiments, the promoter is an inner ear cell type specific promoter (e.g., a cochlear supporting cell specific promoter). In some embodiments, the inner ear cell type specific promoter is a promoter listed in Table 9 (e.g., a promoter capable of directing expression in GJB2-expressing inner ear cells). In some embodiments, the promoter is a GJB2 promoter. In some embodiments, the GJB2 promoter is a promoter as described herein above (e.g., a polynucleotide comprising a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof, and optionally a linker linking the first and second regions). In some embodiments, the GJB2 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: 1 and 8-11. In some embodiments, the GJB2 promoter has the sequence of any one of SEQ ID NOs: 1 and 8-11.In some embodiments, the GJB2 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 the promoters listed in Table 8 (SEQ ID NOs:66-68). In some embodiments, the GJB2 promoter has the sequence of a promoter listed in Table 8.

[0036] In some embodiments of any of the foregoing aspects, the promoter (e.g., GJB2 promoter) is operably linked to a polynucleotide encoding an expression product. In some embodiments, the expression product is a heterologous expression product. In some embodiments, the heterologous expression product is BDNF or NTF3. In some embodiments, the expression product is 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. In some embodiments, the expression product is an expression product that is endogenously expressed in a GJB2-expressing cell. In some embodiments, the expression product is an expression product that is endogenously expressed in a GJB2-expressing inner ear cell. In some embodiments, the expression product is Gjb2 or Gjb6. In some embodiments, the polynucleotide encoding the expression product is a polynucleotide encoding a protein or inhibitory RNA that can induce the differentiation of cochlear supporting cells into cochlear hair cells, a polynucleotide encoding a protein or inhibitory RNA that can induce or increase the proliferation of cochlear supporting cells, or a transgene that corresponds to the wild-type form of a gene that is expressed in cochlear supporting cells and that is mutated in a subject with hearing loss. In some embodiments, the polynucleotide encoding the expression product is a polynucleotide listed in Table 5, or a transgene that corresponds to the wild-type form of a gene listed in Table 6.

[0037] In some embodiments of any of the aforementioned aspects, the polynucleotide further comprises a GJB2 enhancer operably linked to the GJB2 promoter.

[0038] In some embodiments of any of the foregoing aspects, the GJB2 enhancer has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to any one of SEQ ID NOs: 52-63. In some embodiments, the GJB2 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: 52. In some embodiments, the GJB2 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: 53. In some embodiments, the GJB2 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: 54. In some embodiments, the GJB2 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: 55. In some embodiments, the GJB2 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: 56. In some embodiments, the GJB2 enhancer has at least 85% sequence identity to SEQ ID NO:57 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).In some embodiments, the GJB2 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: 58. In some embodiments, the GJB2 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: 59. In some embodiments, the GJB2 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: 60. In some embodiments, the GJB2 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: 61. In some embodiments, the GJB2 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: 62. In some embodiments, the GJB2 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: 63. In some embodiments, the GJB2 enhancer has the sequence of any one of SEQ ID NOs: 52-63. In some embodiments, the GJB2 enhancer has the sequence of SEQ ID NO: 52. In some embodiments, the GJB2 enhancer has the sequence of SEQ ID NO: 53. In some embodiments, the GJB2 enhancer has the sequence of SEQ ID NO: 54. In some embodiments, the GJB2 enhancer has the sequence of SEQ ID NO: 55.In some embodiments, the GJB2 enhancer has a sequence of SEQ ID NO: 56. In some embodiments, the GJB2 enhancer has a sequence of SEQ ID NO: 57. In some embodiments, the GJB2 enhancer has a sequence of SEQ ID NO: 58. In some embodiments, the GJB2 enhancer has a sequence of SEQ ID NO: 59. In some embodiments, the GJB2 enhancer has a sequence of SEQ ID NO: 60. In some embodiments, the GJB2 enhancer has a sequence of SEQ ID NO: 61. In some embodiments, the GJB2 enhancer has a sequence of SEQ ID NO: 62. In some embodiments, the GJB2 enhancer has a sequence of SEQ ID NO: 63.

[0039] In some embodiments of any of the aforementioned aspects, the GJB2 enhancer is located 5' to the promoter.

[0040] In some embodiments of any of the aforementioned aspects, the GJB2 enhancer is located 3' to the promoter.

[0041] In some embodiments of any of the foregoing aspects, the polynucleotide comprises two or more different GJB2 enhancers, each enhancer independently selected from enhancers having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63 (e.g., any one of SEQ ID NOs: 52-59). In some embodiments, each different GJB2 enhancer independently selected from enhancers having a sequence of one of SEQ ID NOs: 52-63 (e.g., any one of SEQ ID NOs: 52-59). In some embodiments, the polynucleotide comprises four different GJB2 enhancers. In some embodiments, the four enhancers are SEQ ID NO: 52, SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59. In some embodiments, the four enhancers are SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0042] In some embodiments of any of the foregoing aspects, the polynucleotide comprises two or more copies of a GJB2 enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63 (e.g., any one of SEQ ID NOs: 52-59). In some embodiments, each copy of the two or more copies of the enhancer has the sequence of one of SEQ ID NOs: 52-63 (e.g., any one of SEQ ID NOs: 52-59).

[0043] In some embodiments of any of the above aspects, the nucleic acid vector is a viral vector, a plasmid, a cosmid, or an artificial chromosome. In some embodiments, the nucleic acid vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, an adenoviral vector, or a lentiviral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector has 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. In some embodiments, the AAV vector has an AAV1 capsid. In some embodiments, the AAV vector has an AAV9 capsid. In some embodiments, the AAV vector has a 7m8 capsid. In some embodiments, the AAV vector has a PHP.S capsid. In some embodiments, the AAV vector has a DJ 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 an AAV2 capsid. In some embodiments, the AAV vector has an AAV2quad(YF) 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 AAV6 capsid. In some embodiments, the AAV vector has an AAV7 capsid. In some embodiments, the AAV vector has an AAV8 capsid. In some embodiments, the AAV vector has a PHP.B capsid.

[0044] In another aspect, the invention provides a composition comprising the nucleic acid vector of any of the preceding aspects and embodiments, hi some embodiments, the composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient.

[0045] In another aspect, the invention provides a cell containing a polynucleotide or vector of any of the preceding aspects and embodiments. In some embodiments, the cell is a GJB2-expressing cell. In some embodiments, the cell is a GJB2-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 a cochlear supporting cell.

[0046] In another aspect, the present invention provides a method for expressing an expression product in a GJB2-expressing cell by contacting the GJB2-expressing cell with a nucleic acid vector or composition of any of the above aspects or embodiments. In some embodiments, the GJB2-expressing cell is a GJB2-expressing inner ear cell (e.g., a cochlear supporting cell). In some embodiments, the contacting is performed inside a subject (e.g., in vivo).

[0047] In another aspect, the invention provides a method of treating a subject having or at risk of developing GJB2-associated hearing loss by administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector composition of any of the preceding aspects and embodiments, wherein the expression product is Gjb2 or Gjb6. In some embodiments, the GJB2-associated hearing loss is DFNB1, DFNA3, or Bart-Pumphrey syndrome-associated hearing loss, hystrixis with hearing loss, keratitis-ichthyosis-deafness syndrome, palmoplantar keratosis with hearing loss, or Vohwinkel syndrome. In some embodiments, the GJB2-associated hearing loss is DFNB1 or DFNA3. In some embodiments, the subject has a mutation in GJB2, a mutation in GJB6, or a mutation in both GJB2 and GJB6.

[0048] In another aspect, the invention provides a method of treating a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss or hearing loss) by 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.

[0049] In another aspect, the invention provides a method of treating a subject having or at risk of developing tinnitus by 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.

[0050] In another aspect, the invention provides a method of inducing or increasing cochlear hair cell regeneration in a subject in need thereof by administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector or composition of any of the preceding aspects and embodiments, 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:59, and the expression product is an expression product that can promote or increase cochlear supporting cell proliferation or differentiation of cochlear supporting cells into cochlear hair cells. In some embodiments, the polynucleotide encoding the expression product is a polynucleotide listed in Table 5. In some embodiments, the expression product is an inhibitory RNA directed to LATS1 and / or LATS2.

[0051] In another aspect, the invention provides a method of treating a subject having or at risk of developing hearing loss associated with damage or loss of cochlear hair cells by administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector or composition of any of the preceding aspects and embodiments, 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:59, and the expression product is an expression product that can promote or increase the proliferation of cochlear supporting cells or the differentiation of cochlear supporting cells into cochlear hair cells. In some embodiments, the polynucleotide encoding the expression product is a polynucleotide listed in Table 5. In some embodiments, the expression product is an inhibitory RNA directed to LATS1 and / or LATS2.

[0052] In another aspect, the invention provides a method of inducing or increasing differentiation of cochlear supporting cells into cochlear hair cells by contacting the cochlear supporting cells with a nucleic acid vector or composition of any of the aforementioned aspects and embodiments, 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: 59, and the expression product is Atoh1 or Atoh1 variant, Pou4F3, Gfi1, or Ikzf2. In some embodiments, the contacting is performed in vivo (e.g., inside a subject).

[0053] In another aspect, the present invention provides a method of inducing or increasing the proliferation of cochlear supporting cells by contacting the cochlear supporting cells with a nucleic acid vector or composition of any of the above aspects and embodiments, 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: 59, and the expression product is an inhibitory RNA directed to Lgr5, Yapl, Tead2, or LATS1 and / or LATS2. In some embodiments, the contacting is performed in vivo (e.g., inside a subject).

[0054] In another aspect, the present invention provides a method of treating a subject having or at risk of developing genetic hearing loss associated with a mutation in a gene endogenously expressed in cochlear supporting cells by administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector or composition of any of the above aspects and embodiments, 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: 59, and the polynucleotide encoding the expression product is the wild-type form of the gene mutated in the cochlear supporting cells. In some embodiments, the genetic hearing loss is associated with a disease listed in Table 6, and the polynucleotide encoding the expression product is a transgene corresponding to the wild-type form of the gene mutated in the disease (e.g., a gene listed in the same row as the disease in Table 6).

[0055] In another aspect, the invention provides a method of improving cochlear supporting cell function or cochlear supporting cell survival by contacting the cochlear supporting cells with a nucleic acid vector or composition of any of the preceding aspects or embodiments. In some embodiments, the contacting is performed in vivo (e.g., within a subject).

[0056] In another aspect, the invention provides a method of improving cochlear supporting cell function or cochlear supporting cell survival in a subject in need thereof by administering to the inner ear of the subject a therapeutically effective amount of a nucleic acid vector or composition of any of the preceding aspects and embodiments.

[0057] In some embodiments of any of the aforementioned aspects, the subject has or is at risk of developing hearing loss (e.g., sensorineural hearing loss or hearing loss).

[0058] In some embodiments of any of the above aspects, the hearing loss is acquired hearing loss.In some embodiments, the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss.In some embodiments, the ototoxic drug is an aminoglycoside, an antitumor drug, ethacrynic acid, furosemide, salicylate, or quinine.

[0059] In some embodiments of any of the foregoing aspects, the hearing loss is genetic hearing loss. In some embodiments, the genetic hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss.

[0060] In some embodiments of any of the aforementioned aspects, the cochlear supporting cells are mammalian cochlear supporting cells. In some embodiments, the mammalian cochlear supporting cells are human cochlear supporting cells.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In some embodiments of any of the foregoing aspects, the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, prevent or reduce tinnitus, delay the onset of hearing loss, slow the progression of hearing loss, improve hearing, increase or induce expression of an expression product in a GJB2 expressing cell, increase the number of cochlear hair cells, increase the regeneration of cochlear hair cells, increase the proliferation of cochlear supporting cells, promote or increase the survival of cochlear supporting cells, induce or increase the differentiation of cochlear supporting cells into hair cells, or improve the function of cochlear supporting cells.

[0065] In some embodiments of any of the aforementioned aspects, the subject is a human subject.

[0066] In another aspect, the invention provides a kit comprising a polynucleotide, nucleic acid vector, or composition of any of the preceding aspects and embodiments.

[0067] definition As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0068] As used herein, "administration" refers to providing or giving a therapeutic agent (e.g., a nucleic acid vector comprising a GJB2 promoter and / or GJB2 enhancer operably linked to a polynucleotide encoding an expression product) to a subject by any effective route. Exemplary administration routes are described herein below.

[0069] As used herein, the phrase "administering to the inner ear" refers to providing or giving a therapeutic agent described herein to a subject by any route that allows 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 GJB2-expressing inner ear cells.

[0070] 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 profiles. 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.

[0071] 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. [Table 1]

[0072] 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 mutation or substitution is one that replaces one amino acid with a member of the same amino acid family (e.g., Ser with Thr or Lys with Arg).

[0073] As used herein, the terms "derived from" and "derivative" refer to a nucleic acid, peptide, or protein, or a variant or analog thereof, that contains one or more mutations and / or chemical modifications compared to a corresponding full-length wild-type nucleic acid, peptide, or protein. Non-limiting examples of chemical modifications involving nucleic acids include, for example, modifications to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or combinations thereof.

[0074] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition, vector construct, or viral vector described herein refer to an amount sufficient to achieve a beneficial or desired result, including a clinical result, when administered to a subject, including a mammal, e.g., a human, and thus "effective amount" or its synonyms depend on the context in which it is applied. For example, in the context of treating sensorineural hearing loss, it is the amount of the composition, vector construct, or viral vector sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition, vector construct, or viral vector. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors, such as a given drug, pharmaceutical formulation, route of administration, type of disease or disorder, characteristics of the subject or host being treated (e.g., age, sex, weight), etc., 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 results in a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of the disclosed composition, vector construct, or viral vector can be readily determined by one skilled in the art by routine methods known in the art. Dosage regimens can be adjusted to provide the optimal therapeutic response.

[0075] 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., humans) 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).

[0076] As used herein, the term "expression" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' 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.

[0077] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell such as a human cell, e.g., a human cochlear supporting cell). Exogenous materials include those provided to an organism from a foreign source or culture materials extracted therefrom.

[0078] As used herein, the term "functional portion," when referring to a promoter sequence described herein (e.g., the GJB2 promoter sequence), refers to a nucleotide sequence that is shorter than SEQ ID NO: 1 or SEQ ID NO: 2 and that is capable of recruiting RNA polymerase to drive transcription of a gene to which it is operably linked. For example, in the context of the present disclosure, a functional portion of SEQ ID NO: 1 can be any one of SEQ ID NOs: 3-19, and a functional portion of SEQ ID NO: 2 can be any one of SEQ ID NOs: 20-28.

[0079] As used herein, the terms "Gjb2" and "GJB2" (also known as connexin 26 and CX26) refer to the protein encoded by the GJB2 gene and the gene encoding this protein, respectively. GJB2 is a member of the connexin gene family. Nearly half of all hearing loss is due to mutations in one of the four members of the connexin gene family, with GJB2 mutations being the most common. Over 100 different mutations in GJB2 have been identified that cause nonsyndromic hearing loss, which is hearing loss that is not associated with other signs and symptoms. The terms "Gjb2" and "GJB2" also refer to a variant 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 99.9% identity or greater) to the amino acid sequence of wild-type Gjb2 (e.g., SEQ ID NO:38 or SEQ ID NO:45), respectively, or a variant 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 99.9% identity or greater) to the amino acid sequence of the wild-type GJB2 gene (e.g., SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:46) or a codon-optimized sequence thereof (e.g., , any one of SEQ ID NOs: 41-44), provided that the encoded Gjb2 analog retains the therapeutic function of wild-type (WT) Gjb2 (e.g., the ability to form hemichannels in supporting cells).

[0080] As used herein, the term "GJB2 expressing cells" refers to cell types in the body that are known to endogenously express GJB2. GJB2 expressing cells include esophageal epithelial cells, cervical cells (cervix vaginalis), cells of the minor salivary glands, skin epithelial cells, vaginal epithelial cells, respiratory epithelial cells, liver hepatocytes, kidney epithelial cells, cells of the testes, luminal epithelial cells of the mammary gland, pancreatic acinar cells, bladder urothelial cells, intestinal epithelial cells, and GJB2 expressing inner ear cells.

[0081] As used herein, the term "GJB2 enhancer" refers to a polynucleotide that can be operably linked to a promoter (e.g., a GJB2 promoter or an inner ear cell type specific promoter such as a cochlear supporting cell specific promoter) to regulate gene expression in a GJB2 expressing cell. A GJB2 enhancer for use in the compositions and methods described herein has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or greater) to any one of SEQ ID NOs: 52-63. The GJB2 enhancer described herein can be operably linked to a promoter that is operably linked to a polynucleotide encoding an expression product, and can increase the expression level of the expression product in a GJB2 expressing cell and increase the number of GJB2 expressing cells in which the expression product is expressed.

[0082] As used herein, the term "GJB2-expressing inner ear cells" refers to cells in the inner ear that endogenously express GJB2. GJB2-expressing cells in the ear are found in both the cochlea and the vestibule. Cochlear GJB2-expressing cells include inner phalangeal cells, inner border cells, inner pillar cells, outer pillar cells, Deiter's cells, Hensen's cells, Claudius cells, interdental cells, inner sulcus cells, outer sulcus cells, spiral lamina margin cells, spiral eminence cells, root cells, stria vascularis basal cells, stria vascularis intermediate cells, fibrocytes of the spiral lamina margin and spiral ligament, and mesenchymal cells lining the scala vestibule. Vestibular GJB2-expressing cells include supporting cells, dark cells, fibrocytes, and mesenchymal cells.

[0083] As used herein, the term "GJB2 promoter" refers to a polynucleotide or variant thereof that can express a transgene specifically in a GJB2-expressing cell, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a GJB2 promoter described herein. The GJB2 promoter of the present disclosure contains one or more regulatory elements from the GJB2 locus and has a sequence comprising a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof. The first region can be directly linked (eg, fused) to the second region, or the first region can be linked to the second region by a nucleic acid linker.

[0084] As used herein, the term GJB2-associated hearing loss refers to diseases and conditions characterized by hearing loss associated with mutations in GJB2, such as DFNB1, which is characterized by moderate to severe prelingual hearing loss and is inherited in an autosomal recessive pattern, and DFNA3, which is characterized by moderate to severe prelingual or perilingual hearing loss that becomes more severe over time and is inherited in an autosomal dominant pattern. GJB2-associated hearing loss also occurs in Bart-Pumphrey syndrome, porcupine ichthyosis with hearing loss, keratitis-ichthyosis-hearing loss syndrome, palmoplantar keratoderma with hearing loss, and Vohwinkel syndrome, all of which are characterized by hearing loss and skin abnormalities and are associated with mutations in GJB2. Two types of GJB2-associated hearing loss, DFNB1 and DFNA3, can also be associated with mutations in GJB6, either alone or in combination with mutations in GJB2. For example, a subject with DFNB1 can have a mutation in GJB2, a mutation in GJB6, or a mutation in both genes.

[0085] 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.

[0086] As used herein, the terms "increasing" and "decreasing" refer to modulating to produce a greater or lesser amount of a metric function, expression, or activity, respectively, relative to a reference. For example, after administering a composition in a manner described herein, the amount of a marker of a metric as described herein (e.g., transgene expression, ABR, or DPOAE) may increase or decrease 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 in a subject relative to the amount of the marker before administration. Generally, the metric is measured after administration at a time when administration has had the recited effect, e.g., at least 1 week, 1 month, 3 months, or 6 months after the treatment regimen has begun.

[0087] As used herein, "locally" or "local administration" refers to administration at a particular site of the body where a local, rather than a systemic, effect is intended. Examples of local administration are epithelial, inhalation, intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intralesional, lymph node, intratumor, administration to the inner ear, and administration to mucous membranes of a subject, where administration is intended to have a local, rather than a systemic, effect.

[0088] As used herein, the term "operably linked" refers to a first molecule bound to a second molecule, where the molecules are positioned such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single continuous molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter regulates the transcription of a 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 may be operably linked to each other without an intervening nucleotide.

[0089] 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 that can transport another nucleic acid to which it is 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 are thereby replicated along with the host genome. Certain plasmids can direct the expression of genes to which they are operatively linked.

[0090] As used herein, the term "polynucleotide" refers to a polymer of nucleosides. Typically, polynucleotides are composed of nucleosides found naturally in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. The term encompasses molecules that contain nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When the application refers to polynucleotides, it is understood that both DNA, RNA, and in each case, both single-stranded and double-stranded forms (as well as the complementary strand of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or to 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.

[0091] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives transcription of the transgene.

[0092] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in a reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways that are 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 over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and Y is the total number of nucleic acids in B. It will be understood 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.

[0093] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharma- ceutically acceptable excipients, diluents, and / or carriers, administered to a subject, such as a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition that is or may be affecting the subject.

[0094] As used herein, the term "pharmacologically 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.

[0095] 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).

[0096] As used herein, the terms "subject" and "patient" refer to an animal (e.g., a mammal, such as a human). A subject treated by the methods described herein may be a subject diagnosed with hearing loss (e.g., sensorineural hearing loss) or at risk for developing this condition (e.g., due to a genetic mutation or risk factors for hearing loss, such as ototoxic drugs, loud noise, head trauma, disease or infection, 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 according to the present disclosure may have been subjected to standard testing or may have been identified, without testing, as a subject at risk due to the presence of one or more risk factors associated with a disease or condition.

[0097] As used herein, the terms "transcriptional regulatory element" and "regulatory sequence" refer to a polynucleotide that controls, at least in part, 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).

[0098] 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, and the like.

[0099] 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 a cell and subsequent transfer and integration of the vector construct or a portion thereof into the cellular genome.

[0100] As used herein, "treatment" and "treating" in reference to a disease or condition refer to an approach to obtain a beneficial or desired result, e.g., a clinical result. Beneficial or desired results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of the disease or condition, a stable (i.e., not worsening) state of the disease, disorder, or condition, prevention of the spread of the disease or condition, delay or slowing of the progression of the disease or condition, remission or palliation of the disease or condition, and remission (whether partial or complete). "Ameliorating" or "alleviating" a disease or condition means that the extent and / or undesirable clinical symptoms of the disease, disorder, or condition are reduced and / or the time course of progression is slowed or extended compared to the extent or time course in the absence of treatment. "Treatment" may also mean extending survival compared to the expected survival in the absence of treatment. Those in need of treatment include those already suffering from the condition or disorder as well as those susceptible to the condition or disorder or those in whom the condition or disorder is to be prevented.

[0101] As used herein, the term "vector" refers to a nucleic acid vector, such as a DNA vector, such as a plasmid, cosmid, or artificial chromosome, an RNA vector, a virus, or any other suitable replicon (e.g., a viral vector). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, Massachusetts, 2006). Expression vectors suitable for use with 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. Certain vectors that can be used for expression of transgenes as described herein include vectors that contain regulatory sequences, such as promoter and enhancer regions that direct gene transcription. Other useful vectors for expression of transgenes contain polynucleotide sequences that increase the translation rate of the transgene or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may contain a polynucleotide encoding a marker for the selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0102] As used herein, the term "wild type" refers to the most frequently occurring genotype for a particular gene in a given organism. [Brief description of the drawings]

[0103] [Figure 1] A, Graph and series of images showing that ubiquitous promoter Gjb2 delivery contributed to elevated ABR thresholds and IHC loss in wild type animals. ABR thresholds for injected (triangles), non-injected contralateral (circles, three frequencies measured), and untreated ears (black diamonds) are shown. B, Graph and series of images showing that ubiquitous promoter Gjb2 delivery contributed to elevated ABR thresholds and IHC loss in wild type animals. Seven of nine wild type ears treated with CMV-Gjb2 showed elevated ABR thresholds (brackets). Inner hair cell loss in wild type ears (Pou4f3: inner hair cells, lower bracket; Prestin: outer hair cells, upper bracket) is shown relative to normal ABR responses. Animals with elevated ABR responses had no remaining inner hair cells. [Diagram 2]A is a series of graphs showing luciferase activity (detected using Nano-Glo luciferase assay) resulting from transfection of HeLa cells with plasmids containing promoter sequences and NanoLuc reporter. GJB2 promoters with sequences of SEQ ID NOs: 4-7, 9, 13, 30-32, 36, and 37 were cloned upstream of the NanoLuc reporter. HeLa cells were transfected with the plasmids containing the promoter and reporter. After 24 hours, NanoLuc expression was detected and quantified using Nano-Glo luciferase assay (Promega® Catalog No. N1110). Results are shown in A. MLP = control minimal promoter (unrelated to GJB2). pGL3 Basic + NanoLuc = promoterless control vector. B is a series of graphs showing luciferase activity (detected using Nano-Glo luciferase assay) resulting from transfection of HeLa cells with plasmids containing promoter sequences and NanoLuc reporter. GJB2 promoters with sequences of SEQ ID NOs: 4-7, 9, 13, 30-32, 36, and 37 were cloned upstream of the NanoLuc reporter. HeLa cells were transfected with plasmids containing the promoter and reporter. After 24 hours, NanoLuc expression was detected and quantified using the Nano-Glo Luciferase Assay (Promega® Cat. No. N1110). Results are shown in B. MLP = control minimal promoter (unrelated to GJB2). pGL3 Basic + NanoLuc = promoterless control vector. [Diagram 3]A is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested were AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP. Samples infected with the GJB2 promoter alone (A and D) showed lower expression of the reporter protein compared to samples infected with vectors containing the GJB2 promoter and enhancer (B, C, and E). B is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested were AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP-Enhancer 9 (SEQ ID NO: 62). Samples infected with the GJB2 promoter alone (A and D) showed lower expression of the reporter protein compared to samples infected with vectors containing the GJB2 promoter and enhancer (B, C, and E). C is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested were AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP-Enhancer GH (SEQ ID NO: 60). Samples infected with the GJB2 promoter alone (A and D) showed lower expression of the reporter protein compared to samples infected with the vector containing the GJB2 promoter and enhancer (B, C, and E). D is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer.Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested were AAV1-p.hGJB2 (SEQ ID NO: 32)-H2B-GFP. Samples infected with the GJB2 promoter alone (A and D) showed lower expression of the reporter protein compared to samples infected with vectors containing the GJB2 promoter and enhancer (B, C, and E). E is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. AAV1-p.hGJB2 (SEQ ID NO: 32)-H2B-GFP-Enhancer9 (SEQ ID NO: 62). Samples infected with the GJB2 promoter alone (A and D) showed lower expression of the reporter protein compared to samples infected with the vector containing the GJB2 promoter and enhancer (B, C, and E). [Figure 4]A is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested were AAV1-p.hGJB2 (SEQ ID NO: 1)-H2B-GFP. Samples infected with the GJB2 promoter of SEQ ID NO: 1 alone showed the lowest expression of the reporter protein. B is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested were: AAV1-p.hGJB2 (SEQ ID NO: 1)-H2B-GFP-Enhancer 9 (SEQ ID NO: 62). For both the promoter and the promoter + spice acceptor (SEQ ID NO: 35), the addition of the enhancer element showed the highest level of reporter protein expression. C is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested were: AAV1-p.hGJB2 (SEQ ID NO: 1)-H2B-GFP-Enhancer GH (SEQ ID NO: 60). For both the promoter and the promoter + spice acceptor (SEQ ID NO: 35), the addition of the enhancer element showed the highest level of reporter protein expression. D is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations.Samples were infected with 2E10 vg / culture. Combinations tested were: AAV1-p.hGJB2 (SEQ ID NO: 35)-H2B-GFP. Addition of the splice acceptor (SEQ ID NO: 22) resulted in increased expression of the promoter alone. E is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. Combinations tested were: AAV1-p.hGJB2 (SEQ ID NO: 35)-H2B-GFP-Enhancer9 (SEQ ID NO: 62). For both the promoter and the promoter+splice acceptor (SEQ ID NO: 35), addition of the enhancer element showed the highest levels of reporter protein expression. F is a series of images showing GFP expression induced by different combinations of the GJB2 promoter and enhancer. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested were: AAV1-p.hGJB2 (SEQ ID NO: 35)-H2B-GFP-Enhancer GH (SEQ ID NO: 60). For both the promoter and promoter + spice acceptor (SEQ ID NO: 35), the addition of the enhancer element showed the highest levels of reporter protein expression. [Figure 5A]A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancer elements. To identify the DNA element within the GH enhancer responsible for enhancer activity, three truncations of the enhancer GH were tested in combination with the GJB2 promoter of SEQ ID NO: 30: GHA, GHB, and GHC. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. Explant cultures infected with AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGH(SEQ ID NO:60) (Figure 5A) or AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHA(SEQ ID NO:61) (Figure 5B) retained strong reporter expression, whereas truncation of EnhancerGH (AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHB(SEQ ID NO:64) (Figure 5C) and AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHC(SEQ ID NO:65) (Figure 5D)) lost enhancer activity. [Figure 5B]A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancer elements. To identify the DNA element within the GH enhancer responsible for enhancer activity, three truncations of the enhancer GH were tested in combination with the GJB2 promoter of SEQ ID NO: 30: GHA, GHB, and GHC. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. Explant cultures infected with AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGH(SEQ ID NO:60) (Figure 5A) or AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHA(SEQ ID NO:61) (Figure 5B) retained strong reporter expression, whereas truncation of EnhancerGH (AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHB(SEQ ID NO:64) (Figure 5C) and AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHC(SEQ ID NO:65) (Figure 5D)) lost enhancer activity. [Figure 5C]A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancer elements. To identify the DNA element within the GH enhancer responsible for enhancer activity, three truncations of the enhancer GH were tested in combination with the GJB2 promoter of SEQ ID NO: 30: GHA, GHB, and GHC. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. Explant cultures infected with AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGH(SEQ ID NO:60) (Figure 5A) or AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHA(SEQ ID NO:61) (Figure 5B) retained strong reporter expression, whereas truncation of EnhancerGH (AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHB(SEQ ID NO:64) (Figure 5C) and AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHC(SEQ ID NO:65) (Figure 5D)) lost enhancer activity. [Figure 5D]A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancer elements. To identify the DNA element within the GH enhancer responsible for enhancer activity, three truncations of the enhancer GH were tested in combination with the GJB2 promoter of SEQ ID NO: 30: GHA, GHB, and GHC. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. Explant cultures infected with AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGH(SEQ ID NO:60) (Figure 5A) or AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHA(SEQ ID NO:61) (Figure 5B) retained strong reporter expression, whereas truncation of EnhancerGH (AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHB(SEQ ID NO:64) (Figure 5C) and AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHC(SEQ ID NO:65) (Figure 5D)) lost enhancer activity. [Figure 6]A is a series of images showing GFP expression induced by the GJB2 promoter with or without the predicted histone marks found within intron 1 of the GJB2 locus. To determine whether methylated DNA elements within the first intron of the GJB2 locus affect promoter activity, the addition of these DNA elements within the GJB2 promoter of SEQ ID NO: 30 was tested. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoters. Samples were infected with 2E10 vg / culture. Similar levels of reporter protein were detected in all samples (AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP-EnhancerGH (SEQ ID NO: 60)), indicating that methylated DNA (histone marks) do not affect expression. B is a series of images showing GFP expression induced by the GJB2 promoter with or without the predicted histone marks found within intron 1 of the GJB2 locus. To determine whether methylated DNA elements within the first intron of the GJB2 locus affect promoter activity, the addition of these DNA elements within the GJB2 promoter of SEQ ID NO: 30 was tested. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoters. Samples were infected with 2E10 vg / culture. C is a series of images showing GFP expression induced by the GJB2 promoter with or without the predicted histone marks found within intron 1 of the GJB2 locus. To determine whether methylated DNA elements within the first intron of the GJB2 locus affect promoter activity, the addition of these DNA elements within the GJB2 promoter of SEQ ID NO: 30 was tested. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoters. Samples were infected with 2E10 vg / culture.This was detected in AAV1-p.hGJB2 (sequence number 33)-H2B-GFP-enhancerGH (sequence number 60) and AAV1-p.hGJB2 (sequence number 34)-H2B-GFP-enhancerGH (sequence number 60), indicating that methylated DNA (histone marks) does not affect expression. [Figure 7A] Figure 1 is a series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancers. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested are listed below: AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP. [Figure 7B] A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancers. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested are listed below: AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP-Enhancer GH (SEQ ID NO: 60). Samples infected with the combination of promoter and full-length enhancer (Enhancer GH and Enhancer 9) showed higher expression of the reporter protein (B and D) when compared to the truncated enhancers (C and E). [Figure 7C]A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancers. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested are listed below: AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP-Enhancer GHA (SEQ ID NO: 61). Samples infected with combinations of the promoter and full-length enhancer (Enhancer GH and Enhancer 9) showed higher expression of the reporter protein (B and D) when compared to the truncated enhancers (C and E). [Figure 7D] A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancers. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested are listed below: AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP-Enhancer 9 (SEQ ID NO: 62). Samples infected with the combination of promoter and full-length enhancer (Enhancer GH and Enhancer 9) showed higher expression of the reporter protein (B and D) when compared to the truncated enhancers (C and E). [Figure 7E]A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancers. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested are listed below: AAV1-p.hGJB2 (SEQ ID NO: 30)-H2B-GFP-Enhancer 9C (SEQ ID NO: 63). Samples infected with the combination of promoter and full-length enhancer (Enhancer GH and Enhancer 9) showed higher expression of the reporter protein (B and D) when compared to the truncated enhancers (C and E). [Figure 7F] A series of images showing GFP expression induced by the GJB2 promoter in combination with various enhancers. Explant cultures of the organ of Corti were established from P4-P6 mice and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. Samples were infected with 2E10 vg / culture. The combinations tested are listed below: AAV1-CMV-H2B-GFP (Figure 7F). Expression of the reporter protein was observed in all cells in the control sample (AAV1-CMV-H2B-GFP, (F)). [Figure 8]A is a series of images showing that enhancer GH in AAV-DJ vector induced more GFP expression in adult mouse cochlea compared to enhancer 9. Representative images of whole specimen cochlea show GFP expression (GFP, white) in supporting cells of the cochlea. B is a series of images showing that enhancer GH in AAV-DJ vector induced more GFP expression in adult mouse cochlea compared to enhancer 9. Representative images of whole specimen cochlea show GFP expression (GFP, white) in supporting cells of the cochlea. C is a series of images showing that enhancer GH in AAV-DJ vector induced more GFP expression in adult mouse cochlea compared to enhancer 9. GFP was excluded from hair cells (Myo7a, grey) in the sensory epithelium (white brackets). A cross-section of the adult mouse cochlea confirmed that GFP expression was restricted to supporting cells (GFP+ nuclei, dark grey, black arrows) and excluded from hair cells in the sensory epithelium (brackets). B A series of images showing that enhancer GH induced more GFP expression in the adult mouse cochlea compared to enhancer 9 in the AAV-DJ vector. GFP was excluded from hair cells (Myo7a, grey) in the sensory epithelium (white brackets). C A cross-section of the adult mouse cochlea confirmed that GFP expression was restricted to supporting cells (GFP+ nuclei, dark grey, black arrows) and excluded from hair cells in the sensory epithelium (brackets). D A series of images showing that enhancer GH induced more GFP expression in the adult mouse cochlea compared to enhancer 9 in the AAV-DJ vector. GFP was excluded from hair cells (Myo7a, grey) in the sensory epithelium (white brackets). E A series of images showing that enhancer GH induced more GFP expression in the adult mouse cochlea compared to enhancer 9 in the AAV-DJ vector. GFP was excluded from hair cells (Myo7a, grey) in the sensory epithelium (white brackets). D A series of images showing that enhancer GH induced more GFP expression in the adult mouse cochlea compared to enhancer 9 in the AAV-DJ vector. GFP was excluded from hair cells (Myo7a, grey) in the sensory epithelium (white brackets). [Figure 9]A, A series of images showing that enhancer GH induced more GFP expression in neonatal mouse cochleae compared to enhancer 9 in AAV-DJ vector. Representative images of whole specimen cochleae show GFP expression in supporting cells of the cochlea (GFP, white). GFP was excluded from hair cells (Myo7a, grey) in the sensory epithelium (white bracket). B, A series of images showing that enhancer GH induced more GFP expression in neonatal mouse cochleae compared to enhancer 9 in AAV-DJ vector. Representative images of whole specimen cochleae show GFP expression in supporting cells of the cochlea (GFP, white). Representative images of whole specimen cochleae show GFP expression in supporting cells of the cochlea (GFP, white). GFP was excluded from hair cells (Myo7a, grey) in the sensory epithelium (white bracket). [Figure 10] A is a series of images and graphs showing that enhancer GH in AAV1 induced GFP expression in supporting cells of the neonatal mouse ear. Representative images of whole specimen cochleae show GFP expression in specific frequency regions (GFP, white; Pou4f3 hair cells, light grey). The top panel is a merged image showing GFP and Pou4f3, and the bottom panel shows GFP only. B is a series of images and graphs showing that enhancer GH in AAV1 induced GFP expression in supporting cells of the neonatal mouse ear. Representative images of whole specimen cochleae show GFP expression in specific frequency regions. GFP expression was excluded from hair cells of the sensory epithelium (brackets). Quantification of GFP+ supporting cells shows more GFP expression in central supporting cells compared to lateral supporting cells. The top panel is a merged image showing GFP and Pou4f3, and the bottom panel shows GFP only. [Figure 11]A, A series of images showing that enhancer GH in AAV1 induced GFP expression in the lateral wall and stria vascularis of neonatal mouse ear. Two representative images of the whole specimen lateral wall show GFP expression in the lateral wall and stria vascularis (GFP, white, DAPI, dark grey) (A of A). Whole specimen preparation with marginal cell layer of the stria vascularis seen. GFP positive nuclei highlighted with arrows. z,y orthogonal projection through the lateral wall (A' and B' of Figures 11A-11B). The basal cell layer of the stria vascularis is on the left and the marginal cell layer is on the right. B, B series of images showing that enhancer GH in AAV1 induced GFP expression in the lateral wall and stria vascularis of neonatal mouse ear. Two representative images of the whole specimen lateral wall show GFP expression in the lateral wall and stria vascularis (GFP, white, DAPI, dark grey) (B of B). Whole specimen preparation with marginal cell layer of the stria vascularis seen. GFP positive nuclei highlighted with arrows. A z,y orthogonal projection through the lateral wall (B' in B). The basal cell layer of the stria vascularis is on the left and the marginal cell layer is on the right. [Figure 12] A, Representative images of whole specimen cochlea, showing GFP expression in specific frequency regions (GFP, white; Pou4f3 hair cells, light grey). Top panel is a merged image showing GFP and Pou4f3, bottom panel shows GFP only. GFP expression was excluded from hair cells of the sensory epithelium (brackets). B, Set of images and graphs showing that enhancer GH in Php.B drives GFP expression in supporting cells of the neonatal mouse ear. B, Quantification of GFP+ supporting cells. Top panel is a merged image showing GFP and Pou4f3, bottom panel shows GFP only. GFP expression was excluded from hair cells of the sensory epithelium (brackets). [Figure 13]A is a series of images showing GFP expression from enhancer GH in Php.B serotype delivered at neonatal age. Cross sections of mouse cochlea show that GFP expression is restricted to supporting cells and lateral wall (GFP+nuclei, dark grey, black arrows) and excluded from hair cells in the sensory epithelium (brackets). B is a series of images showing GFP expression from enhancer GH in Php.B serotype delivered at neonatal age. Cross sections of mouse cochlea show that GFP expression is restricted to supporting cells and lateral wall (GFP+nuclei, dark grey, black arrows) and excluded from hair cells in the sensory epithelium (brackets). [Figure 14] A series of graphs and images showing that the GJB2 promoter of SEQ ID NO: 30 combined with enhancer GH restored hearing and prevented the loss of outer hair cells in gene replacement gene therapy in a GJB2-deficient mouse model. Using the Php.B serotype, delivery of GJB2 to neonatal mice resulted in an average of 40 dB of hearing improvement. The left panel shows the ABR and DPOAE results 4 weeks after injection compared to the contralateral ear. The center panel shows the ABR waveform of a representative responder. The right image shows that outer hair cells (OHCs) were preserved in treated ears compared to the contralateral control (Prestin, white). [Figure 15] A series of graphs and images showing that the GJB2 promoter of SEQ ID NO: 30 combined with enhancer GH restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model. Using the AAV1 serotype to deliver GJB2 to neonatal mice resulted in an average of 20 dB of hearing improvement. The left panel shows the ABR and DPOAE results 4 weeks after injection compared to the contralateral ear. The center panel shows the ABR waveform of a representative responder. The right image shows that outer hair cells (OHCs) were preserved in the treated ear compared to the contralateral control (Prestin, white). Similar results were observed using the AAV-DJ serotype. [Figure 16]A series of images showing GFP expression induced by various enhancers in neonatal cochlear explants. Enhancers driving histone-tagged GFP were screened in the explants using AAV-DJ. Expression patterns, GFP intensity, and exclusion from hair cells were assessed. The sensory epithelium is marked with brackets (GFP, white; Myo7a, grey). High GFP expression was observed for enhancer 1, enhancer 8, and combinations of two enhancers (enhancer 1+6+7+8 and enhancer 2+3+4+5). Complete exclusion from hair cells was observed for enhancers 1, 4, 5, and 6, as well as combinations of two enhancers. GFP was observed in the sensory epithelium region for all enhancers and enhancer combinations tested, except enhancer 7. Enhancer 1: SEQ ID NO: 52, Enhancer 2: SEQ ID NO: 53, Enhancer 3: SEQ ID NO: 54, Enhancer 4: SEQ ID NO: 55, Enhancer 5: SEQ ID NO: 56, Enhancer 6: SEQ ID NO: 57, Enhancer 7: SEQ ID NO: 58, Enhancer 8: SEQ ID NO: 59. [Figure 17] A series of images showing GFP expression induced by different enhancers in vivo. Neonatal mice were injected with various enhancers paired with the GJB2 promoter of SEQ ID NO: 30 using the AAV-DJ serotype. Whole specimens (top row) and sections (bottom row) of the inner ear were collected. With enhancer 1, GFP expression was present in all supporting cells of the sensory epithelium and many non-sensory cell types (top row). For all enhancers tested (GFP, white; Myo7a hair cells, grey), GFP was excluded from hair cells. GFP was detected in the lateral wall with enhancer 4. Section histology confirms the whole specimen expression (bottom row, GFP, dark grey; black arrows indicate examples of GFP+ cells). [Figure 18]A series of images showing GFP expression induced by different enhancers in vivo. Neonatal mice were injected with various enhancers paired with the GJB2 promoter of SEQ ID NO: 30 using the AAV-DJ serotype. Whole specimens (top row) and sections (bottom row) of the inner ear were collected. With enhancer 8, GFP expression was restricted to supporting cells of the sensory epithelium (top row). For all enhancers tested, GFP was excluded from hair cells. GFP expression was observed in Reissner's membrane for enhancer 6 and the combination of enhancers 2+3+4+5. With the combination of enhancers 1+6+7+8, GFP expression was present in all supporting cells of the sensory epithelium, most non-sensory cells of the cochlear duct, cells of the lateral wall and stria vascularis (GFP, white; Myo7a hair cells, grey). Section histology confirms the whole specimen expression (bottom row, GFP, dark grey; black arrows indicate examples of GFP+ cells). [Figure 19]FIG. 1 is a series of schematic diagrams showing the order of elements (e.g., promoter, coding sequence (GFP or mGJB2), WPRE (if present), polyadenylation sequence (polyA), and enhancer (if present)) contained in vectors used in the experiments described herein. FIG. 2 is a series of schematic diagrams showing the order of elements in vectors used to generate the data shown in FIGS. 3A-3E, 4A-4F, 5A-5D, 6A-6C, 7A-7F, 8A-8F, 9A-9B, 10A-10B, 11A-11B, 12A-12B, and 13A-13B. FIG. 3 is a series of schematic diagrams showing the order of elements (e.g., promoter, coding sequence (GFP or mGJB2), WPRE (if present), polyadenylation sequence (polyA), and enhancer (if present)) contained in vectors used in the experiments described herein. FIG. 4 is a series of schematic diagrams showing the order of elements in vectors used to generate the data shown in FIGS. 14 and 15. FIG. 1C is a series of schematic diagrams showing the order of elements (e.g., promoter, coding sequence (GFP or mGJB2), WPRE (if present), polyadenylation sequence (polyA), and enhancer (if present)) contained in vectors used in the experiments described herein. FIG. 1C is a series of schematic diagrams showing the order of elements in vectors used to generate the data shown in FIGS. 16-18. [Figure 20A] A series of graphs showing that overexpression of GJB2 using the GJB2 promoter / enhancer pair is safer than overexpression of GJB2 using the ubiquitous promoter. Wild-type mice were injected with vehicle control or human GJB2 transgene driven by the ubiquitous promoter (CMV) or the GJB2 promoter / enhancer pair (GJB2 promoter of SEQ ID NO: 30 and GJB2 enhancer of SEQ ID NO: 52). ABR thresholds at baseline and 2 weeks after injection are shown. Animals in the GJB2 promoter group had ABR thresholds comparable to baseline measurements, as seen in the vehicle group, while animals in the ubiquitous promoter group showed elevated ABR thresholds. This indicates that non-specific hGJB2 expression leads to hearing loss. [Figure 20B] 1 is a series of graphs showing that overexpression of GJB2 using the GJB2 promoter / enhancer pair is safer than overexpression of GJB2 using the ubiquitous promoter. Wild-type mice were injected with vehicle control or the human GJB2 transgene driven by the ubiquitous promoter (CMV) or the GJB2 promoter / enhancer pair (GJB2 promoter of SEQ ID NO: 30 and GJB2 enhancer of SEQ ID NO: 52). The number of inner and outer hair cells of animals across treatment groups is shown. Loss of inner hair cells was seen in the ubiquitous promoter group, indicating that off-target GJB2 expression results in inner hair cell toxicity, which can be mitigated through the use of the GJB2 promoter. [Figure 21A] A series of graphs showing that AAV1 encoding human GJB2 driven by p.hGJB2 (SEQ ID NO: 30) + enhancer1 (SEQ ID NO: 52) restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model. ABR results are shown 4, 8, and 12 weeks after injection, comparing the contralateral ear to that injected. Hearing recovery was observed in the majority of animals. The variability in response seen is likely due to variability in the amount of virus effectively delivered to the inner ear due to surgical technique. In some responder animals, the contralateral ear also appears to recover some hearing. This is due to the fact that viral crossover can occur after neonatal injection. [Figure 21B]A series of graphs showing that AAV1 encoding human GJB2 driven by p.hGJB2 (SEQ ID NO: 30) + enhancer 1 (SEQ ID NO: 52) restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model. DPOAE results are shown 4, 8, and 12 weeks after injection, comparing with the contralateral ear injected. Hearing recovery was observed in the majority of animals. The variability in response seen is likely due to variability in the amount of virus effectively delivered to the inner ear due to surgical technique. In some responder animals, the contralateral ear also appears to recover some hearing. This is due to the fact that viral crossover can occur after neonatal injection. [Figure 21C]

[0023] Figure 1 is a series of graphs showing that AAV1 encoding human GJB2 driven by p.hGJB2 (SEQ ID NO: 30) + enhancer 1 (SEQ ID NO: 52) restored hearing and prevented outer hair cell loss in a GJB2-deficient mouse model in gene replacement gene therapy, showing that outer hair cells were preserved in the injected ear compared to the uninjected contralateral ear. [Figure 22A] 1 is a series of graphs showing that AAV1 encoding human GJB2 driven by AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 1 + 6 + 7 + 8 (SEQ ID NO: 52, 57, 58, 59) restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model. ABR results are shown 4, 12, and 14 weeks after injection compared to the contralateral ear injected. Hearing restoration was observed in a subset of animals. The variability in response seen is likely due to variability in the amount of virus effectively delivered to the inner ear due to surgical technique. [Figure 22B]1 is a series of graphs showing that AAV1 encoding human GJB2 driven by AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 1 + 6 + 7 + 8 (SEQ ID NO: 52, 57, 58, 59) restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model. DPOAE results are shown 4, 12, and 14 weeks after injection compared to the contralateral ear injected. Hearing restoration was observed in a subset of animals. The variability in response seen is believed to be due to variability in the amount of virus effectively delivered to the inner ear due to surgical technique. [Figure 22C] 1 is a series of graphs showing that AAV1 encoding human GJB2 driven by AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 1 + 6 + 7 + 8 (SEQ ID NOs: 52, 57, 58, 59) restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model, showing that outer hair cells were preserved in the injected ear compared to the uninjected contralateral ear. [Figure 23A] 1 is a series of graphs showing that AAV1 encoding human GJB2 driven by AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 8 (SEQ ID NO: 59) restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model. ABR results are shown 4 and 15 weeks after injection, comparing with the contralateral ear injected. Hearing recovery was observed in a subset of animals. The variability in response seen is likely due to variability in the amount of virus effectively delivered to the inner ear due to surgical technique. In some responder animals, the contralateral ear also appears to recover some hearing. This is due to the fact that viral crossover can occur after neonatal injection. [Figure 23B]1 is a series of graphs showing that AAV1 encoding human GJB2 driven by AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 8 (SEQ ID NO: 59) restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model. DPOAE results are shown 4 and 15 weeks after injection, comparing with the contralateral ear injected. Hearing recovery was observed in a subset of animals. The variability in response seen is likely due to variability in the amount of virus effectively delivered to the inner ear due to surgical technique. In some responder animals, the contralateral ear also appears to recover some hearing. This is due to the fact that viral crossover can occur after neonatal injection. [Figure 23C] 1 is a series of graphs showing that AAV1 encoding human GJB2 driven by AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 8 (SEQ ID NO: 59) restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model, showing that outer hair cells were preserved in the injected ear of responder animals compared to the contralateral ear and non-responder animals. [Figure 24] 1 is a series of graphs showing quantification of GFP expression in supporting cells from gross specimen tissues of non-human primates (NHPs). NHPs were injected with AAV1 virus with GJB2enh corresponding to enhancer 1 (SEQ ID NO: 52), enhancer GH (SEQ ID NO: 60), or one of enhancers 1+6+7+8 (SEQ ID NOs: 52, 57, 58, 59), with nuclear GFP driven by promoter / enhancer combinations AAV1-p.hGJB2 (SEQ ID NO: 30)+GJB2enh-GFP. The gross specimen tissues were used to quantitate the percentage of GFP-expressing central (left) and lateral (right) supporting cells. All constructs were able to drive expression in these cell types across the tonotopic axis. [Figure 25A]A series of images showing that the GJB2 promoter of SEQ ID NO: 30 in combination with GJB2 enhancer 1 (SEQ ID NO: 52), enhancer GH (SEQ ID NO: 60), or enhancer 1+6+7+8 (SEQ ID NO: 52, 57, 58, 59) induced GFP expression in NHP cochleae. Representative images of NHPs injected with AAV1 and various promoter / enhancer combinations driving expression of H2B-GFP are shown. AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 1-GFP. Images of various frequencies are shown and are centered on the sensory epithelium. Minimal expression was seen in hair cells. Various promoter / enhancer combinations drove expression in supporting cells to various degrees. In each figure, left: Myo7a (hair cells), center: native GFP, right: anti-GFP. [Figure 25B] A series of images showing that the GJB2 promoter of SEQ ID NO: 30 in combination with GJB2 enhancer 1 (SEQ ID NO: 52), enhancer GH (SEQ ID NO: 60), or enhancer 1+6+7+8 (SEQ ID NO: 52, 57, 58, 59) induced GFP expression in NHP cochleae. Representative images of NHPs injected with AAV1 and various promoter / enhancer combinations driving expression of H2B-GFP are shown. AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer GH-GFP, AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 1+6+7+8-GFP. Images of various frequencies are shown and are centered on the sensory epithelium. Minimal expression was seen in hair cells. Various promoter / enhancer combinations drove expression in supporting cells to various degrees. In each figure, left: Myo7a (hair cells), center: native GFP, right: anti-GFP. [Figure 25C]A series of images showing that the GJB2 promoter of SEQ ID NO: 30 in combination with GJB2 enhancer 1 (SEQ ID NO: 52), enhancer GH (SEQ ID NO: 60), or enhancers 1+6+7+8 (SEQ ID NO: 52, 57, 58, 59) induced GFP expression in NHP cochleae. Representative images of NHPs injected with AAV1 and various promoter / enhancer combinations driving expression of H2B-GFP are shown. Images of various frequencies are shown and are centered in the sensory epithelium. Minimal expression was seen in hair cells. Various promoter / enhancer combinations drove expression in supporting cells to various degrees. In each figure, left: Myo7a (hair cells), center: native GFP, right: anti-GFP. [Figure 26] 1 is a table showing quantification of GFP expression across cochlear cell types from NHP sections. NHPs were injected with AAV1 virus with GJB2enh corresponding to enhancer 1 (SEQ ID NO: 52), enhancer GH (SEQ ID NO: 60), or one of enhancers 1+6+7+8 (SEQ ID NO: 52, 57, 58, 59), with nuclear GFP driven by promoter / enhancer combination AAV1-p.hGJB2 (SEQ ID NO: 30)+GJB2enh-GFP. Cell types in the sections were scored from 0 to 3, from lowest to highest expression, based on the amount of staining seen with GFP immunohistochemistry (IHC) or in situ hybridization (ISH). Note that the ISH probe labels both the vector genome and mRNA transcripts. GFP expression was detected in various GJB2 expressing cell types, including especially supporting cells (IBC via Claudius / Hensen). Hair cells appeared to have predominantly ISH but no IHC labeling. G1: enhancer GH, G2: enhancer 1, G3: enhancer 1+6+7+8. IHC: inner hair cell, OHC: outer hair cell, SL: spiral lamina border, IBC: inner border cell, IPhC: inner phalangeal cell, IPC: inner pillar cell, OPC: outer pillar cell, SGN: spiral ganglion neuron, SG glia: spiral ganglion glia. [Figure 27A]27A-27C are a series of images and graphs showing that the GJB2 promoter / enhancer combination induced hGJB2-FLAG expression in central and lateral supporting cells of NHPs. NHPs were injected with 60 μL of AAV1 virus (in one or two doses) carrying human FLAG-tagged GJB2 driven by the GJB2 promoter of SEQ ID NO:30 (AAV1-p.hGJB2(SEQ ID NO:30)+enhancer1(SEQ ID NO:52)-hGJB2-FLAG). Representative images from the sensory epithelium region obtained from two different animals (from the two dose group) at the indicated frequencies are shown in FIG. 27A. The central and lateral supporting cell regions are shown in brackets, as defined using DAPI labeling. FLAG staining closely reflected the endogenous GJB2 pattern. [Figure 27B] 27B is a series of images and graphs showing that the GJB2 promoter / enhancer combination induced hGJB2-FLAG expression in central and lateral supporting cells of NHPs. NHPs were injected with 60 μL of AAV1 virus (in one or two doses) carrying human FLAG-tagged GJB2 driven by the GJB2 promoter of SEQ ID NO:30 (AAV1-p.hGJB2(SEQ ID NO:30)+enhancer1(SEQ ID NO:52)-hGJB2-FLAG). Central and lateral supporting cell regions are shown in brackets, as defined using DAPI labeling. FLAG staining closely reflected the endogenous GJB2 pattern. Quantification of the percentage of FLAG-positive Deiters cells in individual animals is shown in FIG. 27B. The transgene could be detected throughout the length of the cochlea, with higher expression levels seen in the higher virus dose groups. [Figure 28]A series of images of neonatal cochlear explants treated with AAV containing GFP driven by two different promoters: the minimal beta globin promoter (B-glob), which is not specific for any cells in the inner ear, and the proximal GJB2 promoter (proxGJB2) of SEQ ID NO:1. The top row shows staining for myosin 7a, a hair cell marker. The second row shows staining for Sox2, a supporting cell marker. The bottom row shows expression of GFP. The B-glob promoter induced little GFP expression by itself, and in combination with either enhancer 1 (SEQ ID NO:52) or enhancer 8 (SEQ ID NO:59), induced expression in many cells of the sensory epithelium, including both hair cells and supporting cells. The proxGJB2 promoter alone did not induce GFP expression in the sensory epithelium of the cochlea. In combination with enhancer 1, proxGJB2 induced strong expression in lateral supporting cells and outer hair cells, and minimal expression in central supporting cells and inner hair cells. ProxGJB2 in combination with enhancer 8 induced strong expression in all supporting cells and was completely excluded from hair cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] Described herein are compositions and methods for specifically inducing transgene expression in GJB2-expressing cells (e.g., GJB2-expressing inner ear cells, such as cochlear supporting cells). The invention features GJB2 promoters that can induce expression of an expression product (e.g., a protein encoded by a transgene or an RNA molecule, such as an inhibitory RNA molecule) in GJB2-expressing cells (e.g., cochlear supporting cells) with minimal or no expression in cochlear hair cells. In addition, the invention features GJB2 enhancers that can be operably linked to the promoter to induce transgene expression in GJB2-expressing cells (e.g., GJB2-expressing inner ear cells) and minimize off-target expression in non-GJB2-expressing cells (e.g., cochlear hair cells). The GJB2 enhancer can also increase gene expression levels and the number of GJB2-expressing cells in which gene expression can be detected. The invention also features a nucleic acid vector comprising a GJB2 promoter as described herein operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein or an inhibitory RNA), and a nucleic acid vector comprising a GJB2 enhancer as described herein 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 an inhibitory RNA molecule). The compositions and methods described herein can be used to specifically express an expression product (e.g., a protein, an inhibitory RNA, a microRNA, or a component of a gene editing system) in a GJB2-expressing cell, and thus the compositions described herein can be administered to a subject (such as a mammalian subject, e.g., a human) to treat a disorder caused by dysfunction of a GJB2-expressing cell, such as hearing loss (e.g., sensorineural hearing loss, such as GJB2-associated hearing loss, other genetic forms of hearing loss associated with mutations in cochlear supporting cell genes, or hearing associated with loss of cochlear hair cells, such as age-related hearing loss, ototoxic drug-induced hearing loss, noise-induced hearing loss, head trauma-related hearing loss, or disease- or infection-related hearing loss).

[0105] supporting cells The sensory epithelium of the inner ear contains two main cell types: hair cells and supporting cells. Hair cells are the sensory cells of the auditory and vestibular systems present in the inner ear. Cochlear hair cells are the sensory cells of the auditory system and are composed of two main cell types: inner hair cells, which are responsible for detecting sound, and outer hair cells, which are thought to amplify low-level sounds. Vestibular hair cells are located in the end organs and otolithic organs of the semicircular canals of the inner ear and are involved in the sense of movement that contributes to balance and spatial orientation. The development, function, and maintenance of the inner ear sensory epithelium are highly dependent on supporting cells, which are nonsensory cells that reside between the hair cells. Supporting cells of the cochlea include Hensen's cells, Deiters' cells, inner and outer pillar cells, Claudius cells, inner phalangeal cells, and border cells. Supporting cells are connected to each other and to hair cells by tight and adherens junctions, and communicate directly with other supporting cells by gap junctions. Gap junctions are composed of connexins encoded by the connexin genes CX26 (also known as GJB2) and CX30 (also known as GJB6). These connexin channels mediate intracellular K + They play important roles in the recycling and regulation of acetylcholine and in pH homeostasis mechanisms, and may provide a route for the rapid removal of ions from the area of ​​the sensory cells during sound conduction to maintain sensitivity. Supporting cells have a rigid cytoskeleton that maintains the structural integrity of the sensory organ during sound stimulation and head movement, expels damaged hair cells from the epithelium after trauma or toxicity, phagocytose hair cell debris, and in some cases, can generate new hair cells.

[0106] Gene therapy has recently emerged as an attractive therapeutic approach for treating hearing loss, especially hearing loss caused by mutations in genes expressed in the inner ear. Mutations in many different genes, including mutations in genes expressed in cochlear supporting cells, have been found to cause hearing loss. For example, mutations in GJB2 are the most common cause of recessive hearing loss, and mutations in other cochlear supporting cell genes, such as GJB6, SLC26A4, and GAS2, have also been associated with hearing loss. Another potential application of gene therapy is to induce the regeneration of cochlear hair cells, which are often lost or damaged in age-related hearing loss, ototoxic drug-induced hearing loss, noise-induced hearing loss, head trauma-related hearing loss, and disease- or infection-related hearing loss, by inducing the differentiation of cochlear supporting cells into cochlear hair cells. However, using gene therapy to treat hearing loss associated with mutations in cochlear supporting cell genes (e.g., genes expressed in cochlear supporting cells) or to promote differentiation of cochlear supporting cells into cochlear hair cells requires methods to induce gene expression in cochlear hair cells rather than in cochlear supporting cells, which are currently very limited.

[0107] GJB2 Gap junction protein beta 2 (Gjb2, also known as connexin 26) is a protein encoded by the GJB2 gene and is a member of the connexin gene family. Connexin oligomerization into hexameric arrangements called connexons or hemichannels often docks with hemichannels from contacting cells to form gap junctions. Nearly half of all hearing loss is due to mutations in one of the four members of the connexin gene family, with GJB2 mutations being the most common. Over 100 different mutations in GJB2 have been identified that cause nonsyndromic hearing loss, which is hearing loss that is not associated with other signs and symptoms. One form of nonsyndromic hearing loss associated with mutations in GJB2 is DFNB1, which is characterized by moderate to severe prelingual hearing loss and is inherited in an autosomal recessive pattern. DFNA3 is another form of non-syndromic hearing loss associated with mutations in GJB2, which is a moderate to severe pre- or post-lingual hearing loss that becomes more severe over time and is inherited in an autosomal dominant pattern. Other health conditions associated with mutations in GJB2 include Bart-Pumphrey syndrome, porcupine ichthyosis with hearing loss, keratitis-ichthyosis-deafness syndrome, palmoplantar keratoderma with hearing loss, and Vohwinkel syndrome, all of which are characterized by hearing loss and skin abnormalities.

[0108] The present invention is based, in part, on the discovery of a region upstream of the GJB2 coding sequence that can be used to drive transgene expression specifically in GJB2-expressing cells (e.g., cochlear supporting cells). After observing inner hair cell loss and elevated ABR thresholds when GJB2 was expressed in wild-type mice using a ubiquitous promoter, the inventors determined that it would be desirable to identify promoters that could drive transgene expression specifically in GJB2-expressing cells. Thus, the compositions and methods described herein can be used to express an expression product (e.g., a polynucleotide encoding a protein or a polynucleotide that can be transcribed to produce an inhibitory RNA molecule) in a GJB2-expressing cell (e.g., a GJB2-expressing inner ear cell, e.g., a cochlear supporting cell), such as a gene endogenously expressed in a GJB2-expressing cell, a gene in a cochlear supporting cell known to be mutated in a subject with hearing loss (e.g., a gene expressed in a cochlear supporting cell), or a gene that can induce differentiation of a cochlear supporting cell into a cochlear hair cell, to treat a subject with or at risk of developing hearing loss (e.g., sensory hearing loss), hearing loss, and / or tinnitus. The discovery of a GJB2 promoter that drives expression in a GJB2-expressing cell while minimizing or eliminating off-target expression in cells that do not express GJB2 (e.g., a cochlear hair cell) can improve the safety and efficacy of gene therapy by reducing the toxicity associated with off-target expression.

[0109] The polynucleotides of the compositions and methods described herein include nucleic acid sequences from regions of the GJB2 locus capable of expressing a transgene specifically in GJB2-expressing cells, or variants thereof, such as nucleic acid sequences having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to regions of the GJB2 locus capable of expressing a transgene specifically in GJB2-expressing cells. The polynucleotides of the compositions and methods described herein can optionally include a linker that operably links the regions of the GJB2 locus capable of expressing a transgene specifically in GJB2-expressing cells, or the regions of the GJB2 locus can be directly linked without an intervening linker.

[0110] In some embodiments, the polynucleotides described herein contain a first region 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 functional portion or derivative thereof, which is linked (e.g., operably linked) to a second region having at least 85% sequence identity (85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2, or a functional portion or derivative thereof. The functional portion of SEQ ID NO:1 may have the sequence of SEQ ID NO:3, the sequence of SEQ ID NO:4, the sequence of SEQ ID NO:5, the sequence of SEQ ID NO:6, the sequence of SEQ ID NO:7, the sequence of SEQ ID NO:8, the sequence of SEQ ID NO:9, the sequence of SEQ ID NO:10, the sequence of SEQ ID NO:11, the sequence of SEQ ID NO:12, the sequence of SEQ ID NO:4 fused to the sequence of SEQ ID NO:12 without the intervening nucleic acid as set forth in SEQ ID NO:13, the sequence of SEQ ID NO:5 fused to the sequence of SEQ ID NO:12 without the intervening nucleic acid as set forth in SEQ ID NO:14, the sequence of SEQ ID NO:6 fused to the sequence of SEQ ID NO:12 without the intervening nucleic acid as set forth in SEQ ID NO:15, the sequence of SEQ ID NO:7 fused to the sequence of SEQ ID NO:12 without the intervening nucleic acid as set forth in SEQ ID NO:16, the sequence of SEQ ID NO:9 fused to the sequence of SEQ ID NO:12 without the intervening nucleic acid as set forth in SEQ ID NO:17, the sequence of SEQ ID NO:10 fused to the sequence of SEQ ID NO:12 without the intervening nucleic acid as set forth in SEQ ID NO:18, or the sequence of SEQ ID NO:11 fused to the sequence of SEQ ID NO:12 without the intervening nucleic acid as set forth in SEQ ID NO:19. A functional portion of SEQ ID NO:1 may also have the sequence of SEQ ID NO:7 fused to the sequence of SEQ ID NO:12 by an endogenous intervening nucleic acid sequence, the sequence of SEQ ID NO:10 fused to the sequence of SEQ ID NO:12 by an endogenous intervening nucleic acid sequence, or the sequence of SEQ ID NO:11 fused to the sequence of SEQ ID NO:12 by an endogenous intervening nucleic acid sequence.In a polynucleotide in which the first region contains the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and the sequence of SEQ ID NO:12, the two sequences (one of SEQ ID NO:4-7 and 9-11 combined with SEQ ID NO:12) may be included in any order (e.g., one of SEQ ID NO:4-7 and 9-11 may be bound to (e.g., before) SEQ ID NO:12, as in SEQ ID NO:13-19, or SEQ ID NO:12 may be bound to (e.g., before) one of SEQ ID NO:4-7 and 9-11). The functional portion of SEQ ID NO:2 may have the sequence of SEQ ID NO:20, the sequence of SEQ ID NO:21, the sequence of SEQ ID NO:22, the sequence of SEQ ID NO:23, the sequence of SEQ ID NO:24, or the sequence of SEQ ID NO:23 or SEQ ID NO:24 fused to the sequence of SEQ ID NO:20 or SEQ ID NO:22 without an intervening nucleic acid as set forth in SEQ ID NO:25-28. In a polynucleotide in which the first region contains the sequence of SEQ ID NO:23 or SEQ ID NO:24, as well as the sequence of SEQ ID NO:20 or SEQ ID NO:22, the two sequences (one of SEQ ID NO:23 and SEQ ID NO:24, and one of SEQ ID NO:20 and SEQ ID NO:22) may be included in any order (for example, one of SEQ ID NO:23 and SEQ ID NO:24 may be linked to (e.g., before) one of SEQ ID NO:20 and SEQ ID NO:22, such as SEQ ID NOs:25-28, or one of SEQ ID NO:20 and SEQ ID NO:22 may be linked to (e.g., before) one of SEQ ID NO:23 and SEQ ID NO:24).

[0111] The first and second regions of the polynucleotide may be directly linked or may be linked by a nucleic acid linker. For example, the polynucleotide may contain the sequence of SEQ ID NO: 1, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-19) fused to the sequence of SEQ ID NO: 2, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-28) without an intervening nucleic acid. For example, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO:1 to SEQ ID NO:2 is set forth in SEQ ID NO:29, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO:3 to SEQ ID NO:20 is set forth in SEQ ID NO:30, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO:3 to SEQ ID NO:2 is set forth in SEQ ID NO:31, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO:3 to SEQ ID NO:21 is set forth in SEQ ID NO:32, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO:3 to SEQ ID NO:25 is set forth in SEQ ID NO:33, the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO:3 to SEQ ID NO:26 is set forth in SEQ ID NO:34, and the nucleic acid sequence of a polynucleotide resulting from the direct fusion of SEQ ID NO:1 to SEQ ID NO:22 is set forth in SEQ ID NO:35. Alternatively, a linker can be used to join the sequence of SEQ ID NO:1 or a functional part or derivative thereof (e.g., any one of SEQ ID NOs:3-19) to the sequence of SEQ ID NO:2 or a functional part or derivative thereof (e.g., any one of SEQ ID NOs:20-28).

[0112] The length of the nucleic acid linker for use in the polynucleotides described herein can be about 5kb or less (e.g., about 5kb, 4.5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 900bp, 800bp, 700bp, 600bp, 500bp, 450bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, 100bp, 90bp, 80bp, 70bp, 60bp, 50bp, 40bp, 30bp, 25bp, 20bp, 15bp, 10bp, 5bp, 4bp, 3bp, 2bp, or less). The nucleic acid linker that can be used in the polynucleotides described herein does not interfere with the ability of the polynucleotides of the present invention to induce transgene expression in GJB2-expressing cells.

[0113] In embodiments in which the first and second regions of the polynucleotide are not directly linked, SEQ ID NO:1, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs:3-12) may be operably linked to SEQ ID NO:2, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs:20-24) by a nucleic acid sequence distinct from the intervening genomic sequence. In some embodiments, the sequence linking SEQ ID NO:1, or a functional portion or derivative thereof, and SEQ ID NO:2, or a functional portion or derivative thereof, is a shorter (e.g., truncated) version of the endogenous genomic sequence.

[0114] In some embodiments, a 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:1, or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs:3-19) is joined (e.g., operably linked) to a 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 a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs:20-28), In a 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 a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs:20-28) is joined (e.g., operably linked) to a 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:1 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs:3-19). Regardless of the order, a sequence having at least 85% sequence identity to SEQ ID NO:1 or a functional part or derivative thereof, and a sequence having at least 85% sequence identity to SEQ ID NO:2 or a functional part or derivative thereof, may be linked by a direct fusion or a nucleic acid linker as described above.

[0115] In some embodiments, the polynucleotide comprises a first region 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 functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3 to 19) and a second region 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 a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20 to 28). %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a second region that is 1 kilobase (kb) or less (e.g., the distance between the 3' end of the first region and the 5' end of the second region is 1 kb or less, e.g., 1 kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, 5 bp, 4 bp, 3 bp, 2 bp, or less). In some embodiments, there is no distance between the first and second regions in the polynucleotide (eg, the 3' end of the first region is directly linked to the 5' end of the second region).

[0116] In some embodiments, the first and second regions of the polynucleotide may be linked by an endogenous intervening nucleic acid sequence. For example, the resulting sequence of SEQ ID NO:3 linked to SEQ ID NO:2 using an endogenous intervening nucleic acid sequence is set forth in SEQ ID NO:36.

[0117] In some embodiments, the polynucleotides described herein have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:37.

[0118] In some embodiments, the polynucleotides described herein have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion or derivative of SEQ ID NO: 1. For example, the polynucleotides 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:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.

[0119] In some embodiments, the polynucleotides described herein have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion or derivative thereof. The functional portion of SEQ ID NO:2 can have the sequence of SEQ ID NO:20, the sequence of SEQ ID NO:21, the sequence of SEQ ID NO:22, the sequence of SEQ ID NO:23, or the sequence of SEQ ID NO:24. The second region can contain the nucleic acid sequence of SEQ ID NO:23 or SEQ ID NO:24 fused to the nucleic acid sequence of SEQ ID NO:20 or SEQ ID NO:22 without an intervening nucleic acid, as set forth in SEQ ID NOs:25-28.

[0120] The aforementioned nucleic acid sequences are summarized in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3]

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

[0121] Additional polynucleotides useful in conjunction with the compositions and methods described herein include nucleic acid molecules 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 nucleic acid sequences set forth in Table 2, as well as functional portions or derivatives of the nucleic acid sequences set forth in Table 2.

[0122] The above-described promoter sequence can be included in a nucleic acid vector and operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein of interest or an inhibitory RNA) in order to express the expression product specifically in GJB2-expressing cells (e.g., GJB2-expressing inner ear cells such as cochlear supporting cells). In some embodiments, the polynucleotide operably linked to the GJB2 promoter described herein (e.g., a polynucleotide containing a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof, and optionally containing a linker joining the first and second regions) is a transgene encoding a wild-type form of the GJB2 gene. In some embodiments, a polynucleotide encoding wild-type Gjb2, such as a polynucleotide sequence encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) Gjb2 (e.g., SEQ ID NO:38 or SEQ ID NO:45), or a variant thereof, is operably linked to a GJB2 promoter described herein.In some embodiments, the polynucleotide sequence encoding the Gjb2 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: 38, provided that the encoded Gjb2 analog retains the therapeutic function of wild-type Gjb2 (e.g., the ability to form functional connexin hemichannels). Not more than 10% of the amino acids in the Gjb2 protein may be replaced with conservative amino acid substitutions. In some embodiments, the polynucleotide sequence encoding Gjb2 is any polynucleotide sequence that encodes SEQ ID NO: 38, due to redundancy in the genetic code. The polynucleotide sequence encoding Gjb2 may be partially or fully codon-optimized for expression (e.g., in human cochlear supporting cells). Exemplary codon-optimized polynucleotide sequences encoding Gjb2 are SEQ ID NOs: 41-44. Gjb2 protein may also be encoded by a polynucleotide having a single nucleotide polymorphism (SNP) known to be non-pathogenic in human subjects (e.g., a SNP that does not result in hearing loss). Human Gjb2 may be encoded by a polynucleotide having the sequence of any one of SEQ ID NOs: 39-44. Mouse Gjb2 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 46. The Gjb2 protein may be a human Gjb2 protein or may be a homolog of a human Gjb2 protein from another mammalian species (e.g., mouse, rat, cow, horse, goat, sheep, donkey, cat, dog, rabbit, guinea pig, or other mammal). Exemplary Gjb2 amino acid and polynucleotide sequences are listed in Table 3 below.A nucleic acid vector (e.g., an AAV vector) containing a GJB2 promoter described herein operably linked to a polynucleotide encoding Gjb2 can be administered to a subject to treat, reduce, or prevent GJB2-associated hearing loss, such as hearing loss in subjects with DFNB1, DFNA3, Bart-Pumphrey syndrome, hystrix ichthyosis with deafness, keratitis-ichthyosis-deafness syndrome, palmoplantar keratosis with deafness, or Vohwinkel syndrome. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0123] In some embodiments, a polynucleotide encoding wild-type Gjb6 (also known as connexin 30), such as a polynucleotide sequence encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of wild-type mammalian (e.g., human or mouse) Gjb6 (e.g., SEQ ID NO:47 or SEQ ID NO:50), or a variant thereof, is a polynucleotide encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of a wild-type mammalian (e.g., human or mouse) Gjb6 (e.g., SEQ ID NO:47 or SEQ ID NO:50), or a variant thereof, is a polynucleotide encoding a protein having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a GJB2 promoter described herein (e.g., SEQ ID NO:1 or a functional portion or derivative thereof). a first region 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 a functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof, and optionally a linker connecting the first and second regions. In some embodiments, the polynucleotide sequence encoding the Gjb6 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) with respect to SEQ ID NO: 47, provided that the encoded Gjb6 analog retains the therapeutic function of wild-type Gjb6 (e.g., the ability to form functional connexin hemichannels). Not more than 10% of the amino acids in the Gjb6 protein may be replaced with conservative amino acid substitutions. In some embodiments, the polynucleotide sequence encoding Gjb6 is any polynucleotide sequence that, due to the redundancy of the genetic code, encodes SEQ ID NO: 47. The polynucleotide sequence encoding Gjb6 may be partially or completely codon-optimized for expression (e.g., in human cochlear supporting cells).Gjb6 protein may also be encoded by a polynucleotide having a single nucleotide polymorphism (SNP) known to be non-pathogenic in human subjects (e.g., a SNP that does not result in hearing loss). Human Gjb6 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 48 or SEQ ID NO: 49. Mouse Gjb6 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 51. The Gjb6 protein may be a human Gjb6 protein or a homologue of the human Gjb6 protein from another mammalian species (e.g., mouse, rat, cow, horse, goat, sheep, donkey, cat, dog, rabbit, guinea pig, or other mammal). Exemplary Gjb6 amino acid and polynucleotide sequences are listed in Table 4 below. Mutations in GJB6 are also associated with DFNB1 and DFNA3, and thus a nucleic acid vector (e.g., an AAV vector) containing the GJB2 promoter described herein operably linked to a polynucleotide encoding Gjb6 can be administered to a subject to treat, reduce, or prevent GJB6-associated hearing loss, such as hearing loss in subjects with DFNB1 or DFNA3. [Table 4-1] [Table 4-2] [Table 4-3]

[0124] Expression of exogenous polynucleotides in mammalian cells Mutations in various genes, such as GJB2, GJB6, SLC26A4, and GAS2, are associated with sensorineural hearing loss. The compositions and methods described herein can be used to construct a GJB2 promoter as described herein (e.g., a first region 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 functional portion or derivative thereof, operably linked to a polynucleotide sequence encoding an expression product (e.g., a protein of interest or an inhibitory RNA), and / or a second region 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 a functional portion or derivative thereof). , or more sequence identity) and optionally a linker linking the first and second regions, may be administered to induce or increase expression of an exogenous polynucleotide (e.g., a gene endogenously expressed in a GJB2-expressing cell, a wild-type form of a gene endogenously expressed in a GJB2-expressing cell that is mutated in a subject with hearing loss, a polynucleotide encoding a protein that regulates differentiation of cochlear supporting cells into cochlear hair cells, or an inhibitory RNA designed to downregulate a gene that inhibits differentiation of cochlear supporting cells into cochlear hair cells) specifically in a GJB2-expressing cell (e.g., a GJB2-expressing inner ear cell, such as a cochlear supporting cell). A wide range of methods have been established for the delivery of proteins to mammalian cells and for the stable expression of polynucleotides encoding proteins in mammalian cells.

[0125] A nucleic acid vector (e.g., an AAV vector) described herein (e.g., a GJB2 promoter described herein (e.g., a first region 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 functional portion or derivative thereof, and / or a first region 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 a functional portion or derivative thereof). A nucleic acid vector comprising a polynucleotide) containing a second region 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), and optionally a linker joining the first and second regions, can be used to express the polynucleotide in one or more GJB2-expressing cells (e.g., GJB2-expressing inner ear cells). Exemplary polynucleotides that can be expressed using the nucleic acid vectors described herein include polynucleotides that encode proteins expressed in healthy GJB2-expressing cells, polynucleotides that encode proteins that promote differentiation of cochlear supporting cells into cochlear hair cells, polynucleotides that correspond to the wild type of genes that are endogenously expressed in GJB2-expressing inner ear cells and that are mutated in subjects with hearing loss, hearing loss, or tinnitus, and other polynucleotides that can be expressed in GJB2-expressing inner ear cells to treat hearing loss, hearing loss, or tinnitus. The nucleic acid vectors described herein can be used to express short hairpin RNAs (shRNAs), antisense oligonucleotides (ASOs), components of a gene editing system (e.g., nucleases such as CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs), or guide RNAs (gRNAs)), or microRNAs (e.g., miR-183, miR-96, or miR-182) in GJB2-expressing cells (e.g., GJB2-expressing inner ear cells, such as cochlear supporting cells).

[0126] In some embodiments, a GJB2 promoter described herein (e.g., a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof) (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), and optionally containing a linker joining the first and second regions, is operably linked to a polynucleotide encoding brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NTF3). A nucleic acid vector (e.g., an AAV vector) comprising a GJB2 promoter operably linked to a polynucleotide encoding BDNF or NTF3 as described herein can be administered to a subject having or at risk of developing hearing loss (e.g., sensory hearing loss such as age-related hearing loss, noise-induced hearing loss, ototoxic drug-induced hearing loss, head trauma-related hearing loss, or disease- or infection-related hearing loss) to treat the subject's hearing loss (e.g., improve hearing), reduce the progression of hearing loss, or delay or prevent the onset of hearing loss (e.g., in a subject at risk of developing hearing loss due to age, infection, or exposure to ototoxic drugs, noise, or head trauma).

[0127] In some embodiments, a GJB2 promoter as described herein (e.g., a polynucleotide containing a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof, and optionally containing a linker joining the first and second regions) is operably linked to a polynucleotide encoding an expression product involved in the regeneration of cochlear hair cells. In some embodiments the expression product is Atoh1 (e.g., wild-type Atoh1 or an Atoh1 variant having one or more amino acid substitutions selected from the group consisting of S328A, S331A, S334A, S328A / S331A, S328A / S334A, S331A / S334A, and S328A / S331A / S334 as described in U.S. Publication No. 20190203210 (A1), which is incorporated by reference herein), Pou4F3, Gfi1, or Ikzf2, or a protein that can promote proliferation of cochlear supporting cells such as Lgr5, Yap1, or Tead2; The expression product is a protein that can induce or increase the differentiation of cochlear supporting cells into cochlear hair cells. In some embodiments, the expression product is an inhibitory RNA directed to a gene that inhibits the differentiation or proliferation of cochlear supporting cells, such as LATS1 and / or LATS2. Additional polynucleotides that can be expressed to promote the regeneration of cochlear hair cells (e.g., polynucleotides that can induce the proliferation or differentiation of cochlear supporting cells) are provided in Table 5 below, along with their reference sequence transcript accession numbers. A GJB2 enhancer having the sequence of SEQ ID NO:59, or a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO:59 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) (e.g., one or more copies of a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO:59) can be operably linked to a GJB2 promoter described herein to regulate expression of an expression product involved in the regeneration of cochlear hair cells. A nucleic acid vector (e.g., an AAV vector) containing a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO:59, operably linked to a GJB2 promoter described herein, operably linked to a polynucleotide encoding an expression product involved in cochlear hair cell regeneration, can be administered to a subject having or at risk of developing hearing loss associated with cochlear hair cell loss (e.g., age-related hearing loss, noise-induced hearing loss, ototoxic drug-induced hearing loss, head trauma-related hearing loss, or disease- or infection-related hearing loss). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

Table 5-5

[0128] In some embodiments, the GJB2 promoter described herein (e.g., a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof) is provided. The polynucleotide (containing a second region having a sequence identity of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and optionally a linker connecting the first and second regions) is operably linked to a transgene that is expressed in cochlear supporting cells and corresponds to a wild type version of a gene known to be mutated in a disease associated with hearing loss (e.g., monogenic hearing loss). For example, the transgene may correspond to a wild type of a mutation in SLC26A4, PAX3, NDP, or COCH associated with Pendred syndrome, Waardenburg syndrome, Norrie disease, and DFNA9, respectively. Table 6 below provides a list of diseases characterized by hearing loss and associated with mutations in genes expressed in cochlear supporting cells, and the accession number of the reference sequence transcript of the gene. A GJB2 enhancer having the sequence of SEQ ID NO:59, or a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO:59 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) (e.g., one or more copies of a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO:59) can be operably linked to a GJB2 promoter described herein to regulate expression of a transgene corresponding to a wild-type form of a gene in cochlear supporting cells that is known to be mutated in subjects with hearing loss (e.g., a gene listed in Table 6 below).A nucleic acid vector (e.g., an AAV vector) containing a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO: 59 operably linked to a GJB2 promoter described herein, operably linked to a transgene corresponding to the wild-type form of any of the genes listed in the table below, or the wild-type form of any cochlear supporting cell gene (e.g., a gene expressed in cochlear supporting cells) known to be mutated in subjects with hearing loss, can be administered to a subject with the mutation to treat the associated disease (e.g., improve or restore hearing). [Table 6-1] [Table 6-2] [Table 6-3]

[0129] A polynucleotide encoding a protein of interest One platform that can be used to achieve therapeutically effective intracellular concentrations of a protein of interest in mammalian cells is by stable expression of a gene encoding the protein of interest (e.g., by integration into the nuclear or mitochondrial genome of the mammalian cell, or by episomal concatemer formation in the nucleus of the mammalian cell). A gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein. To introduce an exogenous gene into a mammalian cell, the gene can be incorporated into a vector. The vector can be introduced into the cell by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and 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 more detail, for example, in Green, et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014), and Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0130] Protein of interest can also be introduced into mammalian cells by targeting the vector containing the gene encoding the protein of interest to cell membrane phospholipids.For example, the vector can target the phospholipids on the extracellular surface of cell membrane by linking 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.

[0131] Recognition and binding of a polynucleotide encoding a protein of interest by mammalian RNA polymerase is important for gene expression. Thus, sequence elements within the polynucleotide that exhibit high affinity for transcription factors that recruit RNA polymerase and promote assembly of a transcription complex at the transcription initiation site may be included. Such sequence elements include, for example, mammalian promoters, the sequences of which may be recognized and bound by specific transcription initiation factors and ultimately 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. In some embodiments, the promoter used in the methods and compositions described herein is a GJB2 promoter as described herein (e.g., a polynucleotide containing a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof, and optionally containing a linker joining the first and second regions).

[0132] 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 an agent that modulates the binding of transcription factors and / or RNA polymerase to a mammalian promoter, thus regulating gene expression. The chemical reagent can serve to promote the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example, by removing a repressor protein bound to the promoter. Alternatively, the chemical reagent can serve to increase the affinity of the mammalian promoter for RNA polymerase and / or transcription factors, such that the transcription rate of genes located downstream of the promoter increases in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, Calif.) and can be administered to mammalian cells to promote gene expression according to established protocols.

[0133] Other DNA sequence elements that may be included in polynucleotides for use in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in polynucleotides containing genes of interest such that the DNA adopts a three-dimensional orientation favorable for the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode proteins of interest and further include mammalian enhancer sequences. Many enhancer sequences are now known from mammalian genes, examples include enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription include the CMV enhancer and the RSV enhancer. The enhancer can be spliced ​​into the vector containing the polynucleotide encoding the protein of interest, for example, at the 5' or 3' position relative to the gene. In a preferred orientation, the enhancer is located 5' to the promoter, which is then located 5' to the polynucleotide encoding the protein of interest.

[0134] The inventors have discovered that a GJB2 promoter as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 200%, 201%, 210%, 211%, 212%, 213%, 214%, 215%, 216%, 217%, 218%, 220%, 2217%, 2221), 223%, 224%, 225%, 226%, 227%, 228%, 229%, 230%, 231%, 232%, 233%, 234%, 235%, 236%, 237%, 238%, 239%, 240%, 241%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 250%, 251%, 252%, 253%, 254%, 255%, 256%, 257%, 258%, 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%, 280%, 281%, 282%, 283%, In one embodiment, the GJB2 enhancer can be operably linked to a polynucleotide having a first region with at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion or derivative thereof, and optionally a linker linking the first region and the second region. Operably linking the GJB2 promoter described herein to the GJB2 enhancer described herein can also reduce or eliminate off-target expression in non-GJB2 expressing cells (e.g., cochlear hair cells). Thus, for use in gene therapy, operably linking one or more GJB2 enhancers to the GJB2 promoters described herein may improve therapeutic efficacy. In some embodiments, the compositions and methods described herein include one or more GJB2 enhancers (one or more of SEQ ID NOs: 52-63) listed in Table 7, such as a polynucleotide sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63. In some embodiments, the GJB2 enhancer has the sequence of any one of SEQ ID NOs: 52-63.In some embodiments, the compositions described herein contain two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) GJB2 enhancers that can have the same sequence (e.g., multiple copies of the same GJB2 enhancer) or different sequences (e.g., one or more copies of at least two different GJB2 enhancers). For example, the compositions and methods described herein can include four GJB2 enhancers that have different sequences, such as SEQ ID NOs: 53, 54, 55, and 56, or SEQ ID NOs: 52, 57, 58, and 59. In some embodiments, the compositions and methods described herein contain two or more copies of the same GJB2 enhancer (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the same GJB2 enhancer) and two or more copies of different GJB2 enhancers (e.g., 2, 3, 4, 5, 6, 7, or 8 different GJB2 enhancers). In embodiments in which the composition contains two or more enhancers (e.g., SEQ ID NOs: 52, 57, 58, and 59), the enhancers may be included in any order, may be positioned directly adjacent to one another (e.g., may be linked without any intervening sequence between the enhancer sequences, e.g., the 3' end of a first enhancer may be positioned immediately before the 5' end of a second enhancer), or may be linked by a nucleic acid linker (e.g., a nucleic acid linker may be positioned between each enhancer sequence included in the composition, or between at least two of the enhancer sequences in the composition). One or more GJB2 enhancers may be located 5' of the promoter, or 3' of the promoter (e.g., 5' of the promoter, or 3' of the coding sequence).

[0135] Exemplary GJB2 enhancer sequences are listed in Table 7. [Table 7-1] [Table 7-2] [Table 7-3]

Table 7-4

Table 7-5

[0136] The aforementioned enhancer sequences (e.g., a GJB2 enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63, such as a GJB2 enhancer having at least 85% sequence identity to any one of SEQ ID NOs: 52-59) can be included in a nucleic acid vector and operably linked to a promoter, which itself can be operably linked to a polynucleotide encoding an expression product (e.g., a polynucleotide encoding a protein of interest or an inhibitory RNA) for expressing the expression product specifically in a GJB2-expressing cell (e.g., a GJB2-expressing inner ear cell such as a cochlear supporting cell). In some embodiments, the polynucleotide encoding the expression product is a transgene encoding a wild-type form of Gjb2 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) Gjb2 (e.g., SEQ ID NO: 38 or SEQ ID NO: 45), such as a transgene having any one of SEQ ID NOs: 39-44 and 46). In some embodiments, the polynucleotide encoding the expression product is a transgene encoding a wild-type form of Gjb6 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) Gjb6 (e.g., SEQ ID NO:47 or SEQ ID NO:50), such as a transgene having the sequence of SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:51).In some embodiments, the polynucleotide encoding the expression product is a polynucleotide encoding a protein that is endogenously expressed in GJB2-expressing cells, a polynucleotide encoding a protein that promotes the differentiation of cochlear supporting cells into cochlear hair cells, a polynucleotide that is endogenously expressed in GJB2-expressing inner ear cells and corresponds to a wild-type form of a gene that is mutated in a subject with hearing loss, hearing loss, or tinnitus, or another polynucleotide that can be expressed in GJB2-expressing inner ear cells to treat hearing loss, hearing loss, or tinnitus. In some embodiments, the polynucleotide encoding the expression product is a transgene encoding BDNF or NTF3. In some embodiments, the polynucleotide encoding the expression product is a polynucleotide encoding shRNA, ASO, a component of a gene editing system (e.g., a nuclease such as Cas9, TALEN, or ZFN, or a gRNA), or a microRNA (e.g., miR-183, miR-96, or miR-182).

[0137] In some embodiments, a GJB2 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:59) is selected from the group consisting of Atoh1 (e.g., wild-type Atoh1 or S328A, S331A, S334A, S328A / S331A, S335A, S336A, S337A, S338A, S339A, S340A, S341A, S342A, S343A, S344A, S345A, S346A, S347A, S348A, S349 ... , S328A / S334A, S331A / S334A, and S328A / S331A / S334), Pou4F3, Gfi1, or Ikzf2, or an expression product that can promote proliferation of cochlear supporting cells such as Lgr5, Yap1, or Tead2, is operably linked to a promoter that is operably linked to a polynucleotide that encodes an expression product that can promote regeneration of cochlear hair cells and / or induce or increase differentiation of cochlear supporting cells into cochlear hair cells, such as an expression product that can promote proliferation of cochlear supporting cells such as LATS1 and / or LATS2. Additional polynucleotides that can be expressed to promote regeneration of cochlear hair cells (e.g., polynucleotides that can induce proliferation or differentiation of cochlear supporting cells) are provided in Table 5 below. A promoter that may be operably linked to a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO:59 for expression of such a polynucleotide is a GJB2 promoter (e.g., a GJB2 promoter described herein above, a GJB2 promoter having at least 85% sequence identity to any one of SEQ ID NOs:1 and 8-11, or a GJB2 promoter having at least 85% sequence identity to a promoter listed in Table 8), or a supporting cell promoter (e.g., a cochlear supporting cell promoter listed in Table 9).A nucleic acid vector (e.g., an AAV vector) containing a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO:59 operably linked to a promoter that is operably linked to a polynucleotide encoding an expression product involved in cochlear hair cell regeneration can be administered to a subject who has or is at risk of developing hearing loss associated with cochlear hair cell loss (e.g., age-related hearing loss, noise-induced hearing loss, ototoxic drug-induced hearing loss, head trauma-related hearing loss, or disease- or infection-related hearing loss).

[0138] In some embodiments, a GJB2 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:59) is expressed in cochlear supporting cells and operably linked to a promoter that is operably linked to a transgene that corresponds to a wild type version of a gene known to be mutated in a disease associated with hearing loss (e.g., a monogenic form of hearing loss, such as a disease listed in Table 6 that can be treated by expressing the wild type of the gene in the same row of Table 6). For example, the transgene can correspond to the wild type of a mutation in SLC26A4, PAX3, NDP, or COCH associated with Pendred syndrome, Waardenburg syndrome, Norrie disease, and DFNA9, respectively. A promoter that may be operably linked to a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO: 59 for expression of such a transgene is a GJB2 promoter (e.g., a GJB2 promoter having at least 85% sequence identity to any one of the GJB2 promoters described herein above, SEQ ID NOs: 1 and 8-11, or a GJB2 promoter having at least 85% sequence identity to a promoter listed in Table 8), or a supporting cell promoter (e.g., a cochlear supporting cell promoter listed in Table 9). A nucleic acid vector (e.g., an AAV vector) containing a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO: 59 operably linked to a promoter that is operably linked to a transgene corresponding to the wild-type form of any of the genes listed in Table 6, or the wild-type form of any cochlear supporting cell gene known to be mutated in a subject with hearing loss (e.g., a gene expressed in cochlear supporting cells), may be administered to a subject with the mutation to treat the associated disease (e.g., improve or restore hearing).

[0139] In some embodiments, the promoter operably linked to the GJB2 enhancer described herein is a GJB2 promoter. For example, the nucleic acid vector may contain the GJB2 enhancer described herein operably linked to the GJB2 promoter operably linked to the polynucleotide encoding Gjb2 (e.g., for the treatment of GJB2-related hearing loss), Gjb6 (e.g., for the treatment of DFNB1 or DFNA3), BDNF (e.g., for the treatment of sensorineural hearing loss), or NTF3 (e.g., for the treatment of sensorineural hearing loss). In another example, the nucleic acid vector may contain the GJB2 enhancer described herein operably linked to the GJB2 promoter operably linked to the polynucleotide listed in Table 5 (e.g., for promoting the regeneration of cochlear hair cells, e.g., for the treatment of hearing loss associated with loss of cochlear hair cells), a transgene corresponding to the wild-type form of the gene listed in Table 6 (e.g., for the treatment of the corresponding disease listed in Table 6), or a polynucleotide encoding a protein or RNA molecule that is endogenously expressed in GJB2-expressing cells. In some embodiments, the GJB2 promoter is a GJB2 promoter described herein (e.g., a GJB2 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 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%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 18 or more sequence identity) to SEQ ID NO:2, and / or a second region 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 a functional portion or derivative thereof, and optionally containing a linker connecting the first and second regions.In some embodiments, the GJB2 promoter operably linked to the GJB2 enhancer described herein is a polynucleotide having at least 85% sequence identity to any one of SEQ ID NOs: 52-63 (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%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178 A polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1 and 8-11 is a portion of the GJB2 promoter described herein, such as a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1 and 8-11. Additional GJB2 promoters that can be operably linked to the GJB2 enhancers described herein (e.g., polynucleotides having at least 85% sequence identity to any one of SEQ ID NOs: 52-59, such as polynucleotides having at least 85% sequence identity to any one of SEQ ID NOs: 52-63 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity)) are provided in Table 8 below. In some embodiments, the GJB2 enhancer described herein is operably linked to a GJB2 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 100% sequence identity) to any one of SEQ ID NOs:66-68. [Table 8-1] [Table 8-2] [Table 8-3]

[0140] In some embodiments, the promoter operably linked to the GJB2 enhancer described herein is an inner ear cell type specific promoter. For example, the nucleic acid vector may contain the GJB2 enhancer described herein operably linked to an inner ear cell type specific promoter operably linked to a polynucleotide encoding Gjb2 (e.g., for the treatment of GJB2-associated hearing loss), Gjb6 (e.g., for the treatment of DFNB1 or DFNA3), BDNF (e.g., for the treatment of sensorineural hearing loss), or NTF3 (e.g., for the treatment of sensorineural hearing loss). In another example, the nucleic acid vector may contain a GJB2 enhancer as described herein operably linked to an inner ear cell type specific promoter operably linked to a polynucleotide listed in Table 5 (e.g., to promote regeneration of cochlear hair cells, e.g., to treat hearing loss associated with loss of cochlear hair cells), a transgene corresponding to a wild type form of a gene listed in Table 6 (e.g., to treat a corresponding disease listed in Table 6), or a polynucleotide encoding a protein or RNA molecule endogenously expressed in GJB2 expressing cells. Cell type specific promoters (e.g., polynucleotides having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs:52-59, such as a polynucleotide having at least 85% sequence identity to any one of SEQ ID NOs:25-29) that can be operably linked to one or more GJB2 enhancers to express an expression product (e.g., GJB6, GJB2, BDNF, NTF3, a polynucleotide listed in Table 5, or a gene listed in Table 6) in one or more GJB2-expressing inner ear cells are provided in Table 9 below. [Table 9]

[0141] Nucleic acid vectors containing the GJB2 promoter and / or GJB2 enhancer described herein may contain a woodchuck post-transcriptional regulatory element (WPRE). The WPRE acts at the mRNA level by facilitating nuclear export of the transcript and / or increasing the efficiency of polyadenylation of the nascent transcript, 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.

[0142] In some embodiments, the nucleic acid vectors containing the GJB2 promoter and / or GJB2 enhancer described herein include reporter sequences that may be useful for verifying expression of a gene operably linked to the GJB2 promoter, for example, in cells and tissues (e.g., GJB2-expressing cells, such as cochlear supporting cells). Reporter sequences that may be provided in a transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When associated with a regulatory element that drives expression, such as the GJB2 promoter, the reporter sequence provides a signal that is detectable by conventional means, including enzymatic, radioactive, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry. For example, if the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the presence of the vector carrying the signal can be visually measured by color or light production in a luminometer.

[0143] Methods for delivery of exogenous polynucleotides to target cells A GJB2 promoter as described herein (e.g., a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof) Techniques that can be used to introduce a polynucleotide operably linked to a promoter operably linked to a GJB2 enhancer described herein, or a polynucleotide operably linked to a promoter operably linked to a GJB2 enhancer described herein, into a target cell (e.g., a mammalian cell) 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 uptake of the exogenous polynucleotide. 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 eukaryotic cell nuclei. Nucleofection™ and protocols useful for carrying out this technique are described, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and US2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0144] Additional 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.

[0145] Lipofection is another technique that is useful for transfection of target cells. This method involves loading polynucleotides into liposomes that often present cationic functional groups, such as quaternary amines or protonated amines, toward the liposome exterior. This promotes electrostatic interactions between the liposome and the cell due to the anionic nature of the cell membrane, ultimately resulting in the uptake of exogenous polynucleotides, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. 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 includes contacting cells with cationic polymer-polynucleotide complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that favors interaction with cell membranes include activated dendrimers (described, for example, 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:I:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to 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.

[0146] Another useful tool for inducing the uptake of exogenous polynucleotide by target cells is laser transfection, also called optical transfection, which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize cells and allow 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.

[0147] Imparefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing genes is intended for intracellular delivery and is attached to the nanostructure surface. A tip with an array of these needles is then pressed against a cell or tissue. Cells impaled 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.

[0148] Magnetofection can also be used to deliver polynucleotides to target cells. The principle of magnetofection is to combine polynucleotides with 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 by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated in the target cells by 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.

[0149] Another useful tool for inducing the uptake of exogenous polynucleotide by target cell is sonoporation, which is a technique that involves using sound waves (typically ultrasonic frequencies) 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.

[0150] 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 the simultaneous overexpression of glycoprotein VSV-G with genome-modifying proteins such as nucleases can be used to efficiently deliver proteins to cells that subsequently catalyze the site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of cells for the covalent integration of polynucleotides of interest, such as genes or regulatory sequences. The use of such vesicles, also called Gesicles, for the genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract], in In:Methylation changes in early embryonic genes in cancer [abstract], and in Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, [abstract] No. 122.

[0151] 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 mammalian cells. A variety of vectors have been developed for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of expression vectors are described, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, Massachusetts, 2006).Expression vectors for use in the compositions and methods described herein include a GJB2 promoter as described herein (e.g., a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof) 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 inhibitory RNA). and optionally a linker linking the first and second regions), or a GJB2 enhancer as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity) to any one of SEQ ID NOs: 52-59) operably linked to a promoter that is 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 inhibitory RNA), as well as additional sequence elements used, for example, for expression of these agents and / or for integration of these polynucleotide sequences into the genome of a mammalian cell.Vectors that may contain the GJB2 promoter and / or GJB2 enhancer 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 inside a cell), cosmids (e.g., pWE or sCos vectors), artificial chromosomes (e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), and viral vectors. Certain vectors that may be used to express an expression product (e.g., a protein of interest) include plasmids that contain regulatory sequences, such as enhancer regions (e.g., the GJB2 enhancer described herein) that direct gene transcription. Other vectors useful for expressing an expression product (e.g., a protein 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, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may contain a polynucleotide encoding a marker for the selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0152] Viral Vectors for Polynucleotide Delivery Viral genomes provide a rich source of vectors that can be used for efficient delivery of genes of interest to the genome of target cells (e.g., mammalian cells, such as human cells).Viral genomes are particularly useful vectors for gene delivery, because the polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction.These processes occur as part of the natural virus replication cycle and do not require additional proteins or reagents to induce gene integration. Examples of viral vectors include negative-stranded RNA viruses such as retroviruses (e.g., retrovirus family viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded 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, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, avian C virus, mammalian C virus, B virus, D virus, oncoretrovirus, HTLV-BLV complex, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon monkey leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which is incorporated herein by reference as it relates to viral vectors for use in gene therapy.

[0153] AAV vectors for polynucleotide delivery In some embodiments, the polynucleotides of the compositions and methods described herein are incorporated into rAAV vectors and / or virions to facilitate their introduction into cells. In some embodiments, rAAV vectors useful in the compositions and methods described herein comprise: (1) a GJB2 promoter described herein (e.g., a first region 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 a functional portion or derivative thereof; and / or a second region 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 a functional portion or derivative thereof; The present invention is a recombinant polynucleotide construct comprising: (1) a polynucleotide that contains a first region having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the first region, and optionally a linker joining the first and second regions; (2) a sequence to be expressed (e.g., a polynucleotide that encodes a protein or a polynucleotide that can be transcribed to produce an inhibitory RNA molecule); and (3) a virus that facilitates integration and expression of the sequence to be expressed. In some embodiments, rAAV vectors useful in the compositions and methods described herein are recombinant polynucleotide constructs that include (1) at least one GJB2 enhancer described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs:52-59), (2) a promoter, (3) a sequence to be expressed, and (4) viral sequences that facilitate integration and expression of the expressed sequence. The viral sequences may include sequences of AAV required in cis for replication and packaging of DNA into virions (e.g., functional ITRs).In a typical application, the expressed sequence encodes a protein that can promote cochlear hair cell regeneration (e.g., differentiation of cochlear supporting cells into cochlear hair cells), cochlear supporting cell survival, cochlear supporting cell proliferation, or a wild-type form of a cochlear supporting cell protein that is mutated in a subject with a form of inherited hearing loss, which can be useful for improving hearing in subjects with mutations associated with hearing loss, hearing loss, tinnitus, or auditory neuropathy. Such rAAV vectors may contain a marker or reporter gene. Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. The 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 by reference herein as they relate to AAV vectors for gene delivery.

[0154] The polynucleotides and vectors described herein (e.g., a GJB2 promoter operably linked to a polynucleotide encoding an expression product, or a GJB2 enhancer operably linked to a promoter operably linked to a polynucleotide encoding an expression product) can be incorporated into rAAV virions to facilitate introduction of the polynucleotide or vector into a cell. 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 virion assembly. Construction of rAAV virions is described, for example, in US5,173,414, US5,139,941, US5,863,541, US5,869,305, US6,057,152, and US6,376,237, as well as 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 relate to AAV vectors for gene delivery.

[0155] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from various AAV serotypes, including AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, and PHP.S. When targeting GJB2-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 transduction of the retina may also be used in the methods and compositions described herein. The construction and use of AAV vectors of different serotypes and AAV proteins are described, 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 relate to AAV vectors for gene delivery.

[0156] 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 from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0157] AAV virions with mutations in the virion capsid can be used to infect specific cell types more effectively 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 virions that can be used in the methods described herein include those capsid hybrids generated by molecular hybridization 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).

[0158] Pharmaceutical Compositions

[0033] The GJB2 promoter described herein (e.g., a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof) and optionally Optionally, a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63) may be operably linked to a polynucleotide encoding an expression product (e.g., a transgene encoding a protein of interest) and incorporated into a vehicle for administration to a patient, such as a human patient suffering from sensorineural hearing loss. Pharmaceutical compositions containing a vector, such as a viral vector, containing a GJB2 promoter and / or GJB2 enhancer described herein operably linked to a polynucleotide encoding an expression product may be prepared using methods known in the art. For example, such compositions can be prepared in a desired form, such as a lyophilized formulation or an aqueous solution, using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013), incorporated herein by reference).

[0159] A GJB2 promoter as described herein operably linked to a polynucleotide encoding an expression product (e.g., a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof). A mixture of nucleic acid vectors (e.g., viral vectors) containing a GJB2 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63) may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the formulation may be sterile and may be fluid to the extent that easy syringability exists. The formulation may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi.The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition may be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0160] For example, the solution containing the pharmaceutical composition described herein may be suitably buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, a single dosage may be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous injection fluid or injected into the proposed injection site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition may be formulated to contain synthetic perilymphatic fluid. An exemplary synthetic perilymph solution contains 20-200 mmol / L NaCl, 1-5 mmol / L KCl, 0.1-10 mmol / L CaCl2, 1-10 mmol / L glucose, and 2-50 mmol / L HEPE, with a pH of about 6-9 and an osmolality of about 300 mOsm / kg. The person administering will, in any event, determine the appropriate dose for the individual subject. Additionally, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0161] Treatment method The compositions described herein can be administered to a subject having or at risk of developing sensorineural hearing loss by a wide variety of routes, including local administration to the middle or inner ear (e.g., administration into the perilymph or endolymph, e.g., administration to or through the oval window, round window, or semicircular canal (e.g., horizontal semicircular canal), or by transtympanic or intratympanic injection, e.g., administration to GJB2-expressing inner ear cells), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most suitable route for administration in any given case will depend on the particular composition being administered, the patient, the pharmaceutical formulation method, the method of administration (e.g., time and route of administration), the patient's age, weight, sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. The compositions may be administered once, or multiple times (eg, once a year, twice a year, three times a year, every other month, once a month, or every other week).

[0162] The subject that may be treated as described herein is the subject that has or is at risk of developing sensorineural hearing loss.In some embodiments, the composition described herein is used to treat GJB2-related hearing loss (e.g., hearing loss associated with DFNB1, DFNA3, or Bart-Pumphrey syndrome, hystrixis with hearing loss, keratitis-ichthyosis-hearing loss syndrome, palmoplantar keratosis with hearing loss, or Vohwinkel syndrome).DFNB1 and DFNA3 can be used in combination with a GJB2 promoter as described herein (e.g., a first region 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 functional portion or derivative thereof, and / or a first region 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 a functional portion or derivative thereof) operably linked to a polynucleotide encoding Gjb2 or Gjb6. a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to a functional portion or derivative thereof, and optionally a linker connecting the first and second regions) and / or a GJB2 enhancer as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63; Gjb2 may be treated by administration of 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) (e.g., a GJB2 enhancer operably linked to a promoter such as the GJB2 promoter or an inner ear cell type specific promoter), while Bart-Pumphrey syndrome, hystrixis with deafness, keratitis-ichthyosis-deafness syndrome, palmoplantar keratosis with deafness, and Vohwinkel syndrome may be treated by administration of a nucleic acid vector containing a GJB2 promoter and / or a GJB2 enhancer described herein (e.g., a GJB2 enhancer operably linked to a promoter such as the GJB2 promoter or an inner ear cell type specific promoter) operably linked to a polynucleotide encoding Gjb2. Subjects may have, or be identified as having, a mutation in GJB2 and / or GJB6 and may have severe, moderate, or mild hearing loss when treatment is initiated, or may be treated prior to the onset of symptoms (e.g., prophylactic treatment).

[0163] In some embodiments, the compositions described herein are used to treat hearing loss associated with a mutation in a gene expressed in a GJB2-expressing inner ear cell (e.g., a cochlear supporting cell). Hearing loss associated with a mutation in a gene expressed in a GJB2-expressing inner ear cell can be treated by administering to a mammalian cell comprising a GJB2 promoter described herein (e.g., a GJB2 promoter having at least 85% sequence identity to any one of SEQ ID NOs: 29-35, or a GJB2 promoter having at least 85% sequence identity to SEQ ID NO: 1 or a functional portion or derivative thereof (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:2 or a functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof, and optionally a linker connecting the first and second regions. 59, a GJB2 enhancer having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1, 8-11, and 66-68, The diseases listed in Table 6 can be treated by administration of a nucleic acid vector containing a GJB2 promoter having at least 85% sequence identity to SEQ ID NO: 59, SEQ ID NOs: 1, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 10-29, 11-29, 12-29, 13-29, 14-29, 15-29, 16-30, 17-31, 18-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-60, 19-61, 19-62, 19-70, 19-71, 19-82, 19-90, 19-100, 19-11, 19-120, 19-130, 19-140, 19-150, 19-160, 19-170, 19-180, 19-190, 19-210, 19-220, 19-230, 19-2and 66-68, or an inner ear cell type specific promoter (e.g., a promoter listed in Table 9) operably linked to a wild-type form of the corresponding gene listed in Table 6. In embodiments in which the compositions described herein are used to deliver a polynucleotide listed in Table 6, the composition or method may improve or restore the function and / or structure of, or improve the health or survival of, GJB2-expressing inner ear cells (e.g., cochlear supporting cells).

[0164] In some embodiments, the compositions described herein are used to treat subjects who have or are at risk of developing hearing loss associated with damage or loss of cochlear hair cells (e.g., damage or loss of cochlear hair cells associated with acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging). Thus, the compositions can be used to treat subjects who have been treated with ototoxic drugs, or who are currently undergoing or will soon begin treatment with ototoxic drugs. Ototoxic drugs are toxic to cells of the inner ear and can cause sensorineural hearing loss, tinnitus, or a combination of these symptoms. Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamicin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, antineoplastic agents (e.g., platinum-containing chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, especially at high doses), and quinine. Diseases associated with damage or loss of cochlear hair cells can be autoimmune diseases or conditions in which an autoimmune response contributes to the damage or death of cochlear hair cells. Autoimmune diseases associated with sensorineural hearing loss include autoimmune inner ear disease (AIED), polyarteritis nodosa (PAN), Cogan's syndrome, relapsing polychondritis, systemic lupus erythematosus (SLE), Wegener's granulomatosis, Sjogren's syndrome, and Behcet's disease. Some infectious conditions, such as Lyme disease and syphilis, can also cause hearing loss (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 hearing loss.Hearing loss associated with damage or loss of cochlear hair cells can be prevented by administering to a patient a therapeutically effective amount of a GJB2 promoter as described herein (e.g., a GJB2 promoter having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:1 or a functional portion or derivative thereof, such as a GJB2 promoter having at least 85% sequence identity to any one of SEQ ID NOs:29-35, and / or a first region 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 functional portion or derivative thereof, and / or a GJB2 promoter having at least 85% sequence identity to any one of SEQ ID NOs:29-35, and a second region 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: 59 operably linked to a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, and optionally a linker connecting the first region and the second region. 9, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1, 8-11, and 66-68; or a polynucleotide encoding an expression product capable of promoting regeneration of cochlear hair cells. The treatment may be by administration of a nucleic acid vector containing an inner ear cell type specific promoter (a promoter listed in Table 9) operably linked to a nucleotide (e.g., a polynucleotide encoding a protein that can promote differentiation of cochlear supporting cells into cochlear hair cells or proliferation of cochlear supporting cells, such as a polynucleotide listed in Table 5, or a polynucleotide that can be transcribed to produce an inhibitory RNA that targets a gene known to suppress or prevent regeneration of cochlear hair cells, such as LATS1 and / or LATS2).

[0165] In some embodiments, the compositions described herein are used to treat sensorineural hearing loss (e.g., acquired sensorineural hearing loss, such as age-related hearing loss, noise-induced hearing loss, ototoxic drug-induced hearing loss, disease- or infection-related hearing loss, or head trauma-related hearing loss, e.g., hearing loss associated with damage or loss of cochlear hair cells). Sensorineural hearing loss can be characterized by the presence of a GJB2 promoter as described herein (a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof, operably linked to a polynucleotide encoding BDNF or NTF3. The subject may be treated by administration of a nucleic acid vector containing a GJB2 enhancer (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63) (e.g., a GJB2 enhancer operably linked to a promoter such as a GJB2 promoter or an inner ear cell type specific promoter).

[0166] The methods described herein may include screening the subject for one or more mutations in genes known to be associated with hearing loss prior to treatment or administration with the compositions described herein. The subject may be screened for genetic mutations using standard methods known to those skilled in the art (e.g., genetic testing). The methods described herein may also include evaluating the hearing in the subject prior to treatment or administration with the compositions described herein. Hearing can be evaluated using standard tests such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions. These tests may also be used to evaluate the hearing of the subject after treatment or administration with the compositions described herein. The compositions and methods described herein may also be administered as a preventative treatment to patients at risk of developing hearing loss, such as patients with a family history of hearing loss (e.g., genetic hearing loss), patients who have a genetic mutation associated with hearing loss but have not yet exhibited hearing impairment, or patients who have been exposed to risk factors for acquired hearing loss (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging).

[0167] The compositions and methods described herein can be used to induce or increase the regeneration of cochlear hair cells in a subject by inducing cochlear supporting cells to differentiate into cochlear hair cells or by inducing the proliferation of cochlear supporting cells. Subjects that can benefit from compositions that induce or increase the regeneration of cochlear hair cells include subjects suffering from hearing loss as a result of cochlear hair cell loss (e.g., trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or age-related cochlear hair cell loss), and subjects with abnormal cochlear hair cells (e.g., cochlear hair cells that do not function properly compared to normal cochlear hair cells), damaged cochlear hair cells (e.g., trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or age-related cochlear hair cell damage), or reduced cochlear hair cell numbers due to genetic mutations or congenital abnormalities.Cochlear hair cell regeneration can be achieved by transfecting cochlear supporting cells with a first region 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 functional portion or derivative thereof, such as a GJB2 promoter as described herein (e.g., a GJB2 promoter having at least 85% sequence identity to any one of SEQ ID NOs:29-35). or a second region 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: 59 operably linked to a polynucleotide having a sequence similar to that of SEQ ID NO: 2 or a functional portion or derivative thereof, and optionally a linker connecting the first and second regions. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 1, 8-11, and 66-68; The expression of cochlear supporting cells, such as leutidine, in the inner ear may be induced or increased by contacting the inner ear with a nucleic acid vector containing an inner ear cell type specific promoter (a promoter listed in Table 9) operably linked to a polynucleotide encoding a protein that can promote differentiation of cochlear supporting cells, such as leutidine, into cochlear hair cells or proliferation of cochlear supporting cells, or a polynucleotide that can be transcribed to produce an inhibitory RNA targeting a gene known to suppress or prevent regeneration of cochlear hair cells, such as LATS1 and / or LATS2. The contacting may occur in vivo.

[0168] A polynucleotide encoding an expression product operably linked to the GJB2 promoter and / or GJB2 enhancer for treating a subject as described herein can be a polynucleotide encoding a protein expressed in healthy cochlear supporting cells (e.g., a protein that plays a role in supporting cochlear supporting cell development, cochlear supporting cell function, cochlear supporting cell structure, or cochlear supporting cell survival, or a wild-type version of a cochlear supporting cell gene (e.g., a gene expressed in cochlear supporting cells) that is mutated in a subject with sensorineural hearing loss), another protein of interest (e.g., a fluorescent protein, The polynucleotide may be a polynucleotide that encodes a reporter protein such as lacZ, or luciferase, a polynucleotide that encodes an expression product that can induce differentiation of cochlear supporting cells into cochlear hair cells or promote proliferation of cochlear supporting cells, or a polynucleotide that can be transcribed to produce an shRNA, an ASO, a component of a gene editing system (e.g., a nuclease such as CRISPR-associated protein 9 (Cas9), transcription activator-like effector nuclease (TALEN), or zinc finger nuclease (ZFN), or a guide RNA (gRNA)), or a microRNA. The polynucleotide may be selected based on the cause of the subject's hearing loss (e.g., if the subject's hearing loss is associated with a particular gene mutation, the polynucleotide may be a wild type of the gene that is mutated in the subject, or if the subject has hearing loss associated with loss of cochlear hair cells, the polynucleotide may encode a protein that promotes regeneration of cochlear hair cells), the severity of the subject's hearing loss, the health of the subject's hair cells, the age of the subject, a family history of hearing loss in the subject, or other factors.

[0169] Treatment may include administration of a composition containing a nucleic acid vector (e.g., AAV vector) containing the GJB2 promoter and / or GJB2 enhancer described herein in various unit doses. Each unit dose typically contains a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route of administration and formulation, are within the skill of the art. The unit dose need not be administered as a single injection, but may include continuous infusion over a 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., an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eb, or PHP.S vector), the viral vector may be, for example, 1 μL to 200 μL (e.g., 1 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, 3×10 9 VG / mL, 4×10 9 VG / mL, 5×10 9 VG / mL, 6×10 9 VG / mL, 7×10 9 VG / mL, 8×10 9 VG / mL, 9×10 9 VG / mL, 1×10 10 VG / mL, 2×10 10 VG / mL, 3×10 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, 3×10 15 VG / mL, 4×10 15 VG / mL, 5×10 15 VG / mL, 6×10 15 VG / mL, 7×10 15 VG / mL, 8×10 15 VG / mL, 9×10 15 VG / mL, or 1×10 16 The AAV vector may be administered to a patient at a dose of approximately 1×10 7 VG / ear ~ approx. 2 x 10 15 VG / ear (e.g. 1×10 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 2×10 15 The subject may be administered a dose of 100 mg / ear (VG / ear).

[0170] The compositions described herein are administered in an amount sufficient to improve hearing, reduce tinnitus, increase or induce expression of an expression product in GJB2 expressing cells (e.g., cochlear supporting cells), increase proliferation of cochlear supporting cells, promote or increase survival of cochlear supporting cells, induce or increase differentiation of cochlear supporting cells into cochlear hair cells (i.e., regeneration of cochlear hair cells), or improve function of cochlear supporting cells. Hearing can be assessed using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions) and can be improved by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or more) compared to hearing measurements obtained before treatment. In some embodiments, the composition is administered in an amount sufficient to improve the subject's ability to understand speech. The compositions described herein may also be administered in an amount sufficient to slow or prevent the onset or progression of sensorineural hearing loss (e.g., in subjects who have a genetic mutation associated with hearing loss, have a family history of hearing loss (e.g., hereditary hearing loss), or have been exposed to risk factors associated with hearing loss (e.g., ototoxic drugs, head trauma, disease or infection, or acoustic trauma), but do not exhibit hearing impairment, or in subjects who exhibit mild to moderate hearing loss). Expression of a protein encoded by a transgene operably linked to the GJB2 promoter and / or GJB2 enhancer described herein in a nucleic acid vector administered to a subject 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%, 125%, 150%, 200% or more) compared to expression prior to administration of a composition described herein.Cochlear supporting cell differentiation, cochlear supporting cell function, or protein function encoded by a transgene operably linked to the GJB2 promoter and / or GJB2 enhancer described herein in a nucleic acid vector administered to a subject may be indirectly assessed based on a hearing test and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to cochlear supporting cell differentiation, cochlear supporting cell function, or protein function prior to administration of a composition described herein. 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 GJB2 promoter and / or GJB2 enhancer described herein (e.g., administration of a nucleic acid vector in which the same transgene is expressed using a ubiquitous promoter). These effects may occur, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 weeks, or more after administration of the compositions described herein. Patients may be evaluated 1, 2, 3, 4, 5, 6, or more months after administration of the compositions, depending on the dose and route of administration used for treatment. Depending on the results of the evaluation, patients may receive additional treatment.

[0171] kit The compositions described herein may be provided in a kit for use in treating sensorineural hearing loss. The compositions may comprise a GJB2 promoter as described herein (e.g., a first region 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 functional portion or derivative thereof, and / or a second region 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 a functional portion or derivative thereof). The composition may comprise a polynucleotide having a first region having a sequence identity with the GJB2-expressing inner ear cell of at least one of the following sequences: a polynucleotide having a sequence identity with the GJB2-expressing inner ear cell of at least one of the following sequences (a polynucleotide having a sequence identity with the GJB2-expressing inner ear cell of at least one of the following sequences) and optionally a linker connecting the first region and the second region, a nucleic acid vector containing such a polynucleotide, or a nucleic acid vector containing a GJB2 promoter as described herein operably linked to a polynucleotide encoding an expression product (e.g., a transgene encoding a protein of interest, such as a protein that can be expressed in GJB2-expressing inner ear cells to treat hearing loss). The GJB2 promoter of any of the foregoing compositions may be operably linked to a GJB2 enhancer as described herein. The composition may also include one or more GJB2 enhancers described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs: 52-63), a nucleic acid vector containing such a polynucleotide, or a nucleic acid vector containing a GJB2 enhancer described herein operably linked to a promoter (e.g., a GJB2 promoter or an inner ear cell type specific promoter) that is operably linked to a polynucleotide encoding an expression product (e.g., a transgene encoding a protein of interest, such as a protein that can be expressed in GJB2-expressing inner ear cells to treat hearing loss).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, 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. EXAMPLES

[0172] 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 illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0173] Example 1. Expression of GJB2 using a ubiquitous promoter resulted in elevated hearing thresholds and inner hair cell death in wild-type mice. Six to eight week old C57BL / 6J mice were injected with an AAV vector (AAV-CMV-mGjb2) containing a mouse Gjb2 coding sequence driven by a ubiquitous promoter via a fenestration in the posterior semicircular canal. Two weeks later, animals were anesthetized with ketamine and xylazine and auditory brainstem responses (ABRs) were measured. After ABR measurements, animals were sacrificed and fixed in 10% NBF via cardiac perfusion, and their temporal bones were harvested and kept in 10% NBF for an additional 16 h. After decalcification in 8% EDTA for 2 days, ears were microdissected and the vesicles and organ of Corti were prepared for immunohistochemistry. Whole specimen tissue preparations of the organ of Corti were counterstained with myosin 7a antibody to visualize all hair cells and imaged under a confocal microscope (Leica® SP8). Seven of nine wild-type ears treated with AAV-CMV-Gjb2 showed elevated ABR thresholds ( Fig. 1<em>A , brackets). As shown in Fig. 1<em>B , animals with elevated hearing thresholds lacked inner hair cells.

[0174] Example 2. Evaluation of luciferase expression driven by the GJB2 promoter in HeLa cells. To test the ability of various GJB2 promoters to drive expression, GJB2 promoters with sequences of SEQ ID NOs: 4-7, 9, 13, 30-32, 36, and 37 were cloned upstream of the NanoLuc reporter. HeLa cells were transfected with plasmids containing the promoter and reporter. After 24 hours, NanoLuc expression was detected and quantified using the Nano-Glo Luciferase Assay (Promega® Catalog No. N1110). Results are shown in Figures 2A-2B. MLP = control minimal promoter (unrelated to GJB2). pGL3 Basic + NanoLuc = promoterless control vector.

[0175] Example 3. The GJB2 promoter alone and paired with the GJB2 enhancer in mouse cochlear explants promotes increased expression and cell type specificity. Cochleae from P0-P5 pups were dissected and cultured on a collagen matrix in DMEM supplemented with 5% FBS and penicillin-G. Explant cultures of the organ of Corti were established and infected with AAV expressing nuclear (H2B)-GFP under the control of various GJB2 promoter and enhancer combinations. 2e10vg / culture was added to the medium. Samples were incubated in the presence of virus for 72 hours and then fixed for fluorescent imaging. All samples were counterstained with anti-Myo7a (hair cells), anti-Tuj1 (neurons), anti-GJB2, and anti-GFP antibodies and imaged on a Zeiss® 810 confocal microscope.

[0176] In the first experiment, samples infected with vectors containing the GJB2 promoter alone (AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP (Figure 3A) and AAV1-p.hGJB2(SEQ ID NO:32)-H2B-GFP (Figure 3D)) showed lower expression of the reporter protein compared to samples infected with vectors containing the GJB2 promoter and enhancer (AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-Enhancer9 (SEQ ID NO:62) (Figure 3B), AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGH (SEQ ID NO:60) (Figure 3C), and AAV1-p.hGJB2(SEQ ID NO:32)-H2B-GFP-Enhancer9 (SEQ ID NO:62) (Figure 3E)). The enhancer was located 3' to the coding sequence in the vector. A schematic showing the order of elements in the vectors used in the experiments of Example 3 is provided in Figure 19A.

[0177] In a second experiment, samples infected with the vector AAV1-p.hGJB2(SEQ ID NO:1)-H2B-GFP, containing the GJB2 promoter alone, showed the lowest expression of the reporter protein (Figure 4A). Addition of a splice acceptor (SEQ ID NO:22) produced a promoter (SEQ ID NO:35) that led to increased expression (AAV1-p.hGJB2(SEQ ID NO:35)-H2B-GFP, Figure 4D). For both promoter and promoter+spice acceptor, the addition of an enhancer element showed the highest levels of reporter protein expression (AAV1-p.hGJB2(SEQ ID NO:1)-H2B-GFP-enhancer9(SEQ ID NO:62) (Figure 4B), AAV1-p.hGJB2(SEQ ID NO:1)-H2B-GFP-enhancerGH(SEQ ID NO:60) (Figure 4C), AAV1-p.hGJB2(SEQ ID NO:35)-H2B-GFP-enhancer9(SEQ ID NO:62) (Figure 4E), and AAV1-p.hGJB2(SEQ ID NO:35)-H2B-GFP-enhancerGH(SEQ ID NO:60) (Figure 4F)). The enhancer was located 3' of the coding sequence in the vector.

[0178] In a third experiment, truncation of Enhancer GH was tested. Explant cultures infected with AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGH(SEQ ID NO:60) (Figure 5A) or AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHA(SEQ ID NO:61) (Figure 5B) retained strong reporter expression, whereas truncation of Enhancer GH (AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHB(SEQ ID NO:64) (Figure 5C) and AAV1-p.hGJB2(SEQ ID NO:30)-H2B-GFP-EnhancerGHC(SEQ ID NO:65) (Figure 5D)) lost enhancer activity. The enhancer was located 3' of the coding sequence in the vector.

[0179] In the fourth experiment, the GJB2 promoter of SEQ ID NO: 30 was modified by the addition of a DNA element corresponding to a predicted histone mark found within intron 1 of the GJB2 locus. Similar levels of reporter protein were detected in all samples (AAV1-p.hGJB2(SEQ ID NO: 30)-H2B-GFP-EnhancerGH(SEQ ID NO: 60) (Figure 6A), AAV1-p.hGJB2(SEQ ID NO: 33)-H2B-GFP-EnhancerGH(SEQ ID NO: 60) (Figure 6B), AAV1-p.hGJB2(SEQ ID NO: 34)-H2B-GFP-EnhancerGH(SEQ ID NO: 60) (Figure 6C)), indicating that methylated DNA (histone mark) does not affect expression. The enhancer was located 3' of the coding sequence in the vector.

[0180] In the fifth experiment, the GJB2 promoter of SEQ ID NO: 30 was tested in combination with different enhancers (full-length and truncated enhancers). Samples infected with the combination of the GJB2 promoter and full-length enhancer (Enhancer GH and Enhancer 9) showed higher expression of the reporter protein (AAV1-p.hGJB2(SEQ ID NO: 30)-H2B-GFP-Enhancer GH (SEQ ID NO: 60) (FIG. 7B) and AAV1-p.hGJB2(SEQ ID NO: 30)-H2B-GFP-Enhancer 9 (SEQ ID NO: 62) (FIG. 7D)) when compared to combinations containing truncated enhancers (AAV1-p.hGJB2(SEQ ID NO: 30)-H2B-GFP-Enhancer GHA (SEQ ID NO: 61) (FIG. 7C) and AAV1-p.hGJB2(SEQ ID NO: 30)-H2B-GFP-Enhancer 9C (SEQ ID NO: 63) (FIG. 7E)). Expression of the reporter protein was observed in all cells in the control sample (AAV1-CMV-H2B-GFP, FIG. 7F). The enhancer was located 3' of the coding sequence in the vector.

[0181] The promoter activities observed in these experiments are summarized in Table 10 below. [Table 10]

[0182] Example 4. GJB2 promoter / enhancer-driven GFP expression is enriched in vivo in mouse GJB2-expressing cells. To test the specificity of the GJB2 promoter of SEQ ID NO:30 in combination with various enhancers disclosed herein in vivo, wild-type C57BL / 6J mice were injected with an AAV vector comprising eGFP operably linked to the GJB2 promoter of SEQ ID NO:30 and the GJB2 enhancer described herein. In some cases, 1 μL of the AAV vector was injected into early postnatal mice via a fenestration of the posterior semicircular canal 2-3 days after birth (see Figures 9A-9B, 10A-10B, 11A-11B, 12A-12B, 13A-13B). In other cases, 1 μL of the AAV vector was injected into 6-8 week-old adult animals via a fenestration of the posterior semicircular canal (see Figures 8A-8D).

[0183] Four weeks after birth, animals were sacrificed and fixed in 10% NBF via cardiac perfusion, and their temporal bones were harvested and kept in 10% NBF for an additional 16 hours. After decalcification in 8% EDTA for 2 days, ears were microdissected and the vesicles and organ of Corti were prepared for immunohistochemistry. Whole specimen tissue preparations of the organ of Corti were counterstained with myosin 7a or Pou4f3 antibodies to visualize all hair cells and imaged under a confocal microscope (Leica® SP8, 20× / 0.75NA, 2 μm step size at 2 AU).

[0184] To visualize GFP expression in cross-sections of mouse cochlea, fixed, decalcified and microdissected temporal bones were embedded in paraffin blocks and then mesodermal sections were obtained. Sections were baked at 65°C overnight, deparaffinized, and tissue hydration was performed with Bound Dewax solution for 30 seconds. In addition, antigen retrieval was utilized via treatment at 94°C for 25 minutes in pH 9.0 EDTA. Primary antibody incubation against GFP (Abcam® No. 183734) was performed for 15 minutes at room temperature, followed by secondary antibody incubation for 30 minutes at room temperature and Red Refine (secondary antibody and color reagent: BOUND Polymer Refine Red Detection) incubation for 10 minutes + 5 minutes at room temperature. Finally, tissues were counterstained with hematoxylin for 7 minutes at room temperature.

[0185] To visualize GFP expression in the lateral wall of the cochlea and the stria vascularis, during cochlear dissociation, the lateral wall of the cochlea was removed by slicing the tissue along the external sulcus where the base of the spiral eminence meets the organ of Corti. After detachment of the lateral wall, fine forceps were used to remove the Reissner's membrane. For immunohistochemistry, fragments of the lateral wall were incubated in 50 mmol / L glycine / 0.01 mol / L phosphate-buffered saline (PBS) for 30 min at room temperature. The tissue was then washed twice for 10 min in 0.1 mol / L PBS. The tissue was then permeabilized with blocking solution (5% bovine serum albumin, 5% normal donkey serum, 0.5% Tx-100, 0.01 M PBS) for 1 h at room temperature. The tissue was then incubated in DAPI solution (2 μg / mL) for 10 min at room temperature, followed by 2 × 10 min washes in 0.1 M PBS each. Finally, the tissue was mounted and coverslipped in SlowFade™ Diamond Antifade Media (S36967). Sidewall sections were scanned using a Zeiss® LSM800 confocal microscope. Z-stacks were acquired using a 40× / 1.4 oil immersion objective with a resolution of 0.3×0.3×1.0 μm per voxel.

[0186] Enhancer GH induced more GFP expression in adult mouse cochleae compared to enhancer 9 in AAV-DJ vector (Figure 8A-8B). Cross-sections of adult mouse cochleae confirmed that GFP expression was restricted to supporting cells (GFP+ nuclei, dark grey, black arrows) and excluded from hair cells in the sensory epithelium (brackets) (Figure 8C-8D). Enhancer GH also induced more GFP expression in neonatal mouse cochleae compared to enhancer 9 in AAV-DJ vector (Figure 9A-9B).

[0187] In the AAV1 vector, enhancer GH induced GFP expression in supporting cells of the neonatal mouse ear (Figure 10A, GFP, white; Pou4f3 hair cells, light gray). GFP expression was excluded from hair cells of the sensory epithelium (brackets). Quantification of GFP+ supporting cells shows more GFP expression in the central supporting cells compared to the lateral supporting cells (Figure 10B). Enhancer GH also induced GFP expression in the lateral wall and stria vascularis (Figures 11A-11B).

[0188] In the Php.B vector, enhancer GH induced GFP expression in supporting cells of the neonatal mouse ear (Figure 12A, GFP, white, Pou4f3 hair cells, light grey). GFP expression was excluded from hair cells of the sensory epithelium (brackets). Figure 12B shows quantification of GFP+ supporting cells. Cross sections of mouse cochlea show that GFP expression was restricted to supporting cells and the lateral wall (Figures 13A-13B, GFP+ nuclei, dark grey, black arrows) and excluded from hair cells in the sensory epithelium (brackets). A schematic showing the order of elements in the vectors used in the experiments of Example 4 is provided in Figure 19A.

[0189] Example 5. Expression of GJB2 driven by the GJB2 promoter / enhancer pair results in restoration of hearing and preservation of outer hair cells in a mouse model of GJB2 deficiency. Neonatal GJB6-LacZ (GJB2-deficient mouse model) mice were unilaterally injected 2-3 days after birth via a fenestration of the posterior semicircular canal with 1 μL of vector encoding mouse Gjb2 driven by the GJB2 promoter / enhancer combination (AAV-p.hGJB2 (SEQ ID NO: 30) + enhancer GH-mGjb2). After 4 weeks, animals were anesthetized with ketamine and xylazine and auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were measured in both ears (injected and non-injected) to test for hearing recovery. Animals were then sacrificed and fixed in 10% NBF via cardiac perfusion, and gross specimen histology of the organ of Corti was performed as described in Example 4, except for GFP, to assess hair cell integrity. As shown in Figures 14 and 15, AAVPhp.Bp.hGJB2 (SEQ ID NO: 30) + enhancer GH-mGjb2 and AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer GH-mGjb2 restored hearing and prevented outer hair cell loss in gene replacement gene therapy in a GJB2-deficient mouse model. Similar results were observed using AAV-DJ serotype vectors. A schematic diagram showing the order of elements in the vectors used in the experiments of Example 5 is provided in Figure 19B.

[0190] Example 6. Characterization of additional GJB2 enhancers in neonatal mice. In the first experiment, the GJB2 promoter of SEQ ID NO:30 was combined with one or more of enhancers 1-8 (Enhancer 1: SEQ ID NO:52, Enhancer 2: SEQ ID NO:53, Enhancer 3: SEQ ID NO:54, Enhancer 4: SEQ ID NO:55, Enhancer 5: SEQ ID NO:56, Enhancer 6: SEQ ID NO:57, Enhancer 7: SEQ ID NO:58, Enhancer 8: SEQ ID NO:59) to evaluate GFP expression in neonatal cochlear explants. Enhancers driving histone-tagged GFP were screened in explants using AAV-DJ vectors. The enhancers were located 5' of the GJB2 promoter in the vector. A schematic showing the order of elements in the vector used in the experiment of Example 6 is provided in Figure 19C. Neonatal cochlear explants were established from P0-P2 wild-type C57BL / 6 mice. Briefly, neonatal mice were euthanized, the inner ear was removed from the head, and the cochlea was carefully removed using microdissection. The apex of the cochlear duct was removed by removing the modiolus from the center of the coil and exposing the sensory epithelium. Cochlear explants were placed on the prepared 3D rat tail collagen matrix with the luminal side of the cochlear duct facing up. Cochlear explant culture medium consisting of DMEM with 7% FBS and 1 U / μL penicillin G was added directly to the cultures. Viruses containing the GJB2 promoter of SEQ ID NO: 30 and one or more GJB2 enhancers (one or more of SEQ ID NOs: 52-59), driving GFP expression, were added to the medium at a concentration of 5e11 vgs / culture. Cultures were incubated with the virus for 48 hours, followed by 72 hours in fresh medium under standard culture conditions at 37°C and 5% CO2. Cultures were then fixed in 4% formaldehyde, permeabilized with 0.01% Triton®X100, blocked with 10% normal donkey serum, and stained with anti-Myo7a antibody (hair cells). Samples were imaged (Myo7a, native GFP) using a confocal microscope (Zeiss® LSM 810) with uniform settings (laser intensity and gain) across samples. Expression patterns, GFP intensity, and exclusion from hair cells were assessed.High GFP expression was observed for enhancer 1, enhancer 8, and two enhancer combinations (enhancer 1+6+7+8 and enhancer 2+3+4+5). Complete exclusion from hair cells was observed for enhancers 1, 4, 5, and 6, as well as two enhancer combinations. GFP was observed in the sensory epithelium region for all enhancers and enhancer combinations tested, except for enhancer 7. The results are shown in Figure 16. The sensory epithelium is marked with brackets (GFP, white; Myo7a, grey).

[0191] In a second experiment, to assess GFP expression in vivo, the same vectors assessed in neonatal cochlear explants, as described above in Example 4, were injected into early postnatal C57BL / 6J mice 2-3 days after birth via a fenestration of the posterior semicircular canal. Whole specimen tissue preparations of the organ of Corti and cross sections of mouse cochleae were also prepared and imaged as described in Example 4. With enhancer 1, GFP expression was present in all supporting cells of the sensory epithelium and many non-sensory cell types (top row, Figure 17). For all enhancers tested (GFP, white; Myo7a hair cells, grey), GFP was excluded from hair cells. GFP was detected in the lateral wall with enhancer 4. Section histology confirms the whole specimen expression (bottom row, GFP, dark grey; black arrows indicate examples of GFP+ cells). With enhancer 8, GFP expression was restricted to supporting cells of the sensory epithelium (top row, Figure 18). For all enhancers tested, GFP was excluded from hair cells. GFP expression was observed in Reissner's membrane for the combinations Enhancer 6 and Enhancer 2+3+4+5. For the combination Enhancer 1+6+7+8, GFP expression was present in all supporting cells of the sensory epithelium, most non-sensory cells of the cochlear duct, cells of the lateral wall and stria vascularis (top row, FIG. 18, GFP, white, Myo7a hair cells, grey). Section histology confirms the overall specimen expression (bottom row, GFP, dark grey, black arrows indicate examples of GFP+ cells). The same promoter / enhancer combinations were tested using AAV1 vectors and similar results were observed.

[0192] Example 7. Expression of GJB2 using the GJB2 promoter / enhancer pair maintained hearing thresholds and hair cells in wild-type mice compared to expression of GJB2 using a ubiquitous promoter, which resulted in elevated hearing thresholds and inner hair cell death. Six to eight week old C57BL / 6J mice were injected via a fenestration in the posterior semicircular canal with vehicle control or an AAV1 vector containing the human GJB2 coding sequence driven by a ubiquitous promoter (AAV-CMV-hGJB2) or the GJB2 enhancer / promoter pair (AAV-p.hGJB2 (SEQ ID NO: 30) + Enhancer 1 (SEQ ID NO: 52)). Enhancer 1 was located 5' to the promoter in the vector. Hearing measurements, extraction, and histological processing of tissues were performed 2 weeks later as described in Example 1. Six of eight wild type ears treated with AAV-CMV-GJB2 showed elevated ABR thresholds, whereas hearing thresholds of animals treated with vehicle control or the human GJB2 coding sequence driven by the GJB2 enhancer / promoter pair were comparable to baseline measurements (Figure 20A). As shown in Figure 20B, animals treated with AAV-CMV-GJB2 had inner hair cell loss, whereas hair cells were preserved in the other groups, demonstrating that overexpression of GJB2 using GJB2 regulatory elements that eliminate expression from hair cells is safe.

[0193] Example 8. Expression of GJB2 driven by the GJB2 promoter / enhancer combination results in hearing restoration and outer hair cell preservation in a mouse model of GJB2 deficiency. Hearing restoration was assessed in a GJB2-deficient mouse model using methods similar to those described in Example 5, with the following modifications. In the first experiment, animals were injected with AAV1 encoding human GJB2 driven by p.hGJB2 (SEQ ID NO: 30) + enhancer 1 (SEQ ID NO: 52). Enhancer 1 was located 5' of the promoter in the vector. Hearing measurements were performed 4, 8, and 12 weeks after injection, after which the animals were enucleated. The results of this experiment are shown in Figures 21A-21C.

[0194] In the second experiment, animals were injected with AAV1 encoding human GJB2 driven by AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 1 + 6 + 7 + 8 (SEQ ID NO: 52, 57, 58, 59). The enhancer was located 5' of the promoter in the vector. Hearing measurements were performed 4, 12, and 14 weeks after injection, after which the animals were enucleated. The results of this experiment are shown in Figures 22A-22C.

[0195] In the third experiment, animals were injected with AAV1 encoding human GJB2 driven by AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer 8 (SEQ ID NO: 59). Enhancer 8 was located 5' of the promoter in the vector. Hearing measurements were performed 4 and 15 weeks after injection, after which the animals were enucleated. The results of this experiment are shown in Figures 23A-23C.

[0196] As shown in Figures 21A-21C, 22A-22C, and 23A-23C, GJB2 gene replacement therapy using various GJB2 enhancer / promoter combinations in AAV1 could restore hearing and prevent outer hair cell loss in a GJB2-deficient mouse model, indicating that successful hearing restoration can be achieved by utilizing a combination of regulatory elements that appropriately restrict GJB2 expression to GJB2-expressing cells.

[0197] Example 9. The GJB2 enhancer / promoter combination can be used to drive expression of GFP in GJB2-expressing cells in non-human primates. Adult non-human primates were locally injected with 60 μL of AAV1-p.hGJB2 (SEQ ID NO: 30) + GJB2enh-GFP into the round window of the cochlea at a flow rate of 6 μL / min. GJB2enh consisted of either: Enhancer 1 (SEQ ID NO: 52), Enhancer GH (SEQ ID NO: 60), or Enhancer 1 + 6 + 7 + 8 (SEQ ID NO: 52, 57, 58, 59). Enhancer 1 and combinations of Enhancers 1, 6, 7, and 8 were located 5' of the promoter in the vector. Enhancer GH was located 3' of the transgene in the vector. Animals' ears were ventilated with the lateral posterior semicircular canal to allow for the outflow of perillin during parturition. Four weeks after injection, animals were sacrificed by cardiac perfusion with 10% neutral buffered formalin (NBF) and their temporal bones were harvested.

[0198] Overall specimen histology and quantification After decalcification in 0.5 M EDTA, ears were microdissected and the organ of Corti was prepared for immunohistochemistry. Whole specimen tissue preparations of the organ of Corti were counterstained with DAPI, Myosin7a antibody, and anti-GFP antibody to visualize cell nuclei, hair cells, and low GFP signal. These were imaged together with virus-mediated native GFP expression under a confocal microscope (Zeiss® LSM880, 40× / 0.95NA, 1 μm step size at 2 AU). To quantify the percentage of GFP-positive central and lateral supporting cells, regions of interest in frequencies spanning the length of the cochlea were selected and the total number of central and lateral supporting cells was counted using DAPI and the number of GFP-positive supporting cells was counted. The results are shown in Figure 24. GFP expression was found in approximately 20% of the central supporting cells and approximately 50% of the lateral supporting cells, and was relatively similar between groups. Representative images are shown in Figures 25A-25C. Minimal expression was detected in hair cells of all groups.

[0199] Section histology and quantification After decalcification with 0.5m EDTA, the temporal bones were embedded in paraffin and then midlimb sections were obtained. Sections were baked at 65°C overnight to deparaffinize and tissue hydration was performed with Bound Dewax solution for 30 seconds. In addition, antigen retrieval via 2 hours of 70°C treatment in pH 9.0 EDTA was utilized. Primary antibody incubation against GFP (Abcam® No. 183734) was performed for 15 minutes at room temperature, followed by a 30 minute secondary antibody incubation at room temperature and a 10 minute + 5 minute room temperature incubation with FastRed chromagen (secondary antibody and color reagent: BOND Polymer Refine Red Detection). Finally, the tissue was counterstained with hematoxylin for 7 minutes at room temperature. Slides were imaged with a 20x objective using a Lecia® Aperio slide scanner.

[0200] For RNAscope on NHP sections, epitope retrieval was performed using BOND ER solution (pH 9) for 10 min at 90°C. After subsequent washing with BOND wash (1x), slides were incubated in RNAscope 2.5LSx protease for 10 min at 40°C, followed by additional washing. Sections were incubated in hydrogen peroxide to block endogenous peroxidase activity. Following additional washing with BOND wash (1x), probe hybridization was performed for 2 h at 42°C, followed by 6 amplification steps. Sections were then incubated with color development reagent (BOND Polymer Refine Red) for 1 min + 15 min at room temperature. Finally, tissues were counterstained with hematoxylin for 10 min at room temperature. Slides were imaged with a 40x objective using a Leica® Aperio slide scanner.

[0201] Sections were used to score the magnitude of GFP from both IHC and ISH readouts across various cochlear cell types. Scores ranged from 0 to 3, from lowest to highest expression, and are summarized in Figure 26. Animals that showed a complete lack of labeling for both readouts were assumed to be the result of failed viral delivery and were excluded from the analysis. Consistent with the fact that the GFP ISH probe labeled both the viral genome and GFP mRNA, the GFP ISH signal was broadly detected across cochlear cell types, including hair cells. However, quantification of the GFP IHC signal appeared to be more restricted to GJB2-expressing cell types. Specifically, protein expression was largely absent from hair cells but was detected in Deiters cells and various groups of supporting cells, including the inner and outer sulcus, spiral eminence, and fibrocytes.

[0202] Example 10. The GJB2 enhancer / promoter pair drives expression of a FLAG-tagged human GJB2 transgene in GJB2-expressing cells in non-human primates. Adult non-human primates were locally injected with 60 μL of AAV1-p.hGJB2 (SEQ ID NO: 30) + enhancer1 (SEQ ID NO: 52)-hGJB2-FLAG in one or two doses at a flow rate of 6 μL / min into the round window of the inner ear. Enhancer1 was located 5' of the promoter in the vector. Animal ears were ventilated with the lateral posterior semicircular canal to allow outflow of perillin during parturition. Tissue processing was performed as in Example 9 with the following modifications. Gross specimens were counterstained with DAPI, GJB2 antibody, FLAG antibody, and Sox2 antibody to label cell nuclei, endogenous GJB2 protein, tagged GJB2 transgene, and supporting cells, respectively. FLAG staining was utilized to quantify the percentage of transgene-positive Deiters cells (Figure 27B). Transgene expression was detected in Deiters cells and was biased toward higher frequencies. Representative images centered on the sensory epithelium are shown in Figure 27A. The transgene (FLAG staining) was detected in both the central and lateral supporting cell regions, and the transgene staining pattern reflected endogenous GJB2, thus indicating that the transgene was appropriately expressed and integrated into plasma membrane plaques. Minimal transgene expression was seen in hair cells.

[0203] Example 11. Enhancer 8 retains specificity when paired with the GJB2 promoter of SEQ ID NO:1 in mouse cochlear explants. Mouse cochlear explant cultures were established as previously described in Example 6, except for the AAV dose, which in this case was 5e11vg / culture. To evaluate the contribution of the proximal promoter region to the specificity of the enhancer / promoter pair, enhancer 1 (SEQ ID NO: 52) and enhancer 8 (SEQ ID NO: 59) were paired with either the proximal GJB2 promoter sequence (proxGJB2-SEQ ID NO: 1) or the minimal beta-globin promoter (B-glob), which drives nuclear GFP expression. Both promoters were tested alone as controls. Expression patterns, GFP intensity, and exclusion from hair cells were evaluated and compared to results from the same enhancer paired with the GJB2 promoter of SEQ ID NO: 30 (Figure 16). The results are shown in Figure 28. Only the proxGJB2 and B-glob promoters drove minimal GFP expression overall. The use of the B-glob promoter eliminated specificity from both enhancers 1 and 8, and expression was seen broadly in hair cells, supporting cells, and beyond the sensory epithelium. Enhancer 1 paired with proxGJB2 resulted in positive expression in outer hair cells compared to the GJB2 promoter of SEQ ID NO: 30, which completely excluded GFP expression from hair cells. Enhancer 8 paired with proxGJB2 excluded expression from hair cells, but supporting cell expression was not restricted to the sensory epithelium compared to the GJB2 promoter of SEQ ID NO: 30. This indicates that enhancer 8 can exclude GFP expression from hair cells when paired with an alternative GJB2 promoter.

[0204] Example 12. Administration of a composition containing a nucleic acid vector containing the GJB2 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., sensorineural hearing loss, such as GJB2-associated hearing loss) to improve or restore hearing. To this end, one skilled in the art can provide a GJB2 promoter as described herein (e.g., a promoter of any one of SEQ ID NOs:29-35, including a first region 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 functional portion or derivative thereof, operably linked to a polynucleotide encoding an expression product, such as a polynucleotide encoding a wild-type form of Gjb2 (e.g., a polynucleotide encoding the amino acid sequence of SEQ ID NO:38), and / or a second region 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 a functional portion or derivative thereof. A composition containing an AAV vector (e.g., an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vector) containing a polynucleotide that contains a second region having sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), and optionally a linker joining the first and second regions may be administered to a human patient. A composition containing an AAV vector may be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or through the round window membrane) to treat sensorineural hearing loss.

[0205] After administering the composition to the patient, a practitioner in the art can monitor the improvement of the patient in response to the treatment by a variety of methods.For example, the practitioner can monitor the hearing of the patient by carrying out standard tests such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions after administering the composition.The finding that the patient shows improvement in hearing in one or more of the tests after administering the composition compared to the hearing test results before administering the composition indicates that the patient is responding favorably to the treatment.Subsequent doses can be determined and administered as necessary.

[0206] Example 13. Administration of a composition containing a nucleic acid vector containing a GJB2 enhancer to a subject with sensorineural hearing loss. According to the methods disclosed herein, a practitioner of the art can treat a patient, e.g., a human patient, with hearing loss (e.g., sensorineural hearing loss) to improve or restore hearing. To this end, one skilled in the art can administer a gene encoding the present invention operably linked to a promoter (e.g., a GJB2 promoter, such as the GJB2 promoter described herein, or a GJB2 promoter having at least 85% sequence identity to any one of SEQ ID NOs: 1, 8-11, and 66-68, or an inner ear cell type specific promoter, such as a promoter listed in Table 9) operably linked to a polynucleotide encoding an expression product (e.g., a wild type of a gene for cochlear supporting cells associated with hearing loss that is mutated in a subject, such as GJB2, GJB6, GAS2, or a gene listed in Table 6, or a polynucleotide encoding an expression product that can induce differentiation of cochlear supporting cells into cochlear hair cells, such as a polynucleotide listed in Table 5). A composition containing an AAV vector (e.g., an AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vector) containing a GJB2 enhancer as described herein (e.g., a polynucleotide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to any one of SEQ ID NOs:52-63) may be administered to a human patient. A composition containing an AAV vector can be administered to a patient, for example, by local administration to the inner ear (e.g., injection into the perilymph or through the round window membrane) to treat sensorineural hearing loss.

[0207] After administering the composition to the patient, a practitioner in the art can monitor the improvement of the patient in response to the treatment by a variety of methods.For example, the practitioner can monitor the hearing of the patient by carrying out standard tests such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions after administering the composition.The finding that the patient shows improvement in hearing in one or more of the tests after administering the composition compared to the hearing test results before administering the composition indicates that the patient is responding favorably to the treatment.Subsequent doses can be determined and administered as necessary.

[0208] Exemplary embodiments of the present invention are described in the following enumerated paragraphs.

[0209] E1. 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 functional portion or derivative thereof comprising at least one (e.g., one of many) of SEQ ID NOs: 3-12, operably linked to a second region 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 a functional portion or derivative thereof comprising at least one (e.g., one of many) of SEQ ID NOs: 20-24, the polynucleotide comprising a GJB2 promoter comprising a first region 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), wherein the distance between the first region and the second region in the polynucleotide (e.g., the distance between the 3' end of the first region and the 5' end of the second region) is 1 kb or less, and optionally comprising a linker comprising 1 to 100 nucleotides between the first region and the second region.

[0210] E2. The polynucleotide of E1, wherein the distance between the first region and the second region in the polynucleotide is 0.5 kb or less.

[0211] E3. The polynucleotide of E2, wherein the distance between the first region and the second region in the polynucleotide is 0.25 kb or less.

[0212] E4. A polynucleotide comprising at least one of SEQ ID NO:1, or at least one of SEQ ID NOs:3-12 (e.g., one of many thereof) operably linked to a second region 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 a functional portion or derivative thereof comprising at least one of SEQ ID NOs:20-24 (e.g., one of many thereof). 97%, 98%, 99% or more sequence identity) to a functional portion or derivative thereof comprising a GJB2 promoter comprising a first region 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), and if said first region and said second region are not directly linked, said first region is operably linked to said second region by a nucleic acid sequence that is distinct from an intervening genomic sequence.

[0213] E5. A polynucleotide comprising a GJB2 promoter operably linked to a heterologous polynucleotide, wherein the GJB2 promoter is operably linked to a second region 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 a functional portion or derivative thereof comprising at least one (e.g., one of many) of SEQ ID NOs:20-24. the polynucleotide comprises a first region 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 functional part or derivative thereof comprising at least one of SEQ ID NOs:3 to 12 (e.g., one or more of them), linked to a functional group, and optionally comprising a linker comprising 1 to 100 nucleotides between the first region and the second region.

[0214] E6. The polynucleotide of E5, wherein the heterologous polynucleotide is a codon-optimized nucleic acid sequence encoding wild-type GJB2 (eg, a nucleic acid sequence set forth in SEQ ID NOs: 41-44).

[0215] E7. The polynucleotide of E5, wherein said heterologous polynucleotide encodes a Gjb2 protein comprising one or more conservative amino acid substitutions relative to SEQ ID NO: 38 that retain the therapeutic function of wild-type Gjb2.

[0216] E8. The polynucleotide of E5, wherein the heterologous polynucleotide encodes Gjb6 (eg, encodes SEQ ID NO:47).

[0217] E9. The polynucleotide of E5, wherein said heterologous polynucleotide encodes BDNF or NTF3.

[0218] E10. The polynucleotide of E5, wherein said heterologous polynucleotide is a polynucleotide encoding a protein or inhibitory RNA that can induce differentiation of cochlear supporting cells into cochlear hair cells or induce or increase proliferation of cochlear supporting cells (e.g., a polynucleotide listed in Table 5), or a transgene that is expressed in cochlear supporting cells and corresponds to a wild-type form of a gene that is mutated in a subject with hearing loss (e.g., a transgene that corresponds to a wild-type form of a gene listed in Table 6).

[0219] E11. The polynucleotide of E5, wherein the heterologous polynucleotide encodes (e.g., can be transcribed to produce) 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.

[0220] E12. The polynucleotide of any one of E1 to E11, wherein the first region has at least 85% sequence identity to SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

[0221] E13. The polynucleotide of E12, wherein said first region has the sequence of SEQ ID NO:1.

[0222] E14. The polynucleotide of any one of E1 to E11, wherein the first region 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: 3 to 12.

[0223] E15. The polynucleotide of E14, wherein the first region 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.

[0224] E16. The polynucleotide of E15, wherein said first region has the sequence of SEQ ID NO:3.

[0225] E17. The polynucleotide of E14, wherein the first region 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.

[0226] E18. The polynucleotide of E17, wherein said first region has the sequence of SEQ ID NO:4.

[0227] E19. The polynucleotide of E14, wherein the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:5.

[0228] E20. The polynucleotide of E19, wherein said first region has the sequence of SEQ ID NO:5.

[0229] E21. The polynucleotide of E14, wherein the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:6.

[0230] E22. The polynucleotide of E21, wherein said first region has the sequence of SEQ ID NO:6.

[0231] E23. The polynucleotide of E14, wherein the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:7.

[0232] E24. The polynucleotide of E23, wherein said first region has the sequence of SEQ ID NO:7.

[0233] E25. The polynucleotide of E14, wherein the first region 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.

[0234] E26. The polynucleotide of E25, wherein said first region has the sequence of SEQ ID NO:8.

[0235] E27. The polynucleotide of E14, wherein the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:9.

[0236] E28. The polynucleotide of E27, wherein said first region has the sequence of SEQ ID NO:9.

[0237] E29. The polynucleotide of E14, wherein the first region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:10.

[0238] E30. The polynucleotide of E29, wherein said first region has the sequence of SEQ ID NO:10.

[0239] E31. The polynucleotide of E14, wherein the first region 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:11.

[0240] E32. The polynucleotide of E31, wherein said first region has the sequence of SEQ ID NO:11.

[0241] E33. The polynucleotide of E14, wherein the first region 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:12.

[0242] E34. The polynucleotide of E33, wherein said first region has the sequence of SEQ ID NO:12.

[0243] E35. The polynucleotide according to any one of E1 to E11, wherein the functional part of SEQ ID NO:1 comprises the sequence of SEQ ID NO:4 and the sequence of SEQ ID NO:12.

[0244] E36. The polynucleotide of E35, wherein the first region 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: 13.

[0245] E37. The polynucleotide of E36, wherein said first region has the sequence of SEQ ID NO:13.

[0246] E38. The polynucleotide according to any one of E1 to E11, wherein the functional part of SEQ ID NO:1 comprises the sequence of SEQ ID NO:5 and the sequence of SEQ ID NO:12.

[0247] E39. The polynucleotide of E38, wherein the first region 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:14.

[0248] E40. The polynucleotide of E39, wherein said first region has the sequence of SEQ ID NO:14.

[0249] E41. The polynucleotide according to any one of E1 to E11, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6 and the sequence of SEQ ID NO: 12.

[0250] E42. The polynucleotide of E41, wherein the first region 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:15.

[0251] E43. The polynucleotide of E42, wherein said first region has the sequence of SEQ ID NO: 15.

[0252] E44. The polynucleotide according to any one of E1 to E11, wherein the functional part of SEQ ID NO:1 comprises the sequence of SEQ ID NO:7 and the sequence of SEQ ID NO:12.

[0253] E45. The polynucleotide of E44, wherein the first region 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: 16.

[0254] E46. The polynucleotide of E45, wherein the first region has the sequence of SEQ ID NO:16.

[0255] E47. The polynucleotide according to any one of E1 to E11, wherein the functional part of SEQ ID NO:1 comprises the sequence of SEQ ID NO:9 and the sequence of SEQ ID NO:12.

[0256] E48. The polynucleotide of E47, wherein the first region 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.

[0257] E49. The polynucleotide of E48, wherein the first region has the sequence of SEQ ID NO:17.

[0258] E50. The polynucleotide according to any one of E1 to E11, wherein the functional portion of SEQ ID NO:1 comprises the sequence of SEQ ID NO:10 and the sequence of SEQ ID NO:12.

[0259] E51. The polynucleotide of E50, wherein the first region 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:18.

[0260] E52. The polynucleotide of E51, wherein said first region has the sequence of SEQ ID NO:18.

[0261] E53. The polynucleotide according to any one of E1 to E11, wherein the functional part of SEQ ID NO:1 comprises a sequence of SEQ ID NO:11 and a sequence of SEQ ID NO:12.

[0262] E54. The polynucleotide of E53, wherein the first region 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:19.

[0263] E55. The polynucleotide of E54, wherein said first region has the sequence of SEQ ID NO:19.

[0264] E56. The polynucleotide of any one of E35, E38, E41, E44, E47, E50, and E53, wherein the sequence of SEQ ID NO:12 precedes the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0265] E57. The polynucleotide of any one of E1-E56, wherein the second region 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.

[0266] E58. The polynucleotide of E57, wherein the second region has the sequence of SEQ ID NO:2.

[0267] E59. The polynucleotide of any one of E1 to E56, wherein the second region 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: 20 to 24.

[0268] E60. The polynucleotide of E59, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:20.

[0269] E61. The polynucleotide of E60, wherein said second region has the sequence of SEQ ID NO: 20.

[0270] E62. The polynucleotide of E59, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:21.

[0271] E63. The polynucleotide of E62, wherein said second region has the sequence of SEQ ID NO: 21.

[0272] E64. The polynucleotide of E59, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:22.

[0273] E65. The polynucleotide of E64, wherein said second region has the sequence of SEQ ID NO: 22.

[0274] E66. The polynucleotide of E59, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:23.

[0275] E67. The polynucleotide of E66, wherein said second region has the sequence of SEQ ID NO: 23.

[0276] E68. The polynucleotide of E59, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:24.

[0277] E69. The polynucleotide of E68, wherein said second region has the sequence of SEQ ID NO: 24.

[0278] E70. The polynucleotide according to any one of E1 to E56, wherein the functional part of SEQ ID NO:2 comprises the sequence of SEQ ID NO:23 and the sequence of SEQ ID NO:20.

[0279] E71. The polynucleotide of E70, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:25.

[0280] E72. The polynucleotide of E71, wherein said second region has the sequence of SEQ ID NO: 25.

[0281] E73. The polynucleotide according to any one of E1 to E56, wherein the functional part of SEQ ID NO:2 comprises the sequence of SEQ ID NO:24 and the sequence of SEQ ID NO:20.

[0282] E74. The polynucleotide of E73, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:26.

[0283] E75. The polynucleotide of E74, wherein said second region has the sequence of SEQ ID NO:26.

[0284] E76. The polynucleotide according to any one of E1 to E56, wherein the functional part of SEQ ID NO:2 comprises the sequence of SEQ ID NO:23 and the sequence of SEQ ID NO:22.

[0285] E77. The polynucleotide of E76, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:27.

[0286] E78. The polynucleotide of E77, wherein the second region has the sequence of SEQ ID NO: 27.

[0287] E79. The polynucleotide according to any one of E1 to E56, wherein the functional part of SEQ ID NO:2 comprises the sequence of SEQ ID NO:24 and the sequence of SEQ ID NO:22.

[0288] E80. The polynucleotide of E79, wherein the second region has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to SEQ ID NO:28.

[0289] E81. The polynucleotide of E80, wherein said second region has the sequence of SEQ ID NO:28.

[0290] E82. The polynucleotide of any one of E70, E73, E76, and E79, wherein the sequence of SEQ ID NO:20 precedes the sequence of SEQ ID NO:23 or SEQ ID NO:24, or the sequence of SEQ ID NO:22 precedes the sequence of SEQ ID NO:23 or SEQ ID NO:24.

[0291] E83. The polynucleotide of any one of E1-E82, wherein the first region is directly linked (i.e., fused) to the second region without a linker (e.g., the 3' end of the first region is immediately preceding the 5' end of the second region).

[0292] E84. The polynucleotide of E83, wherein said GJB2 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:29.

[0293] E85. The polynucleotide of E84, wherein said GJB2 promoter has the sequence of SEQ ID NO:29.

[0294] E86. The polynucleotide of E83, wherein said GJB2 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:30.

[0295] E87. The polynucleotide of E86, wherein said GJB2 promoter has the sequence of SEQ ID NO: 30.

[0296] E88. The polynucleotide of E83, wherein said GJB2 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:31.

[0297] E89. The polynucleotide of E88, wherein said GJB2 promoter has the sequence of SEQ ID NO:31.

[0298] E90. The polynucleotide of E83, wherein said GJB2 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:32.

[0299] E91. The polynucleotide of E90, wherein said GJB2 promoter has the sequence of SEQ ID NO:32.

[0300] E92. The polynucleotide of E83, wherein said GJB2 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: 33.

[0301] E93. The polynucleotide of E92, wherein said GJB2 promoter has the sequence of SEQ ID NO: 33.

[0302] E94. The polynucleotide of E83, wherein said GJB2 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:34.

[0303] E95. The polynucleotide of E94, wherein said GJB2 promoter has the sequence of SEQ ID NO:34.

[0304] E96. The polynucleotide of E83, wherein said GJB2 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:35.

[0305] E97. The polynucleotide of E96, wherein said GJB2 promoter has the sequence of SEQ ID NO:35.

[0306] E98. A polynucleotide comprising a GJB2 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:13.

[0307] E99. The polynucleotide of E98, wherein said GJB2 promoter has the sequence of SEQ ID NO:13.

[0308] E100. A polynucleotide comprising a GJB2 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:14.

[0309] E101. The polynucleotide of E100, wherein said GJB2 promoter has the sequence of SEQ ID NO:14.

[0310] E102. A polynucleotide comprising a GJB2 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:15.

[0311] E103. The polynucleotide of E102, wherein said GJB2 promoter has the sequence of SEQ ID NO:15.

[0312] E104. A polynucleotide comprising a GJB2 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:16.

[0313] E105. The polynucleotide of E104, wherein said GJB2 promoter has the sequence of SEQ ID NO:16.

[0314] E106. A polynucleotide comprising a GJB2 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.

[0315] E107. The polynucleotide of E106, wherein said GJB2 promoter has the sequence of SEQ ID NO:17.

[0316] E108. A polynucleotide comprising a GJB2 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:18.

[0317] E109. The polynucleotide of E108, wherein said GJB2 promoter has the sequence of SEQ ID NO:18.

[0318] E110. A polynucleotide comprising a GJB2 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:19.

[0319] E111. The polynucleotide of E110, wherein said GJB2 promoter has the sequence of SEQ ID NO:19.

[0320] E112. A polynucleotide comprising a GJB2 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:25.

[0321] E113. The polynucleotide of E112, wherein said GJB2 promoter has the sequence of SEQ ID NO: 25.

[0322] E114. A polynucleotide comprising a GJB2 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:26.

[0323] E115. The polynucleotide of E114, wherein said GJB2 promoter has the sequence of SEQ ID NO:26.

[0324] E116. A polynucleotide comprising a GJB2 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:27.

[0325] E117. The polynucleotide of E116, wherein said GJB2 promoter has the sequence of SEQ ID NO:27.

[0326] E118. A polynucleotide comprising a GJB2 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:28.

[0327] E119. The polynucleotide of E118, wherein said GJB2 promoter has the sequence of SEQ ID NO:28.

[0328] E120. A polynucleotide comprising a GJB2 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 ...

Claims

1. A polynucleotide comprising: (i) a first region having at least 85% sequence identity to SEQ ID NO:1 or to a functional portion or derivative of SEQ ID NO:1, including at least one of SEQ ID NOs:3-12; (ii) a second region to which the first region is operably linked, the second region having at least 85% sequence identity to SEQ ID NO: 2 or to a functional portion or derivative of SEQ ID NO: 2, including at least one of SEQ ID NOs: 20-24; wherein the distance between the first region and the second region in the polynucleotide is 1 kilobase (kb) or less.

2. The first region is (i) has at least 85% sequence identity to SEQ ID NO:1 or any one of SEQ ID NOs:3-19; or (ii) having the sequence of SEQ ID NO: 1 or any one of SEQ ID NOs: 3 to 19; The polynucleotide of claim 1.

3. The polynucleotide of claim 2, wherein the first region has the sequence of SEQ ID NO:

3.

4. The second region is (i) has at least 85% sequence identity to SEQ ID NO:2 or any one of SEQ ID NOs:20-28; or (ii) having the sequence of SEQ ID NO: 2 or any one of SEQ ID NOs: 20 to 28; The polynucleotide of claim 1.

5. The polynucleotide of claim 4, wherein the second region has a sequence selected from the group consisting of SEQ ID NOs: 20 to 22, 25, and 26.

6. The polynucleotide of claim 1 , wherein the first region is fused directly to the second region without a linker.

7. The GJB2 promoter is (i) has at least 85% sequence identity to any one of SEQ ID NOs: 30-35; or (ii) having any one of the sequences set forth in SEQ ID NOs: 30 to 35; The polynucleotide of claim 6.

8. The polynucleotide of claim 1 , wherein the GJB2 promoter is operably linked to a polynucleotide encoding an expression product.

9. The expression product (i) a protein, a short hairpin RNA (shRNA), an antisense oligonucleotide (ASO), a component of a gene editing system, or a microRNA; (ii) Gjb2 or Gjb6 endogenously expressed in GJB2-expressing cells or GJB2-expressing inner ear cells; or (iii) is a heterologous expression product; The polynucleotide of claim 8.

10. The polynucleotide of claim 1 , wherein the polynucleotide further comprises a GJB2 enhancer operably linked to the GJB2 promoter.

11. The GJB2 enhancer (i) has at least 85% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 52-63; or (ii) The polynucleotide of claim 10, having any one of the sequences of SEQ ID NOs: 52 to 63.

12. A nucleic acid vector comprising the polynucleotide of claim 1.

13. A composition comprising the nucleic acid vector of claim 12 and a pharmaceutically acceptable carrier, diluent, or excipient.

14. A nucleic acid vector described in claim 12 or a composition described in claim 13 for use in a method for expressing an expression product in a GJB2-expressing cell or a GJB2-expressing inner ear cell, the method comprising contacting the GJB2-expressing cell with the nucleic acid vector described in claim 12 or the composition described in claim 13.

15. 14. The nucleic acid vector of claim 12 or the composition of claim 13 for use in a method for treating a subject having or at risk of developing GJB2-associated hearing loss, the method comprising administering to the inner ear of the subject a therapeutically effective amount of the nucleic acid vector of claim 12 or the composition of claim 13, wherein the polynucleotide encodes an expression product that is Gjb2 or Gjb6.

16. The nucleic acid vector or composition of claim 15, wherein the GJB2-associated hearing loss is DFNB1 or DFNA3.

17. 17. The nucleic acid vector or composition of claim 16, wherein the subject has a mutation in GJB2, a mutation in GJB6, or a mutation in both GJB2 and GJB6.

18. A nucleic acid vector as described in claim 12 or a composition as described in claim 13 for use in a method for (i) inducing or increasing cochlear hair cell regeneration in a subject in need thereof or (ii) treating a subject having or at risk of developing hearing loss associated with damage or loss of cochlear hair cells, said method comprising administering to the inner ear of said subject a therapeutically effective amount of the nucleic acid vector as described in claim 12 or the composition as described in claim 13, wherein said nucleic acid vector comprises a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO: 59, and said polynucleotide encodes an expression product capable of promoting or increasing the proliferation of cochlear supporting cells or the differentiation of said cochlear supporting cells into said cochlear hair cells.

19. 19. The nucleic acid vector or composition of claim 18, wherein the polynucleotide encoding the expression product is a polynucleotide listed in Table 5.

20. 14. The nucleic acid vector of claim 12 or the composition of claim 13 for use in a method of treating a subject having or at risk of developing an inherited hearing loss associated with a mutation in a gene endogenously expressed in cochlear supporting cells, or a disease listed in Table 6, said method comprising administering to the inner ear of said subject a therapeutically effective amount of the nucleic acid vector of claim 12 or the composition of claim 13, wherein said nucleic acid vector comprises a GJB2 enhancer having at least 85% sequence identity to SEQ ID NO:59, and wherein said polynucleotide encodes an expression product which is a wild-type form of the gene that is mutated in said cochlear supporting cells or said disease.