Gene Therapy Delivery Compositions and Methods for Treating Hearing Loss - Patent application

Abstract

The present disclosure provides a construct comprising a coding sequence operably linked to a promoter, the coding sequence encoding a polypeptide (e.g., a therapeutic polypeptide). Exemplary constructs include AAV constructs. Methods of using the disclosed constructs for the treatment of hearing loss and / or hearing loss are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 188,450, filed May 13, 2021, U.S. Provisional Patent Application No. 63 / 251,025, filed September 30, 2021, and U.S. Provisional Patent Application No. 63 / 277,549, filed November 9, 2021, which are incorporated by reference herein in their entireties.

[0002] Reference to an electronically submitted sequence listing The contents of the electronically submitted Sequence Listing as an ASCII text file (Name: 4833_008CP03_Seqlisting_ST25.TXT; Size: 269,049 bytes; Creation Date: May 9, 2022) have been submitted with this application and are incorporated herein by reference in their entirety. [Background technology]

[0003] Hearing loss can be conductive (originating from the ear canal or middle ear), sensorineural (originating from the inner ear or auditory nerve), or mixed. Most forms of non-syndromic hearing loss are associated with permanent hearing loss caused by damage to the structures of the inner ear (sensorineural hearing loss), although some forms may involve changes in the middle ear (conductive hearing loss). The majority of human sensorineural hearing loss is caused by abnormalities of the hair cells of the organ of Corti in the cochlea (poor hair cell function). The hair cells may be abnormal at birth or may be damaged during an individual's lifetime (e.g., as a result of noise trauma or infection).

[0004] Sensorineural hearing loss (SNHL) is the most common congenital sensory disorder, and the most common genetic cause is a mutation in the gap junction β2 gene (GJB2), which encodes the connexin 26 (Cx26) protein. Summary of the Invention

[0005] Certain aspects of the present disclosure relate to promoters, e.g., cell-specific promoters, derived from portions of the GDF6, PARM1, MMP15, or VIM promoters, and capable of directing transcription of a coding sequence (e.g., encoding a connexin 26 polypeptide or a functional fragment thereof) in inner ear supporting cells.

[0006] Certain embodiments of the present disclosure relate to a polynucleotide comprising a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, or 90-99. In some embodiments, the polynucleotide is a promoter.

[0007] Certain aspects of the present disclosure relate to polynucleotides comprising a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 40, 90, 96, or 99.

[0008] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:90.

[0009] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:40.

[0010] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:96.

[0011] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:99.

[0012] In some embodiments, the polynucleotide is capable of directing transcription of a coding sequence for a connexin 26 polypeptide or a functional fragment thereof.

[0013] Certain aspects of the present disclosure relate to constructs comprising a polynucleotide disclosed herein and a nucleic acid sequence comprising a coding sequence for a connexin 26 polypeptide or a functional fragment thereof. In some aspects, the construct is an expression cassette.

[0014] In some embodiments, the polynucleotide of the construct is a promoter and is operably linked to a coding sequence. In some embodiments, the polynucleotide is capable of directing transcription of the coding sequence in inner ear supporting cells.

[0015] In some embodiments, the polypeptide of the construct is a connexin 26 polypeptide or a functional fragment thereof.

[0016] Certain aspects of the present disclosure relate to constructs, including constructs comprising a polynucleotide. In some aspects, the construct further comprises a nucleic acid sequence encoding a polypeptide. In some aspects, the polynucleotide is operably linked to the nucleic acid sequence encoding the polypeptide. In some aspects, the polynucleotide promotes expression of the nucleic acid in inner ear supporting cells.

[0017] Certain aspects of the present disclosure relate to constructs comprising a polynucleotide encoding a therapeutic polypeptide operably linked to a promoter that expresses the polynucleotide in inner ear supporting cells. In some aspects, the polynucleotide encodes a therapeutic polypeptide or a reporter polypeptide. In some aspects, the promoter selectively expresses the polynucleotide in inner ear supporting cells.

[0018] Certain aspects of the present disclosure relate to constructs comprising a polynucleotide encoding a polypeptide operably linked to a promoter that expresses the polynucleotide in an inner ear supporting cell, wherein the promoter is heterologous to the polynucleotide.

[0019] Certain aspects of the present disclosure relate to expression constructs comprising a coding sequence for a connexin 26 polypeptide or a functional fragment thereof operably linked to a promoter, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 40, 90, 96 or 99, and wherein the promoter is capable of directing transcription of the coding sequence.

[0020] In some embodiments, the promoter of the expression construct comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:90.

[0021] In some embodiments, the promoter of the expression construct comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:40.

[0022] In some embodiments, the promoter of the expression construct comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:96.

[0023] In some embodiments, the promoter of the expression construct comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:99.

[0024] In some embodiments, the expression construct further comprises a second promoter operably linked to the coding sequence, wherein the second promoter is heterologous or homologous to the coding sequence.

[0025] In some embodiments, the promoter of the expression construct is capable of directing transcription of the coding sequence in inner ear supporting cells.

[0026] In some embodiments, the inner ear supporting cells are selected from one or more of medial phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of corikers cells (KOs), greater ridge epithelial cells (GERs) (including lateral greater ridge epithelial cells (LGERs)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall.

[0027] In some aspects, the polynucleotides, constructs, or expression constructs disclosed herein further comprise a minimal GJB2 promoter operably linked to the coding sequence for a connexin 26 polypeptide or a functional fragment thereof.

[0028] In some aspects, a construct or expression construct disclosed herein comprises a GJB2 nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 117-126.

[0029] Certain aspects of the present disclosure relate to expression constructs comprising a coding sequence for a connexin 26 polypeptide or functional fragment thereof operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter, wherein the polynucleotide is expressed in inner ear supporting cells. In some aspects, the inner ear supporting cell-selective promoter is heterologous to the coding sequence for the connexin 26 polypeptide or functional fragment thereof.

[0030] In some embodiments, the inner ear supporting cell-selective promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 40, 90, 96, or 99.

[0031] In some embodiments, the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:90.

[0032] In some embodiments, the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:40.

[0033] In some embodiments, the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:96.

[0034] In some embodiments, the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:99.

[0035] In some embodiments, the inner ear supporting cell-selective promoter comprises a nucleic acid sequence having at least 95% identity to a sequence selected from one or more of SEQ ID NOs: 90, 40, 96, or 99.

[0036] In some embodiments, the inner ear supporting cells are selected from one or more of medial phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of corikers cells (KOs), greater ridge epithelial cells (GERs) (including lateral greater ridge epithelial cells (LGERs)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall.

[0037] In some aspects, a polynucleotide, construct, or expression construct of the present disclosure comprises a minimal GJB2 promoter comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:86.

[0038] In some embodiments, the expression construct comprises a GJB2 nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 117-126.

[0039] Certain aspects of the present disclosure relate to viral vector constructs comprising (i) a 5' inverted terminal repeat (ITR), (ii) a coding sequence for a connexin 26 polypeptide or a functional fragment thereof operably linked to a promoter capable of directing transcription of the coding sequence in inner ear supporting cells, and (iii) a 3' ITR, wherein the promoter is heterologous to the coding sequence. In some aspects, the viral construct promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 40, 90, 96, or 99.

[0040] In some embodiments, the viral construct promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:90.

[0041] In some embodiments, the viral construct promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:40.

[0042] In some embodiments, the viral construct promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:96.

[0043] In some embodiments, the viral construct promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:99.

[0044] In some embodiments, the viral vector construct further comprises a 5' untranslated region (UTR).

[0045] In some embodiments, the viral vector construct further comprises a 3' untranslated region (UTR).

[0046] In some embodiments, the viral vector construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a 5' untranslated region (UTR), (iii) a coding sequence for a connexin 26 polypeptide or a functional fragment thereof operably linked to a promoter that expresses the polynucleotide in inner ear supporting cells, (iv) a 3' UTR, and (v) a 3' ITR.

[0047] In some embodiments, the viral vector construct comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 117-126.

[0048] In some embodiments, the viral vector construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a coding sequence for a connexin 26 polypeptide or a functional fragment thereof operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter, and (iii) a 3' ITR, wherein the inner ear supporting cell-selective promoter is heterologous to the coding sequence.

[0049] In some embodiments, the viral vector construct comprises an inner ear supporting cell-selective promoter comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 40, 90, 96, or 99.

[0050] In some embodiments, the viral vector construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a 5' untranslated region (UTR), (iii) a coding sequence for a connexin 26 polypeptide or a functional fragment thereof operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter, (iv) a 3' UTR, and (v) a 3' ITR.

[0051] In some embodiments, the viral vector construct comprises a GJB2 nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 117-126.

[0052] In some embodiments, the viral vector construct comprises a minimal GJB2 promoter comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:86.

[0053] In some aspects, the promoter can express the coding sequence for a connexin 26 polypeptide or functional fragment thereof in inner ear supporting cells selected from one or more of medial phalangeal / border cells (IPhC), inner pillar cells (IPC), outer pillar cells (OPC), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSC), interdental cells (Idc), inner sulcus cells (ISC), organ of corikers cells (KO), greater ridge epithelial cells (GER) (including outer greater ridge cells (LGER)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall.

[0054] In some embodiments, the 5'UTR comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to any one of SEQ ID NOs: 20, 21, or 66.

[0055] In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to any one of SEQ ID NOs: 22, 67, 68, or 69.

[0056] In some embodiments, the polynucleotides, constructs, expression constructs, or viral vector constructs disclosed herein further comprise a polyA tail. In some embodiments, the polyA tail is bovine growth hormone, mouse β-globin, mouse α-globin, human collagen, polyoma virus, herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy chain gene, human growth hormone, or SV40 late and early poly(A). In some embodiments, the polyA tail is bovine growth hormone polyA.

[0057] In some embodiments, the viral vector constructs disclosed herein further comprise 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the 5' and 3' ITRs flank the promoter and coding sequence. In some embodiments, the 5' and 3' ITRs are AAV ITRs from a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV Anc80 ITRs. In some embodiments, the AAV ITRs are from serotype AAV2.

[0058] In some embodiments, the 5' AAV ITR comprises the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:52.

[0059] In some embodiments, the 3' AAV ITR comprises the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:53.

[0060] In some embodiments, the viral vector constructs disclosed herein comprise: a) the 5' ITR comprises a nucleic acid sequence according to SEQ ID NO: 8 and the 3' ITR comprises a nucleic acid sequence according to SEQ ID NO: 9; and / or b) the 5' ITR comprises a nucleic acid sequence according to SEQ ID NO: 52 and the 3' ITR comprises a nucleic acid sequence according to SEQ ID NO: 53.

[0061] In some embodiments, the viral vector comprises: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 8 or 52; (ii) a 5' UTR comprising any one of the nucleic acids of SEQ ID NO: 20, 21, or 66; (iii) a promoter comprising any one of the nucleic acid sequences of SEQ ID NO: 10-16, 28, 40, 57, 90-99; (iv) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 22, 67, 68, or 69; and (v) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 9 or 53.

[0062] In some embodiments, the viral vector comprises: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 8 or 52; (ii) a 5' UTR comprising the nucleic acid sequence of any one of SEQ ID NOs: 20, 21, or 66; (iii) an inner ear supporting cell-selective promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 10-16, 28, 40, 57, 90-99; a minimal GJB2 promoter comprising the sequence of SEQ ID NO: 86; (v) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 22, 67, 68, or 69; and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 9 or 53.

[0063] In some aspects, the construct, expression construct, or viral vector construct disclosed herein comprises a nucleic acid sequence according to any one of SEQ ID NOs: 7, 17, 38, 45-51, 54, 61, 82-84, 87-88, and 100-107.

[0064] In some aspects, the construct, expression construct, or viral vector construct is selectively expressed in inner ear supporting cells.

[0065] In some embodiments, the construct, expression construct, or viral vector construct comprises nucleotides 12 to 4557 of SEQ ID NO:7, nucleotides 12 to 4338 of SEQ ID NO:17, nucleotides 12 to 3976 of SEQ ID NO:38, nucleotides 12 to 4754 of SEQ ID NO:54, nucleotides 12 to 4429 of SEQ ID NO:61, nucleotides 12 to 4645 of SEQ ID NO:100, nucleotides 12 to 4708 of SEQ ID NO:101, nucleotides 12 to 4993 of SEQ ID NO:102, nucleotides 12 to 4496 of SEQ ID NO:103, nucleotides 12 to 4253 of SEQ ID NO:104, nucleotides 12 to 4320 of SEQ ID NO:105, nucleotides 12 to 4464 of SEQ ID NO:106, or nucleotides 12 to 4328 of SEQ ID NO:107.

[0066] Certain aspects of the present disclosure relate to viral vectors or AAV particles comprising the polynucleotides, constructs, expression constructs, or viral vector constructs disclosed herein. In some aspects, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, or lentivirus vectors. In some aspects, the viral vector is an AAV vector.

[0067] In some embodiments, the viral vector or AAV particle comprises an AAV capsid, which is or is derived from an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-rh8, AAV-rh10, AAV-rh39, AAV-rh43, or AAV Anc80 serotype capsid. In some embodiments, the AAV vector or AAV particle comprises an AAV capsid that is an AAV Anc80 capsid.

[0068] Certain aspects of the present disclosure relate to compositions comprising the polynucleotides, constructs, expression constructs, viral vector constructs, or AAV particles disclosed herein. In some aspects, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition is a synthetic perilymph solution.

[0069] Certain aspects of the present disclosure relate to ex vivo cells comprising a polynucleotide, construct, expression construct, viral vector construct, viral vector, or AAV particle disclosed herein.

[0070] In some embodiments, the ex vivo cells are inner ear cells. In some embodiments, the ex vivo cells are inner ear supporting cells. In some embodiments, the supporting cells are selected from one or more of medial phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of corikers cells (KOs), greater ridge epithelial cells (GERs) (including lateral greater ridge cells (LGERs)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall.

[0071] Certain aspects of the present disclosure relate to methods comprising transducing ex vivo cells with: a. a polynucleotide, construct, expression construct, viral vector construct, viral vector, or AAV particle disclosed herein; and b. one or more helper plasmids collectively comprising an AAV Rep gene, an AAV Cap gene, an AAV VA gene, an AAV E2a gene, and an AAV E4 gene.

[0072] Certain aspects of the present disclosure relate to methods for expressing a connexin 26 polypeptide or a functional fragment thereof in inner ear supporting cells, comprising administering a polynucleotide, construct, expression construct, viral vector construct, viral vector, AAV particle, or ex vivo cell disclosed herein.

[0073] Certain aspects of the present disclosure relate to methods of increasing expression of a connexin 26 polypeptide or a functional fragment thereof in inner ear supporting cells, comprising administering to a subject a polynucleotide, construct, expression construct, viral vector construct, viral vector, AAV particle, or ex vivo cell disclosed herein.

[0074] In some embodiments, expression of the connexin 26 polypeptide or functional fragment thereof in inner ear supporting cells is increased compared to endogenous expression of the polypeptide in inner ear supporting cells.

[0075] Certain aspects of the present disclosure relate to methods of treating hearing loss in a subject suffering from or at risk of hearing loss, comprising administering to the subject a polynucleotide, construct, expression construct, viral vector construct, viral vector, AAV particle, or ex vivo cell disclosed herein.

[0076] In some embodiments, (i) the connexin 26 polypeptide or functional fragment thereof is predominantly expressed in inner ear supporting cells, (ii) the connexin 26 polypeptide or functional fragment thereof is selectively expressed at higher levels in inner ear supporting cells than in inner ear hair cells, (iii) the connexin 26 polypeptide or functional fragment thereof is not expressed at a level sufficient to cause toxicity in inner ear hair cells, or (iv) any combination thereof.

[0077] In some embodiments, the inner ear supporting cells are selected from one or more of medial phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of corlicer cells (KOs), and OC90+ cells (OC90s).

[0078] In some embodiments, administration is to the inner ear of the subject.

[0079] In some embodiments, administration is to the cochlea of ​​the subject.

[0080] In some embodiments, administration is by round window membrane injection.

[0081] Certain aspects relate to the use of a polynucleotide, construct, expression construct, viral vector construct, viral vector, AAV particle, or ex vivo cell disclosed herein for the treatment of hearing loss in a subject suffering from or at risk of hearing loss.

[0082] Certain aspects relate to the use of a polynucleotide, construct, expression construct, viral vector construct, viral vector, AAV particle, or ex vivo cell disclosed herein in the manufacture of a medicament for treating hearing loss.

[0083] In some aspects, a polynucleotide, construct, expression construct, viral vector construct, viral vector, AAV particle, or ex vivo cell disclosed herein for use as a medicament.

[0084] In some aspects, the polynucleotide, construct, expression construct, viral vector construct, viral vector, AAV particle, or ex vivo cell disclosed herein for use in treating hearing loss.

[0085] In some embodiments, the construct, vector, AAV particle, composition, or ex vivo cells are pre-loaded into a device for administration. In some embodiments, the device is a microcatheter. In some embodiments, the microcatheter is shaped to enter the middle ear cavity via the ear canal and allow the end of the microcatheter to contact the RWM. In some embodiments, the distal end of the microcatheter is comprised of at least one microneedle having a diameter of 10 to 1,000 microns. In some embodiments, the kit further comprises a device. In some embodiments, the device is a device described in any one of Figures 5-8. In some embodiments, the device comprises a needle comprising a bent portion and a beveled tip.

[0086] Certain aspects relate to kits comprising the polynucleotides, constructs, expression constructs, viral vector constructs, viral vectors, AAV particles, or ex vivo cells disclosed herein. In some aspects, the kits further comprise a device disclosed herein.

[0087] Certain aspects of the present disclosure relate to constructs comprising a polynucleotide encoding a polypeptide operably linked to a promoter, wherein the promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, 90-99. In some aspects, the promoter is heterologous to the polynucleotide.

[0088] Certain aspects of the present disclosure relate to a construct comprising a polynucleotide encoding a polypeptide, an inner ear supporting cell-selective promoter and a minimal GJB2 promoter, wherein the polynucleotide is operably linked to the inner ear supporting cell-selective promoter and the minimal GJB2 promoter such that the polynucleotide is expressed in an inner ear supporting cell, and the inner ear supporting cell-selective promoter is heterologous to the polynucleotide.

[0089] Certain aspects of the present disclosure relate to constructs comprising a polynucleotide encoding a polypeptide, an inner ear supporting cell-selective promoter, and a minimal GJB2 promoter, wherein the polynucleotide is operably linked to the inner ear supporting cell-selective promoter and the minimal GJB2 promoter, and the inner ear supporting cell-selective promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, 90-99. In some aspects, the inner ear supporting cell-selective promoter is heterologous to the polynucleotide. In some aspects, the minimal GJB2 promoter comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 86.

[0090] Certain aspects of the present disclosure relate to constructs comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a polynucleotide encoding a polypeptide operably linked to a promoter that expresses the polynucleotide in an inner ear supporting cell; and (iii) a 3' ITR, wherein the promoter is heterologous to the polynucleotide.

[0091] Certain aspects of the present disclosure relate to constructs comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a polynucleotide encoding a polypeptide operably linked to a promoter; and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, or 90-99. In some aspects, the construct further comprises a minimal GJB2 promoter. In some aspects, the minimal GJB2 promoter comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 86.

[0092] In some aspects, the promoter is selected from one or more of the GJB6 promoter, GDF6 promoter, IGFBP2 promoter, RBP7 promoter, PARM1 promoter, GFAP promoter, BACE2 promoter, DBI2 promoter, FABP3 promoter, KLHL14 promoter, MMP15 promoter, SPARC promoter, TSPAN8 promoter, VIM promoter, derivatives thereof, or fragments thereof.

[0093] In some embodiments, the promoter is a GJB2 promoter or a minimal GJB2 promoter.

[0094] In some embodiments, the construct comprises two or more promoters. In some embodiments, a first promoter is selected from a GJB6 promoter, a GDF6 promoter, an IGFBP2 promoter, an RBP7 promoter, a PARM1 promoter, a GFAP promoter, a BACE2 promoter, a DBI2 promoter, a FABP3 promoter, a KLHL14 promoter, an MMP15 promoter, a SPARC promoter, a TSPAN8 promoter, a VIM promoter, or any combination thereof. In some embodiments, a second promoter is selected from a GJB2 promoter or a minimal GJB2 promoter.

[0095] Certain aspects of the present disclosure relate to constructs comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a polynucleotide encoding a polypeptide operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter, wherein the polynucleotide is expressed in an inner ear supporting cell; and (iii) a 3' ITR, wherein the inner ear supporting cell-selective promoter is heterologous to the polynucleotide.

[0096] Certain aspects of the present disclosure relate to constructs comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a polynucleotide encoding a polypeptide operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter; and (iii) a 3' ITR, wherein the inner ear supporting cell-selective promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, or 90-99.

[0097] In some embodiments, inner ear supporting cells include, but are not limited to, inner phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of corikers cells (KOs), greater ridge epithelial cells (GERs) (including lateral greater ridge epithelial cells (LGERs)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall.

[0098] In some embodiments, the promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, 90-99.

[0099] In some embodiments, the construct comprises a miRNA regulatory target site (miRTS) for a microRNA expressed in an inner ear cell. In some embodiments, the microRNA is expressed in an inner ear hair cell. In some embodiments, the microRNA is one or more of miR-194, miR-140, miR-18a, miR-99a, miR-30b, miR-15a, miR182, miR-183, or any combination thereof.

[0100] Certain aspects of the present disclosure relate to constructs comprising a polynucleotide encoding a polypeptide operably linked to a promoter, the construct comprising a miRNA regulatory target site (miRTS) for a microRNA expressed in an inner ear cell.

[0101] In some embodiments, the polynucleotide encodes a therapeutic polypeptide (eg, a connexin 26 polypeptide) or a reporter polypeptide.

[0102] In some embodiments, the microRNA is expressed in one or more of inner ear hair cells, spiral ganglion cells, outer supporting cells, basilar membrane cells, inner supporting cells, spiral border cells, or inner sulcus cells.

[0103] In some embodiments, the microRNA is expressed in inner ear hair cells.

[0104] In some embodiments, the microRNA is one or more of miR-194, miR-140, miR-18a, miR-99a, miR-30b, miR-15a, miR182, or miR-183.

[0105] In some embodiments, the construct comprises 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the construct comprises a 5' untranslated region (UTR). In some embodiments, the construct comprises a 3' untranslated region (UTR).

[0106] Certain aspects of the present disclosure relate to compositions comprising vectors, viral particles (e.g., AAV), ex vivo cells, and constructs disclosed herein.

[0107] Certain aspects of the present disclosure relate to adeno-associated virus (AAV) particles comprising the constructs disclosed herein.

[0108] Certain embodiments relate to adeno-associated virus (AAV) particles comprising a construct comprising (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide operably linked to a promoter that expresses the polynucleotide in an inner ear supporting cell, and (iii) a 3' ITR, wherein the promoter is heterologous to the polynucleotide. In some embodiments, the promoter comprises a nucleic acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 16, 28, 40, 57, or 90-99. In some embodiments, the construct further comprises a minimal GJB2 promoter. In some embodiments, the minimal GJB2 promoter comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 86.

[0109] Certain aspects relate to adeno-associated virus (AAV) particles comprising a construct comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a 5' untranslated region (UTR); (iii) a polynucleotide encoding a polypeptide operably linked to a promoter that expresses the polynucleotide in an inner ear supporting cell; (iv) a 3' UTR; and (v) a 3' ITR, wherein the promoter is heterologous to the polynucleotide.

[0110] Certain aspects of the present disclosure relate to adeno-associated virus (AAV) particles comprising a construct comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a 5' untranslated region (UTR); (iii) a polynucleotide encoding a polypeptide operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter, wherein the polynucleotide is expressed in an inner ear supporting cell; (iv) a 3' UTR; and (v) a 3' ITR, wherein the inner ear supporting cell-selective promoter is heterologous to the polynucleotide.

[0111] Certain aspects of the present disclosure relate to adeno-associated virus (AAV) particles comprising a construct comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a 5' untranslated region (UTR); (iii) a polynucleotide encoding a polypeptide operably linked to a promoter; (iv) a 3' UTR; and (v) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, or 90-99.

[0112] Certain adeno-associated virus (AAV) particles include a construct comprising: (i) a 5' inverted terminal repeat (ITR), (ii) a 5' untranslated region (UTR), (iii) a polynucleotide encoding a polypeptide operably linked to a promoter, (iv) a miRNA regulatory target site (miRTS) for a microRNA expressed in an inner ear cell, (v) a 3' UTR, and (vi) a 3' ITR.

[0113] Certain aspects of the present disclosure relate to adeno-associated virus (AAV) particles comprising a construct comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a 5' untranslated region (UTR); (iii) a polynucleotide encoding a polypeptide operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter; (iv) a 3' UTR; and (v) a 3' ITR, wherein the inner ear supporting cell-selective promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, or 90-99.

[0114] Certain adeno-associated virus (AAV) particles include a construct comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a 5' untranslated region (UTR); (iii) a polynucleotide encoding a polypeptide operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter; (iv) miRNA regulatory target sites (miRTS) for microRNAs expressed in inner ear cells; (v) a 3' UTR; and (vi) a 3' ITR.

[0115] In some embodiments, the inner ear supporting cell-selective promoter is selected from one or more of the following: GJB6 promoter, GDF6 promoter, IGFBP2 promoter, RBP7 promoter, PARM1 promoter, GFAP promoter, BACE2 promoter, DBI2 promoter, FABP3 promoter, KLHL14 promoter, MMP15 promoter, SPARC promoter, TSPAN8 promoter, VIM promoter, derivatives thereof, or fragments thereof.

[0116] In some embodiments, the minimal GJB2 promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:86.

[0117] Certain aspects of the present disclosure relate to methods of using the constructs, vectors, viral particles (e.g., AAV), ex vivo cells, and compositions disclosed herein to express polypeptides in inner ear cells (e.g., supporting cells).

[0118] Certain aspects of the present disclosure relate to methods of using the constructs, vectors, viral particles (e.g., AAV), ex vivo cells, and compositions disclosed herein to increase expression of a polypeptide (e.g., a therapeutic polypeptide, a connexin 26 polypeptide) in inner ear cells (e.g., supporting cells). In some aspects, the increased expression is compared to the corresponding endogenous polypeptide expression in inner ear cells (e.g., supporting cells).

[0119] Certain aspects of the present disclosure relate to methods of using the constructs, vectors, viral particles (e.g., AAV), ex vivo cells, and compositions disclosed herein to reduce expression of a polypeptide (e.g., a therapeutic polypeptide) in non-inner ear supporting cells (e.g., inner ear hair cells). In some aspects, the reduction in expression is compared to the corresponding endogenous polypeptide expression in non-inner ear supporting cells (e.g., inner ear hair cells).

[0120] Certain aspects of the present disclosure relate to methods of using the constructs, vectors, viral particles (e.g., AAV), ex vivo cells, and compositions disclosed herein to reduce toxicity associated with expression of a polypeptide (e.g., a therapeutic polypeptide) in inner ear cells.

[0121] Certain aspects of the present disclosure relate to methods of using the constructs, vectors, viral particles (e.g., AAV), ex vivo cells, and compositions disclosed herein to treat hearing loss in a subject suffering from or at risk of hearing loss. [Brief explanation of the drawings]

[0122] [Figure 1A] Panel (1A) shows a simplified endogenous AAV genome, and panel (1B) shows a simplified recombinant AAV (rAAV) construct capable of expressing a therapeutic polypeptide (e.g., the GJB2 gene). [Figure 1B] Panel (1A) shows a simplified endogenous AAV genome, and panel (1B) shows a simplified recombinant AAV (rAAV) construct capable of expressing a therapeutic polypeptide (e.g., the GJB2 gene).

[0123] [Figure 2A]Alternative exemplary rAAV constructs containing a therapeutic polypeptide are shown. Figure 2A shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a bGH polyA, and a 3'ITR. Figure 2B shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2C shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2D shows an exemplary rAAV construct containing a 5'ITR, an smCBA promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2E shows an exemplary rAAV construct including a 5'ITR, a promoter including a CMV promoter and an hGJB2 promoter, a 5'UTR, a nucleic acid encoding the hGJB2 gene, a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2F shows an exemplary rAAV construct including a 5'ITR, a CAG promoter, a 5'UTR, an hGJB2 promoter, a FLAG tag, a microRNA regulatory target site, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2G shows an exemplary rAAV construct including a 5'ITR, a promoter including an inner ear supporting cell-selective promoter and an hGJB2 minimal promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 5'UTR, a bGH polyA, and a 3'ITR. Figure 2H shows an exemplary rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a T2A element, a nucleic acid encoding eGFP, bGH polyA, and a 3' ITR. [Figure 2B]Alternative exemplary rAAV constructs containing a therapeutic polypeptide are shown. Figure 2A shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a bGH polyA, and a 3'ITR. Figure 2B shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2C shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2D shows an exemplary rAAV construct containing a 5'ITR, an smCBA promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2E shows an exemplary rAAV construct including a 5'ITR, a promoter including a CMV promoter and an hGJB2 promoter, a 5'UTR, a nucleic acid encoding the hGJB2 gene, a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2F shows an exemplary rAAV construct including a 5'ITR, a CAG promoter, a 5'UTR, an hGJB2 promoter, a FLAG tag, a microRNA regulatory target site, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2G shows an exemplary rAAV construct including a 5'ITR, a promoter including an inner ear supporting cell-selective promoter and an hGJB2 minimal promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 5'UTR, a bGH polyA, and a 3'ITR. Figure 2H shows an exemplary rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a T2A element, a nucleic acid encoding eGFP, bGH polyA, and a 3' ITR. [Figure 2C]Alternative exemplary rAAV constructs containing a therapeutic polypeptide are shown. Figure 2A shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a bGH polyA, and a 3'ITR. Figure 2B shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2C shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2D shows an exemplary rAAV construct containing a 5'ITR, an smCBA promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2E shows an exemplary rAAV construct including a 5'ITR, a promoter including a CMV promoter and an hGJB2 promoter, a 5'UTR, a nucleic acid encoding the hGJB2 gene, a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2F shows an exemplary rAAV construct including a 5'ITR, a CAG promoter, a 5'UTR, an hGJB2 promoter, a FLAG tag, a microRNA regulatory target site, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2G shows an exemplary rAAV construct including a 5'ITR, a promoter including an inner ear supporting cell-selective promoter and an hGJB2 minimal promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 5'UTR, a bGH polyA, and a 3'ITR. Figure 2H shows an exemplary rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a T2A element, a nucleic acid encoding eGFP, bGH polyA, and a 3' ITR. [Figure 2D]Alternative exemplary rAAV constructs containing a therapeutic polypeptide are shown. Figure 2A shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a bGH polyA, and a 3'ITR. Figure 2B shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2C shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2D shows an exemplary rAAV construct containing a 5'ITR, an smCBA promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2E shows an exemplary rAAV construct including a 5'ITR, a promoter including a CMV promoter and an hGJB2 promoter, a 5'UTR, a nucleic acid encoding the hGJB2 gene, a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2F shows an exemplary rAAV construct including a 5'ITR, a CAG promoter, a 5'UTR, an hGJB2 promoter, a FLAG tag, a microRNA regulatory target site, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2G shows an exemplary rAAV construct including a 5'ITR, a promoter including an inner ear supporting cell-selective promoter and an hGJB2 minimal promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 5'UTR, a bGH polyA, and a 3'ITR. Figure 2H shows an exemplary rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a T2A element, a nucleic acid encoding eGFP, bGH polyA, and a 3' ITR. [Figure 2E]Alternative exemplary rAAV constructs containing a therapeutic polypeptide are shown. Figure 2A shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a bGH polyA, and a 3'ITR. Figure 2B shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2C shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2D shows an exemplary rAAV construct containing a 5'ITR, an smCBA promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2E shows an exemplary rAAV construct including a 5'ITR, a promoter including a CMV promoter and an hGJB2 promoter, a 5'UTR, a nucleic acid encoding the hGJB2 gene, a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2F shows an exemplary rAAV construct including a 5'ITR, a CAG promoter, a 5'UTR, an hGJB2 promoter, a FLAG tag, a microRNA regulatory target site, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2G shows an exemplary rAAV construct including a 5'ITR, a promoter including an inner ear supporting cell-selective promoter and an hGJB2 minimal promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 5'UTR, a bGH polyA, and a 3'ITR. Figure 2H shows an exemplary rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a T2A element, a nucleic acid encoding eGFP, bGH polyA, and a 3' ITR. [Figure 2F]Alternative exemplary rAAV constructs containing a therapeutic polypeptide are shown. Figure 2A shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a bGH polyA, and a 3'ITR. Figure 2B shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2C shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2D shows an exemplary rAAV construct containing a 5'ITR, an smCBA promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2E shows an exemplary rAAV construct including a 5'ITR, a promoter including a CMV promoter and an hGJB2 promoter, a 5'UTR, a nucleic acid encoding the hGJB2 gene, a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2F shows an exemplary rAAV construct including a 5'ITR, a CAG promoter, a 5'UTR, an hGJB2 promoter, a FLAG tag, a microRNA regulatory target site, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2G shows an exemplary rAAV construct including a 5'ITR, a promoter including an inner ear supporting cell-selective promoter and an hGJB2 minimal promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 5'UTR, a bGH polyA, and a 3'ITR. Figure 2H shows an exemplary rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a T2A element, a nucleic acid encoding eGFP, bGH polyA, and a 3' ITR. [Figure 2G]Alternative exemplary rAAV constructs containing a therapeutic polypeptide are shown. Figure 2A shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a bGH polyA, and a 3'ITR. Figure 2B shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2C shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2D shows an exemplary rAAV construct containing a 5'ITR, an smCBA promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2E shows an exemplary rAAV construct including a 5'ITR, a promoter including a CMV promoter and an hGJB2 promoter, a 5'UTR, a nucleic acid encoding the hGJB2 gene, a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2F shows an exemplary rAAV construct including a 5'ITR, a CAG promoter, a 5'UTR, an hGJB2 promoter, a FLAG tag, a microRNA regulatory target site, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2G shows an exemplary rAAV construct including a 5'ITR, a promoter including an inner ear supporting cell-selective promoter and an hGJB2 minimal promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 5'UTR, a bGH polyA, and a 3'ITR. Figure 2H shows an exemplary rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a T2A element, a nucleic acid encoding eGFP, bGH polyA, and a 3' ITR. [Figure 2H]Alternative exemplary rAAV constructs containing a therapeutic polypeptide are shown. Figure 2A shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a bGH polyA, and a 3'ITR. Figure 2B shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2C shows an exemplary rAAV construct containing a 5'ITR, a CAG promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2D shows an exemplary rAAV construct containing a 5'ITR, an smCBA promoter, a 5'UTR, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2E shows an exemplary rAAV construct including a 5'ITR, a promoter including a CMV promoter and an hGJB2 promoter, a 5'UTR, a nucleic acid encoding the hGJB2 gene, a FLAG tag, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2F shows an exemplary rAAV construct including a 5'ITR, a CAG promoter, a 5'UTR, an hGJB2 promoter, a FLAG tag, a microRNA regulatory target site, a 3'UTR, a bGH polyA, and a 3'ITR. Figure 2G shows an exemplary rAAV construct including a 5'ITR, a promoter including an inner ear supporting cell-selective promoter and an hGJB2 minimal promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a 5'UTR, a bGH polyA, and a 3'ITR. Figure 2H shows an exemplary rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a therapeutic polypeptide (hGJB2 gene), a FLAG tag, a T2A element, a nucleic acid encoding eGFP, bGH polyA, and a 3' ITR.

[0124] [Figure 3A]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3B]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3C]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3D]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3E]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3F]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3G]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3H]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3I]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3J]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3K]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3L]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3M]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3N]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3O]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3P]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR. [Figure 3Q]Figure 3 shows in vitro or ex vivo expression of a transgene in HEK293FT cells transfected or transduced with a construct containing a microRNA targeting site (miRTS) in the presence or absence of a microRNA that recognizes the site. Figure 3A is a schematic diagram depicting constructs containing a gene of interest and miRTS. Figure 3B is a Venn diagram depicting the selection of miRTS based on the expression of microRNAs expressed in different inner ear cell types. Figure 3C is a graph showing GFP expression in cells transfected with a miRNA expression plasmid (pITR.CAG.mScarlet.miRNA) and a plasmid containing a gene of interest and a microRNA target site (pITR.CAG.GOI.miRTS). Figure 3D is a graph showing GFP expression measured by flow cytometry in HEK293FT cells transduced with an AAVAnc80 vector containing GFP and a microRNA target site (AAVAnc0-CAG.GOI.miRTS) and transfected with a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3E is a graph showing gene of interest expression measured by RT-qPCR in cells transduced with AAVAnc80 expressing a gene of interest with a microRNA target site (AAVAnc80-CAG.GOI.miRTS) after transfection with either of two doses of a plasmid expressing a plasmid encoding a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3F is a protein Western blot showing expression of a gene of interest in cells transduced with AAVAnc80 (AAVAnc80-CAG.GOI.miRTS), which contains the gene of interest and a microRNA targeting site, after transfection with either of two amounts of a plasmid expressing a miRNA targeting miRTS (pITR.CAG.mScarlet.miRNA). Figure 3G is a graph showing quantification of protein levels determined from the Western blot in Figure 3F.Figure 3H is a heat map of gene expression following in vitro transduction of a gene of interest with a microRNA targeting site compared to transduction with the gene of interest alone. Figure 3I is a volcano plot showing differential gene expression between samples. Figure 3J shows the expression of a gene of interest in untreated cochlear explants (left panel) and after transduction with an AAV encoding a FLAG-tagged gene of interest without a microRNA targeting site (right panel). Immunostaining for the FLAG tag is shown in green. Immunostaining for MYO7A was used to label hair cells in red. White arrowheads indicate hair cells expressing connexin26-FLAG. Figure 3K shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3L shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS1, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figure 3M shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS2, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3N shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS3, which contains a FLAG-tagged gene of interest and a microRNA targeting site recognized by a microRNA expressed in hair cells. Figure 3O shows a cochlear explant transduced with AAVAnc80-CAG-GOI.miRTS4, which contains a FLAG-tagged gene of interest and a microRNA targeting site for a microRNA expressed in hair cells. Figures 3P and 3Q show in vitro expression of GJB2 protein in HEK293FT cells transfected with the CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.CAG.5UTR.hGJB2.FLAG.miRTS.3UTR contains miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR, allowing knockdown of exogenous hGJB2 in the presence of regulatory miR-182 and / or miR-183. To confirm miRNA regulation of the construct, HEK293FT cells were transfected with a hGJB2-containing plasmid and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR-183. Seventy-two hours after transfection, cells were harvested for protein and RNA analysis. Figure 3P shows exemplary GJB2 protein levels analyzed using Western blot. Figure 3Q shows exemplary GJB2 mRNA levels analyzed using qPCR.

[0125] [Figure 4A] Figure 4 shows FLAG protein expression in mouse cochlear explants transduced at P2 with exemplary rAAVAnc80 particles containing constructs driven by the CAG, CMVe-GJB2p, or smCBA promoter / enhancer sequences, as indicated. Explants were fixed 72 hours later. Immunostaining for FLAG is shown in green, immunostaining for the hair cell marker Myo7a is shown in red, and the nuclear marker DAPI is shown in blue. Panel (4A) shows an exemplary explant transduced with AAVAnc80-CAG.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 82) at 5.8E9 vg / explant. Panel (4B) shows an exemplary explant transduced with AAVAnc80-smCBA.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 83) at 1.4E10 vg / explant. Panel (4C) shows exemplary explants transduced with AAVAnc80-CMVeGFAPp.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 84) at 1.8E10 vg / explant. [Figure 4B]Figure 4 shows FLAG protein expression in mouse cochlear explants transduced at P2 with exemplary rAAVAnc80 particles containing constructs driven by the CAG, CMVe-GJB2p, or smCBA promoter / enhancer sequences, as indicated. Explants were fixed 72 hours later. Immunostaining for FLAG is shown in green, immunostaining for the hair cell marker Myo7a is shown in red, and the nuclear marker DAPI is shown in blue. Panel (4A) shows an exemplary explant transduced with AAVAnc80-CAG.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 82) at 5.8E9 vg / explant. Panel (4B) shows an exemplary explant transduced with AAVAnc80-smCBA.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 83) at 1.4E10 vg / explant. Panel (4C) shows exemplary explants transduced with AAVAnc80-CMVeGFAPp.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 84) at 1.8E10 vg / explant. [Figure 4C] Figure 4 shows FLAG protein expression in mouse cochlear explants transduced at P2 with exemplary rAAVAnc80 particles containing constructs driven by the CAG, CMVe-GJB2p, or smCBA promoter / enhancer sequences, as indicated. Explants were fixed 72 hours later. Immunostaining for FLAG is shown in green, immunostaining for the hair cell marker Myo7a is shown in red, and the nuclear marker DAPI is shown in blue. Panel (4A) shows an exemplary explant transduced with AAVAnc80-CAG.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 82) at 5.8E9 vg / explant. Panel (4B) shows an exemplary explant transduced with AAVAnc80-smCBA.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 83) at 1.4E10 vg / explant. Panel (4C) shows exemplary explants transduced with AAVAnc80-CMVeGFAPp.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 84) at 1.8E10 vg / explant.

[0126] [Figure 5]1 illustrates a perspective view of a device for delivering fluid to the inner ear, according to aspects of the present disclosure.

[0127] [Figure 6] FIG. 1 illustrates a side view of a curved needle subassembly according to an aspect of the present disclosure.

[0128] [Figure 7] 1 illustrates a perspective view of a device for delivering fluid to the inner ear, according to aspects of the present disclosure.

[0129] [Figure 8] FIG. 10 shows a perspective view of a curved needle subassembly coupled to the distal end of a device, according to an aspect of the present disclosure.

[0130] [Figure 9A]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9B]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9C]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9D]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9E]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9F]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9G]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9H]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9I]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9J]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9K]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9L]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9M]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9N]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red. [Figure 9O]Figure 9A shows the in vivo expression of connexin 26 in wild-type mice. Wild-type mice (p20) were cochlearly administered rAAVAnc80 particles containing CAG.hGJB2.FLAG.GFP (a schematic diagram is provided in Figure 2H) (Figure 9A). Connexin 26 expression in supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, GFP in green, FLAG in purple, and endogenous connexin 26 in red. SC - supporting cells; IHC - inner hair cells; OHC - outer hair cells. Young mice were transfected with AAVAnc80-CMVeGFAPp.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9B), AAVAnc80-GDF6p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9C and 9I) (a schematic diagram is provided in Figure 2G), AAVAnc80-IGFBP2p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figure 9D) (a schematic diagram is provided in Figure 2G), AAV rAAVAnc80 particles containing Anc80-PARM1p.mGJB2p.5UTR.hGJB2.FLAG.3UTR (Figures 9E and 9J) (a schematic diagram is provided in Figure 2G), AAVAnc80-GFAPp.mGJB2p.hGJB2 (Figure 9F), AAVAnc80-MMP15p.mGJB2p.hGJB2 (Figures 9G and 9L), and AAVAnc80-VIMp.mGJB2p-hGJB2 (Figures 9H and 9K) were administered into the cochlea. (VIM is also referred to as VIM1 in Figure 9K.) Connexin 26 expression was detected 2 weeks after administration. Immunostaining of actin filaments and hair cell stereocilia bundles with phalloidin is shown in blue, FLAG is shown in green, and endogenous Connexin 26 or Myo7a is shown in red. Figure 9M shows in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GFAP.mGJB2p.hGJB2.FLAG. Endogenous connexin 26 is shown in white, flag-tagged connexin 26 is shown in green, and hair cells are shown in blue with phalloidin staining.Figures 9N-9O show the in vivo expression of connexin 26 in wild-type mice administered AAVAnc80 particles containing AAVAnc80.CMVe.GDF6.mGJB2p.hGJB2.FLAG or AAVAnc80.CMVe.PARM1.mGJB2p.hGJB2.FLAG. Flag-tagged connexin 26 is shown in green, phalloidin staining is shown in blue, and Myo7a, which marks hair cells, is shown in red.

[0131] [Figure 10A] Figure 10 shows in vitro expression of GJB2 mRNA and detection of connexin 26 protein from constructs containing support cell-selective promoters. Figure 10A shows connexin 26-FLAG protein levels ("GJB2-FLAG") in HEK293FT cells transduced with exemplary rAAVAnc80 particles containing constructs driven by the GJB6, IGFBP2, RPB7, PARM1, or GDF6 promoter in combination with the minimal GJB2 promoter. GAPDH is shown as a loading control. Figure 10B shows GJB2 mRNA levels in HEK293FT cells transduced with rAAVAnc80 particles containing constructs driven by GFAP and the minimal GJB2 promoter, the CMV enhancer / GFAP, the GJB2 enhancer / GJB2, the CMV enhancer / GJB2, or the CAG promoter. Figure 10C shows connexin26-FLAG protein levels (GJB2-FLAG) in HEK293FT cells transfected with plasmids containing constructs driven by the FABP3, KLHL14, DBI2, TSPAN8, MMP15, SPARC, or VIM promoters in combination with the minimal GJB2 promoter. FLAG was used to distinguish protein levels between endogenous and transduced connexin26 expression. GAPDH is shown as a loading control. [Figure 10B]Figure 10 shows in vitro expression of GJB2 mRNA and detection of connexin 26 protein from constructs containing support cell-selective promoters. Figure 10A shows connexin 26-FLAG protein levels ("GJB2-FLAG") in HEK293FT cells transduced with exemplary rAAVAnc80 particles containing constructs driven by the GJB6, IGFBP2, RPB7, PARM1, or GDF6 promoter in combination with the minimal GJB2 promoter. GAPDH is shown as a loading control. Figure 10B shows GJB2 mRNA levels in HEK293FT cells transduced with rAAVAnc80 particles containing constructs driven by GFAP and the minimal GJB2 promoter, the CMV enhancer / GFAP, the GJB2 enhancer / GJB2, the CMV enhancer / GJB2, or the CAG promoter. Figure 10C shows connexin26-FLAG protein levels (GJB2-FLAG) in HEK293FT cells transfected with plasmids containing constructs driven by the FABP3, KLHL14, DBI2, TSPAN8, MMP15, SPARC, or VIM promoters in combination with the minimal GJB2 promoter. FLAG was used to distinguish protein levels between endogenous and transduced connexin26 expression. GAPDH is shown as a loading control. [Figure 10C]Figure 10 shows in vitro expression of GJB2 mRNA and detection of connexin 26 protein from constructs containing support cell-selective promoters. Figure 10A shows connexin 26-FLAG protein levels ("GJB2-FLAG") in HEK293FT cells transduced with exemplary rAAVAnc80 particles containing constructs driven by the GJB6, IGFBP2, RPB7, PARM1, or GDF6 promoter in combination with the minimal GJB2 promoter. GAPDH is shown as a loading control. Figure 10B shows GJB2 mRNA levels in HEK293FT cells transduced with rAAVAnc80 particles containing constructs driven by GFAP and the minimal GJB2 promoter, the CMV enhancer / GFAP, the GJB2 enhancer / GJB2, the CMV enhancer / GJB2, or the CAG promoter. Figure 10C shows connexin26-FLAG protein levels (GJB2-FLAG) in HEK293FT cells transfected with plasmids containing constructs driven by the FABP3, KLHL14, DBI2, TSPAN8, MMP15, SPARC, or VIM promoters in combination with the minimal GJB2 promoter. FLAG was used to distinguish protein levels between endogenous and transduced connexin26 expression. GAPDH is shown as a loading control.

[0132] [Figure 11] Figure 1 shows GJB2 mRNA levels in mouse cochlear explants transduced with rAAVAnc80 particles containing constructs driven by the CAG promoter, the CMV enhancer / GFAP promoter, or the GFAP and minimal GJB2 promoter. GJB2 mRNA levels were determined by qPCR.

[0133] definition The scope of the present disclosure is defined by the claims appended hereto and is not limited by the specific embodiments described herein. Those skilled in the art will recognize, upon reading this specification, various modifications that may be equivalent to such described embodiments or that may otherwise be within the scope of the claims. Generally, terms used herein adhere to the meanings understood in the art unless expressly indicated otherwise. Clear definitions of certain terms are provided below. The meaning of these and other terms in specific instances throughout this specification will be apparent to those skilled in the art from the context.

[0134] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, of itself, imply any priority, precedence, or ordering of one claim element relative to another, or the chronological order in which acts of a method are performed, but is merely used as a label to distinguish one claim element having a certain name from another element having the same name (other than the use of ordinal terms).

[0135] The articles "a" and "an," as used herein, should be understood to include plural referents unless clearly indicated to the contrary. A claim or description including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. In some embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In some embodiments, more than one or all group members are present in, employed in, or relevant to a given product or process. It should be understood that the present disclosure encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims introduced into another claim dependent on the same base claim (or any other claim, as related), unless otherwise indicated or unless a contradiction or inconsistency would arise apparent to one skilled in the art. When elements are presented as lists (e.g., in a Markush group or similar format), it should be understood that each subgroup of elements is also disclosed, and that any element can be removed from the group. Generally, when an aspect or aspects are referred to as "comprising" certain elements, features, etc., it should be understood that the particular aspect or aspects "consist" or "consist essentially of" such elements, features, etc. For simplicity, those aspects have not in all instances been specifically described in this specification in so many words. It should also be understood that any embodiment or aspect can be explicitly excluded from the claims, regardless of whether a specific exclusion is set forth herein.

[0136] Throughout this specification, whenever a polynucleotide or polypeptide is represented by a sequence of letters (e.g., in the case of polynucleotides, A, C, G, and T, which represent adenosine, cytidine, guanosine, and thymidine, respectively), such polynucleotide or polypeptide is presented from left to right in 5' to 3' or N-terminal to C-terminal order.

[0137] Administration: As used herein, the term "administration" typically refers to the administration of a construct or composition to a subject or system to achieve delivery of the agent to the subject or system. In some embodiments, the agent is a composition or is contained in a composition. In some embodiments, the agent is produced through metabolism of the composition or one or more of its components. Those of skill in the art will recognize various routes that may be utilized for administration to a subject (e.g., a human) in appropriate circumstances. For example, in some embodiments, administration may be systemic or local. In some embodiments, systemic administration may be intravenous. In some embodiments, administration may be local. Local administration may include delivery to the cochlear perilymph, for example, by injection through the round window membrane or into the scala tympani, through the endolymph, perilymph, and / or endolymph after ductoplasty. In some embodiments, administration may include only a single dose. In some embodiments, administration may include the application of a fixed number of doses. In some embodiments, administration may include administration that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) administration. In some embodiments, administration may include continuous administration (e.g., perfusion) for at least a selected period of time.

[0138] Allele: As used herein, the term "allele" refers to one of two or more existing genetic variants at a particular polymorphic genomic locus.

[0139] Amelioration: As used herein, the term "amelioration" refers to the prevention, reduction, or alleviation of a condition, or an improvement in a subject's condition. Amelioration may include, but does not require, complete reversal or complete prevention of a disease, disorder, or symptom.

[0140] Amino acid: In its broadest sense, as used herein, the term "amino acid" refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure, e.g., HN-C(H)(R)-COOH. In some embodiments, an amino acid is a natural amino acid. In some embodiments, an amino acid is a non-natural amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal and / or amino-terminal amino acids in a polypeptide, can include structural modifications compared to the general structures shown above. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to a polypeptide containing an otherwise identical, unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to a polypeptide containing an otherwise identical, unmodified amino acid.

[0141] Approximately or About: As used herein, the term "approximately" or "about" may apply to one or more values ​​of interest, including values ​​similar to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within ±10% (greater or less) of a stated reference value, unless otherwise stated or apparent from the context (except where such number exceeds 100% of possible values). For example, in some embodiments, the term "approximately" or "about" may encompass a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0142] Associated: As used herein, the term "associated" describes two events or entities as "associated" with one another when the presence, level, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another when they interact directly or indirectly such that they are in and / or remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently bound by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0143] Biological activity: As used herein, the term "biological activity" refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or extent of a biological activity is assessed by detection of a direct or indirect product produced by the biological pathway or event of interest.

[0144] Cell-selective promoter: As used herein, the term "cell-selective promoter" refers to a promoter that is primarily active in a particular cell type (e.g., transcription of a particular gene occurs only in cells that express a transcriptional regulatory protein and / or control protein that binds to the tissue-specific promoter). In some embodiments, an inner ear supporting cell-selective promoter is a promoter that is primarily active in one or more supporting cells of the inner ear.

[0145] Characteristic portion: As used herein, the term "characteristic portion" refers in its broadest sense to a portion of a substance whose presence (or absence) correlates with the presence (or absence) of a particular characteristic, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion found in a given substance and related substances that share a particular characteristic, attribute, or activity, but not in substances that do not share the particular characteristic, attribute, or activity. In some embodiments, a characteristic portion shares at least one functional property with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is one that contains a contiguous stretch of amino acids, or a collection of contiguous stretches of amino acids, that together are characteristic of the protein or polypeptide. In some embodiments, such contiguous stretches each contain approximately at least 2, 5, 10, 15, 20, 50, or more amino acids. Generally, a characteristic portion of a substance (e.g., a protein, antibody, etc.) is one that shares at least one functional characteristic with the related intact substance, in addition to the sequence and / or structural identity identified above. In some embodiments, the characteristic moiety may be biologically active.

[0146] Characteristic sequence: As used herein, the term "characteristic sequence" is a sequence that is found in all members of a family of polypeptides or nucleic acids and therefore can be used by those skilled in the art to define the members of the family.

[0147] Characteristic sequence element: As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., sequentially linked monomers). In some embodiments, a characteristic sequence element comprises at least first and second stretches of consecutive monomers separated by one or more spacer regions whose length may or may not vary across the polymer sharing the sequence element.

[0148] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is subjected to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more agents may be administered simultaneously. In some embodiments, two or more agents may be administered sequentially. In some embodiments, two or more agents may be administered in an overlapping dosing regimen.

[0149] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, circumstances, sets of symptoms, subjects, populations, etc. that may not be identical to one another, but are sufficiently similar to permit comparisons between them, so that one of skill in the art would understand that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, equivalent agent sets, entities, circumstances, sets of symptoms, subjects, populations, etc. are characterized by multiple substantially identical characteristics and one or a few diverse characteristics. One of skill in the art would understand what degree of identity is required in any given situation for two or more such agents, entities, circumstances, sets of symptoms, subjects, populations, etc. to be considered equivalent in context. For example, one of skill in the art would understand that agent sets, entities, circumstances, sets of symptoms, subjects, populations, etc. are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a valid conclusion that differences in results obtained or phenomena observed under or with different sets of environments, stimuli, agents, entities, circumstances, sets of symptoms, subjects, populations, etc. are caused by or indicate variation in those characteristics that vary.

[0150] Construct: As used herein, the term "construct" refers to a composition comprising a polynucleotide that can carry at least one heterologous polynucleotide. In some embodiments, the construct can be a plasmid, transposon, cosmid, artificial chromosome (e.g., human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), or a viral vector, capsid, viral particle, and any Gateway® plasmid. The construct can, for example, contain sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host primate cell or in an in vitro expression system. The construct can contain any genetic element (e.g., a plasmid, transposon, cosmid, artificial chromosome, or viral vector, capsid, viral particle, etc.) that can replicate when associated with the appropriate control elements. Thus, in some embodiments, a "construct" may include a cloning and / or expression construct and / or a viral construct (e.g., an adeno-associated viral (AAV) construct, an adenoviral construct, a lentiviral construct, or a retroviral construct).

[0151] Conservative: As used herein, the term "conservative" refers to cases describing conservative amino acid substitutions, which involve replacing an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional property of interest of a protein (e.g., the ability of a receptor to bind a ligand). Examples of groups of amino acids having side chains with similar chemical properties include aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I); aliphatic-hydroxyl side chains such as serine (Ser, S) and threonine (Thr, T); amide-containing side chains such as asparagine (Asn, N) and glutamine (Gln, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains such as cysteine ​​(Cys, C) and methionine (Met, M). Conservative amino acid substitution groups include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamic acid / aspartic acid (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, conservative amino acid substitutions can be alanine substitutions of any naturally occurring residue in a protein, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., 1992, Science 256:1443-1445 (incorporated herein by reference in its entirety). In some aspects, the substitution is a moderately conservative substitution that has a non-negative value in the PAM250 log-likelihood matrix.Those skilled in the art will understand that changes (e.g., substitutions, additions, deletions, etc.) of amino acids that are not conserved between the same proteins from different species are unlikely to affect the function of the protein, and therefore these amino acids should be selected for mutation. Amino acids that are conserved between the same proteins from different species should not be altered (e.g., deleted, added, substituted, etc.), as these mutations are more likely to result in changes in the function of the protein. Exemplary conservative amino acid substitutions are shown in Table 1.

[0152] [Table 1]

[0153] Control: As used herein, the term "control" refers to the art-understood meaning of "control," which is a standard against which results are compared. Typically, controls are used to increase integrity in experiments by isolating a variable in order to draw conclusions about such a variable. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a point of comparison. For example, in one experiment, the "test" (i.e., the variable being tested) is administered. In a second experiment, the "control" (the variable being tested) is not administered. In some embodiments, a control is a historical control (e.g., of a previously performed test or assay, or of a previously known amount or result). In some embodiments, a control is or includes a printed or otherwise kept record. In some embodiments, a control is a positive control. In some embodiments, a control is a negative control.

[0154] Determining, Measuring, Evaluating, Assessing, Assaying, and Analyzing: As used herein, the terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" may be used interchangeably to refer to any form of measurement, including determining whether an element is present or not. These terms include both quantitative and / or qualitative determinations. Assaying can be relative or absolute. For example, in some embodiments, "assaying for the presence" can be determining the amount of something present and / or determining whether it is present or not.

[0155] Endogenous: As used herein with respect to a substance or process, refers to a naturally occurring substance or process that originates within a system such as an organism, tissue, or cell.

[0156] Engineered: Generally, as used herein, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a cell or organism is considered "engineered" if it has been manipulated so that its genetic information has been altered (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutations or by mating protocols). As is common practice and understood by those of skill in the art, the progeny of an engineered polynucleotide or cell will typically still be referred to as "engineered," even if the actual manipulation was performed on the earlier entity.

[0157] Excipient: As used herein, the term "excipient" refers to an inert (e.g., non-therapeutic) agent that may be included in a pharmaceutical composition to, for example, provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.

[0158] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product (e.g., a transcript, e.g., mRNA, e.g., a polypeptide, etc.) from the nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (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 formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0159] Adjacent: As used herein, the term "adjacent" refers to a position relative to the ends of a reference nucleic acid sequence. More specifically, when referring to a reference nucleic acid sequence, "adjacent" refers to having sequences upstream and downstream of the reference nucleic acid sequence. In some embodiments, an adjacent reference nucleic acid sequence has a first sequence or series of nucleotide residues located adjacent to the 5' end of the reference nucleic acid and a second sequence or series of nucleotide residues located adjacent to the 3' end of the reference nucleic acid. In some embodiments, the upstream and / or downstream flanking sequences are immediately adjacent to the reference nucleic acid sequence. In some embodiments, there is an intervening nucleic acid between the upstream and / or downstream flanking sequences and the reference nucleic acid sequence.

[0160] Functional: As used herein, the term "functional" describes something that exists in a form in which it exhibits a property and / or activity by which it is characterized. For example, in some embodiments, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized. In some such embodiments, a functional biomolecule is characterized relative to another biomolecule that is non-functional in that the "non-functional" version does not exhibit the same or equivalent property and / or activity as the "functional" molecule. A biological molecule may have one function, two functions (i.e., bifunctional), or many functions (i.e., multifunctional).

[0161] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a gene product (e.g., an RNA product, e.g., a polypeptide product). In some embodiments, a gene comprises coding sequence (i.e., a sequence that encodes a specific product). In some embodiments, a gene comprises non-coding sequence. In some particular embodiments, a gene may comprise both coding sequence (e.g., exon sequence) and non-coding sequence (e.g., intron sequence). In some embodiments, a gene may comprise one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that can, for example, control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.). As used herein, the term "gene" generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof. The term may optionally encompass regulatory sequences, as will be clear to one of skill in the art from the context. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but rather to clarify that in most cases the term as used herein refers to a polypeptide-encoding nucleic acid. In some embodiments, a gene may encode a polypeptide, but the polypeptide may not be functional, e.g., a gene variant may encode a polypeptide that does not function as well as, or does not function at all, compared to a wild-type gene. In some embodiments, a gene may encode a transcript that may be toxic in some embodiments above a threshold level. In some embodiments, a gene may encode a polypeptide, but the polypeptide may not be functional and / or may be toxic above a threshold level.

[0162] Hearing Loss: As used herein, the term "hearing loss" may refer to the partial or complete inability of an organism to hear. In some embodiments, the hearing loss may be acquired. In some embodiments, the hearing loss may be hereditary. In some embodiments, the hearing loss may be genetic. In some embodiments, the hearing loss may be the result of disease or trauma (e.g., physical trauma, treatment with one or more drugs that result in hearing loss, etc.). In some embodiments, the hearing loss may be due to one or more known genetic causes and / or syndromes. In some embodiments, the hearing loss may be of unknown etiology. In some embodiments, the hearing loss may or may not be alleviated by the use of hearing aids or other treatments.

[0163] Heterologous: As used herein, the term "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. In some embodiments, a promoter operably linked to a nucleic acid encoding a Therapeutic protein may be derived from a different gene than the gene encoding the Therapeutic protein.

[0164] Identity: As used herein, the term "identity" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence; then, the nucleotides at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the two molecules (i.e., the first and second) are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17, incorporated herein by reference in its entirety) as incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons performed using the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0165] Improve, increase, enhance, inhibit, or reduce: As used herein, the terms "improve," "increase," "enhance," "inhibit," "reduce," or their grammatical equivalents refer to a value relative to a baseline or other reference measurement. In some embodiments, the value is statistically significantly different from the baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in an equivalent system known or expected to respond in a particular way in the presence of the relevant agent or treatment. In some embodiments, a suitable reference is a negative reference. In some embodiments, a suitable reference is a positive reference.

[0166] Knockdown: As used herein, the term "knockdown" refers to a reduction in expression of one or more gene products. In some embodiments, an inhibitory nucleic acid achieves the knockdown. In some embodiments, the genome editing system described herein achieves the knockdown.

[0167] Knockout: As used herein, the term "knockout" refers to the elimination of expression of one or more gene products. In some embodiments, the genome editing systems described herein achieve the knockout.

[0168] Minimal promoter: As used herein, unless otherwise indicated, the term "minimal promoter" refers to a promoter that contains less than the complete naturally occurring promoter sequence that is still capable of directing transcription of a coding sequence (e.g., a heterologous or homologous coding sequence).

[0169] In some embodiments, a minimal promoter can comprise one or more regions (including all regions) of a complete naturally occurring promoter capable of directing transcription of a coding sequence.

[0170] In some embodiments, a minimal promoter can comprise a portion of the region of a complete naturally occurring promoter that is capable of directing transcription of a coding sequence.

[0171] MicroRNA: As used herein, the term "microRNA" or "miRNA" refers to a class of biomolecules involved in the regulation of gene expression. Mature miRNAs are typically 18-25 nucleotide non-coding RNAs that regulate the expression of mRNAs containing sequences complementary to the miRNA. These small RNA molecules are known to control gene expression by regulating mRNA stability and / or translation. For example, miRNAs bind to the 3'UTR of target mRNAs and repress translation. MiRNAs can also bind to target mRNAs and mediate gene silencing via the RNAi pathway. MiRNAs can also regulate gene expression by inducing chromatin condensation.

[0172] In some embodiments, the microRNA is from about 10 nucleotides to about 30 nucleotides in length (e.g., from about 10 nucleotides to about 28 nucleotides, from about 10 nucleotides to about 26 nucleotides, from about 10 nucleotides to about 24 nucleotides, from about 10 nucleotides to about 22 nucleotides, from about 10 nucleotides to about 20 nucleotides, from about 10 nucleotides to about 18 nucleotides, from about 10 nucleotides to about 16 nucleotides, from about 10 nucleotides to about 14 nucleotides, from about 10 nucleotides to about 12 nucleotides, from about 12 nucleotides to about 30 nucleotides, from about 12 nucleotides to about 30 nucleotides, Nucleotide to about 28 nucleotides, about 12 nucleotides to about 26 nucleotides, about 12 nucleotides to about 24 nucleotides, about 12 nucleotides to about 22 nucleotides, about 12 nucleotides to about 20 nucleotides, about 12 nucleotides to about 18 nucleotides, about 12 nucleotides to about 16 nucleotides, about 12 nucleotides to about 14 nucleotides, about 16 nucleotides to about 30 nucleotides, about 16 nucleotides to about 28 nucleotides, about 16 nucleotides to about 26 nucleotides, about 16 nucleotides to about 24 nucleotides, about 16 nucleotides to about 22 nucleotides nucleotides, about 16 nucleotides to about 20 nucleotides, about 16 nucleotides to about 18 nucleotides, about 18 nucleotides to about 30 nucleotides, about 18 nucleotides to about 28 nucleotides, about 18 nucleotides to about 26 nucleotides, about 18 nucleotides to about 24 nucleotides, about 18 nucleotides to about 22 nucleotides, about 18 nucleotides to about 20 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 28 nucleotides, about 20 nucleotides to about 26 nucleotides, about 20 nucleotides to about 24 nucleotides, about 20 nucleotides nucleotides to about 22 nucleotides, about 22 nucleotides to about 30 nucleotides, about 22 nucleotides to about 28 nucleotides, about 22 nucleotides to about 26 nucleotides, about 22 nucleotides to about 24 nucleotides, about 24 nucleotides to about 30 nucleotides, about 24 nucleotides to about 28 nucleotides, about 24 nucleotides to about 26 nucleotides, about 26 nucleotides to about 30 nucleotides, about 26 nucleotides to about 28 nucleotides, about 28 nucleotides to about 30 nucleotides, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides).

[0173] MicroRNA regulatory target site: As used herein, the term "microRNA regulatory target site" or "miRTS" refers to a sequence on an mRNA transcript that directly interacts with a miRNA. Often, miRTSs are present in the 3' untranslated region (UTR) of an mRNA, but they can also be present in the coding sequence or 5' UTR. A miRTS is not necessarily a perfect complement to a miRNA; it usually has only a few bases of complementarity to the miRNA and often contains one or more mismatches. A miRTS may be any sequence that can be bound by a miRNA sufficiently such that translation of a gene to which the miRTS is operably linked is suppressed by a miRNA silencing mechanism such as RISC. In some embodiments, inclusion of a miRTS in a nucleic acid construct containing a polynucleotide (e.g., a therapeutic polynucleotide) can result in degradation of the therapeutic polynucleotide after transcription.

[0174] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA. In some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), regeneration in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 residues or longer. In some embodiments, the nucleic acid is partially or completely single-stranded. In some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide, or is complementary to a sequence that encodes a polypeptide.In some embodiments, the nucleic acid has enzymatic activity.

[0175] Operably linked: As used herein, refers to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated such that expression and / or activity of the functional element is achieved under conditions compatible with the control elements. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest. In some embodiments, the control element acts in trans on or otherwise from the functional element of interest. In some embodiments, "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. In some embodiments, for example, functional linkage can include transcriptional control. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, in the same reading frame.

[0176] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for administration of injectable formulations, e.g., aqueous or non-aqueous solutions or suspensions or drops designed to be administered into the ear canal. In some embodiments, pharmaceutical compositions may be formulated for administration via injection, either directly into a specific organ or compartment, e.g., into the ear, or systemically, e.g., intravenously. In some embodiments, a formulation may be or include a drench (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granules, paste, capsule, powder, etc. In some embodiments, the active agent may be or include an isolated, purified, or pure compound.

[0177] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable," which may be used in reference to a carrier, diluent, or excipient used, for example, to formulate a pharmaceutical composition disclosed herein, means the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0178] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle (e.g., a liquid or solid filler, diluent, excipient, or solvent encapsulating material) that is involved in the transport or delivery of a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0179] Polyadenylation: As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or its modified variants to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. In some embodiments, the 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to a pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, a poly(A) tail can be added to transcripts containing a specific sequence, a polyadenylation signal or "poly(A) sequence." The poly(A) tail and its associated proteins help protect the mRNA from degradation by exonucleases. Polyadenylation can affect transcription termination, mRNA export from the nucleus, and translation. Typically, polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also occur later in the cytoplasm. After transcription is completed, the mRNA strand can be cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site can be characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, adenosine residues can be added to the free 3' end of the cleavage site. As used herein, a "poly(A) sequence" is a sequence that triggers endonuclease cleavage of mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0180] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an engineered amino acid sequence, in that it has been designed and / or produced by the action of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modified or attached to one or more amino acid side chains at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be acetylated, amidated, lipidated, methylated, pegylated, etc. (including combinations thereof). In some embodiments, a polypeptide may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. In some embodiments, useful modifications may be or include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is used to generally refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the polypeptide may be a therapeutic polypeptide (e.g., a connexin 26 polypeptide). In some embodiments, the polypeptide may be a support cell polypeptide (e.g., a connexin 26 polypeptide). In some embodiments, the polypeptide may be a reporter polypeptide.

[0181] Polynucleotide: As used herein, the term "polynucleotide" refers to any polymeric chain of nucleic acid. In some embodiments, a polynucleotide is or comprises RNA. In some embodiments, a polynucleotide is or comprises DNA. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, polynucleotide analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a polynucleotide has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, a polynucleotide comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, a polynucleotide has a nucleotide sequence that encodes a functional gene product such as RNA or a protein.In some embodiments, the polynucleotide comprises one or more introns. In some embodiments, the polynucleotide is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, the polynucleotide is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 residues or longer. In some embodiments, the polynucleotide is partially or completely single-stranded. In some embodiments, the polynucleotide is partially or completely double-stranded. In some embodiments, the polynucleotide has a nucleotide sequence comprising at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, the polynucleotide has enzymatic activity.

[0182] Promoter: As used herein, the term "promoter" refers to a nucleic acid sequence that functions to control transcription of one or more coding sequences (e.g., a gene or transgene that encodes a polypeptide (e.g., a therapeutic polypeptide)) located upstream in the direction of transcription of the coding sequence's transcription start site. In some aspects, a promoter is structurally identified by the presence of binding sites for a DNA-dependent RNA polymerase, a transcription start site, or other DNA sequences (e.g., a transcription factor binding site, a repressor and / or activator protein binding site, or other sequences of nucleotides that act directly or indirectly to regulate the amount of transcription from the promoter). In some aspects, a promoter can comprise a naturally occurring promoter sequence, a functional fragment thereof, or a variant of a naturally occurring promoter sequence or a functional fragment thereof.

[0183] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be processed or modified in other ways. Those of skill in the art will understand that a "protein" may be an entire polypeptide chain (with or without a signal sequence) as produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may comprise multiple polypeptide chains, for example, linked by one or more disulfide bonds or associated by other means.

[0184] Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide designed, engineered, prepared, expressed, produced, manufactured, and / or isolated by recombinant means (e.g., a polypeptide expressed using a recombinant expression construct transfected into a host cell); a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise engineered to express one or more genes or genetic components that encode and / or direct the expression of a polypeptide or one or more components, portions, elements, or domains thereof; and / or a polypeptide prepared, expressed, produced, or isolated by any other means, including splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs the expression of a polypeptide or one or more components, portions, elements, or domains thereof. In some aspects, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from a natural or synthetic source, such as, for example, the germline of a source organism of interest (e.g., human, mouse, etc.).

[0185] Reference: As used herein, the term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a prior reference or control, optionally embodied in a tangible medium. Typically, as understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control. In some embodiments, the reference is a negative control reference. In some embodiments, the reference is a positive control reference. In some embodiments, the reference can be a compound, protein, polypeptide, or polynucleotide disclosed in the present disclosure.

[0186] Regulatory element: As used herein, the term "regulatory element" or "regulatory sequence" refers to a non-coding region of DNA that regulates in some way the expression of one or more specific genes. In some embodiments, such genes are juxtaposed to or "proximal" to a given regulatory element. In some embodiments, such genes are located at a considerable distance from a given regulatory element. In some embodiments, a regulatory element impairs or enhances transcription of one or more genes. In some embodiments, a regulatory element may be located in cis with respect to the gene being regulated. In some embodiments, a regulatory element may be located in trans with respect to the gene being regulated. For example, in some embodiments, a regulatory sequence refers to a nucleic acid sequence that regulates the expression of a gene product operably linked to the regulatory sequence. In some such embodiments, this sequence may be an enhancer sequence or other regulatory element that regulates expression of the gene product.

[0187] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism (e.g., a virus), a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal fluid, vitreous humor, vomit, and / or combinations or components thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or intercellular fluid. In some embodiments, the biological fluid may be or include plant exudates. In some embodiments, the biological tissue or sample may be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, lavage, or irrigation (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other lavage or irrigation). In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample), such as filtration using a semi-permeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of specific components, etc.

[0188] Selective expression: As used herein, the term "selective expression" or "selectively express" refers to the expression of a gene or polypeptide of interest primarily in a particular cell type (e.g., inner ear cells, e.g., inner ear supporting cells).

[0189] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including prenatal human forms in some embodiments). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a person with one or more characteristics characteristic of susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or who has been diagnosed and / or treated.

[0190] Substantially: As used herein, the term "substantially" refers to the qualitative condition of indicating the entire or nearly entire extent or degree of a characteristic or property of interest. Those skilled in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed perfectly, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0191] Supporting Cell: As used herein, the terms "support cell," "supporting cell," "inner ear support cell," or "inner ear supporting cell" refer to cells of the inner ear that maintain the structure of the inner ear and the environment of the sensory epithelium of the inner ear. In some embodiments, inner ear supporting cells include, but are not limited to, inner phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of Coriker cells (KOs), greater ridge epithelial cells (GERs) (including greater ridge epithelial cells (LGERs)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall.

[0192] Supporting cell polypeptide: As used herein, the term "supporting cell polypeptide" or "support cell polypeptide" refers to a polypeptide that is endogenously expressed in supporting cells of the inner ear.

[0193] Reporter polypeptide: As used herein, the term "reporter polypeptide" refers to a polypeptide that confers a detectable or selectable phenotype on an organism or cell. The detectable phenotype can be, for example, colorimetric, fluorescent, or luminescent. Reporter polypeptides can include enzymes that mediate luminescent reactions (luxA, luxB, luxAB, luc, ruc, nluc), enzymes that mediate colorimetric reactions (lacZ, HRP), fluorescent proteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent protein), affinity peptides (His tag, 3X-FLAG), and selection markers (ampC, tet(M), CAT, erm). Reporter polypeptides can be used as markers for successful uptake of nucleic acid molecules or exogenous sequences (plasmids) into cells. Reporter polypeptides can also be used to indicate the presence of target genes, target nucleic acid molecules, target polypeptides, target intracellular molecules, or cells, as described herein.

[0194] Therapeutic Polypeptide: As used herein, the term "therapeutic polypeptide" refers to a polypeptide having biological activity that can be used for the prevention and / or treatment of a disease (e.g., hearing loss). Examples of therapeutic polypeptides include those that can prevent, inhibit, stabilize, or reverse inherited or non-inherited genetic defects in metabolism, immunoregulation, hormone regulation, enzyme, or membrane-associated structural function. For example, a therapeutic protein can replace an absent or defective cellular protein or enzyme, or can compensate for the production of a defective or under-expressed cellular protein or enzyme.

[0195] Treatment: As used herein, the term "treatment" (also referred to as "treat" or "treating") refers to any administration of therapy that partially or completely alleviates, ameliorate, eliminate, reverse, relieve, inhibit, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of a subject who does not exhibit signs of the relevant disease, disorder, and / or condition and / or a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment of a subject who exhibits established signs of one or more of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who is known to have one or more susceptibility factors that statistically correlate with an increased risk of developing a given disease, disorder, and / or condition.

[0196] Variant: As used herein, the term "variant" refers to a version of something (e.g., a gene sequence) that differs in some way from another version. To determine whether something is a variant, typically a reference version is selected, and the variant differs relative to that reference version. In some embodiments, a variant can have the same or a different (e.g., increased or decreased) level of activity or functionality as the wild-type sequence. For example, in some embodiments, a variant can have improved functionality compared to the wild-type sequence, e.g., if it is codon-optimized to resist degradation, e.g., by an inhibitory nucleic acid, e.g., miRNA. Such variants are referred to herein as gain-of-function variants. In some embodiments, a variant has reduced or eliminated activity or functionality, or an altered activity that has a negative consequence (e.g., increased electrical activity resulting in chronic depolarization leading to cell death). Such variants are referred to herein as loss-of-function variants. In some embodiments, a gain-of-function variant is a codon-optimized sequence that encodes a transcript or polypeptide that may have improved properties (e.g., less susceptible to degradation, e.g., less susceptible to miRNA-mediated degradation) over its corresponding wild-type (e.g., non-codon-optimized) version. In some aspects, a loss-of-function variant has one or more changes that result in a transcript or polypeptide that is defective in some way (e.g., reduced function, non-functional) compared to the wild-type transcript and / or polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0197] In certain aspects, the present disclosure relates to promoters for selective transgene expression, for example, preferential expression in inner ear supporting cells.

[0198] In some aspects, the present disclosure relates to constructs comprising polynucleotides encoding therapeutic polypeptides (e.g., connexin 26 polypeptides), and compositions comprising the same, designed for selective transgene expression, e.g., preferential expression in inner ear supporting cells and / or reduced expression in other inner ear cells, e.g., hair cells.

[0199] In some aspects, the present disclosure also relates to constructs comprising polynucleotides encoding polypeptides, and compositions comprising the same, designed for selective transgene expression, e.g., preferential expression in inner ear supporting cells and / or reduced expression in other inner ear cells, e.g., hair cells.

[0200] In some aspects, the present disclosure relates to constructs comprising polynucleotides encoding therapeutic polypeptides (e.g., connexin 26 polypeptides), and compositions comprising the same, designed for transgene expression in inner ear supporting cells, e.g., preferential expression in inner ear supporting cells and / or reduced expression in other inner ear cells, e.g., hair cells. In some aspects, the preferential expression and / or reduced expression is compared to the corresponding endogenous expression.

[0201] In some aspects, the present disclosure relates to an AAV particle comprising a promoter or construct disclosed herein.

[0202] In some aspects, the present disclosure relates to methods of using the promoters, constructs and AAV particles disclosed herein to treat hearing loss.

[0203] hearing loss Generally, the ear can be described as including the outer ear, middle ear, inner ear, auditory (acoustic) nerve, and auditory system (which processes sound as it travels from the ear to the brain). In addition to detecting sound, the ear also helps maintain balance. Thus, in some aspects, disorders of the inner ear can cause hearing loss, tinnitus, dizziness, imbalance, or a combination thereof.

[0204] Hearing loss can be the result of genetic factors, environmental factors, or a combination of genetic and environmental factors. Approximately half of all people with tinnitus (phantom noise in the auditory system (ringing, buzzing, chirping, humming, or beating)) also have an oversensitivity / decreased tolerance to certain sound frequencies and volume ranges, known as hyperacusis (also called hyperacusis). Various non-syndromic and syndromic associated hearing loss are known to those skilled in the art (e.g., DFNB1 and DFNA3, respectively; and Bart-Pumphrey syndrome, histricoid ichthyosis with hearing loss (HID), palmar keratoderma with hearing loss, keratitis-ichthyosis-hearing loss (KID) syndrome, and Vohwinkel syndrome). Environmental causes of hearing impairment or loss may include, for example, certain medications, certain prenatal or postnatal infections, and / or prolonged exposure to loud noise. In some aspects, hearing loss can result from noise, ototoxins, presbycusis, disease, infection, or cancer affecting specific parts of the ear. In some aspects, ischemic injury can cause hearing loss through pathophysiological mechanisms. In some aspects, intrinsic abnormalities, such as congenital mutations in genes that play important roles in the anatomy or physiology of the cochlea, or genetic or anatomical changes in supporting cells and / or hair cells, can cause or contribute to hearing loss.

[0205] Hearing loss and / or hearing loss is one of the most common human sensory defects and can occur for many reasons. In some embodiments, subjects may be born with hearing loss or without hearing loss, while others may slowly lose their hearing over time. Approximately 36 million American adults report some degree of hearing loss, with one in three people over the age of 60 and half of people over the age of 85 experiencing hearing loss. Approximately 1.5 out of 1,000 children are born with profound hearing loss, and an additional 2-3 out of 1,000 children are born with partial hearing loss (Smith et al., 2005, Lancet 365:879-890, incorporated herein by reference in its entirety). More than half of these cases are due to genetic causes (Di Domenico, et al., 2011, J. Cell. Physiol. 226:2494-2499, incorporated herein by reference in its entirety).

[0206] Treatments for hearing loss currently consist of hearing amplification for mild to severe hearing loss and cochlear implants for severe to profound hearing loss (Kral and O'Donoghue, 2010, N. Engl. J. Med. 363:1438-1450, incorporated herein by reference in their entireties). Recent research in this field has focused on cochlear hair cell regeneration, which is applicable to the most common forms of hearing loss, including presbycusis, noise injury, infectious, and toxic hearing loss. There remains a need for effective treatments, such as gene therapy, that can repair and / or alleviate the cause of hearing problems (see, e.g., WO 2018 / 039375, WO 2019 / 165292, and PCT application US2019 / 060328, each of which is incorporated herein by reference in its entirety).

[0207] In some embodiments, nonsyndromic hearing loss and / or hearing loss is not associated with other signs and symptoms. In some embodiments, syndromic hearing loss and / or hearing loss occurs along with abnormalities in other parts of the body. Approximately 70% to 80% of cases of genetic hearing loss and / or hearing loss are nonsyndromic. The remaining cases are often caused by specific genetic syndromes. Nonsyndromic hearing loss and / or hearing loss can have different patterns of inheritance and can occur at any age. Types of nonsyndromic hearing loss and / or hearing loss are generally named according to their inheritance pattern. For example, the autosomal dominant type is called DFNA, the autosomal recessive type is DFNB, and the X-linked type is DFN. Each type is also numbered in the order in which it was first described. For example, DFNA1 was the first autosomal dominant type of nonsyndromic hearing loss described. Seventy-five to eighty percent of cases of genetically caused hearing loss and / or hearing loss are inherited in an autosomal recessive pattern, meaning that both copies of the gene in each cell carry the mutation. Typically, each parent of an individual with autosomal recessive hearing loss and / or hearing loss carries one copy of the mutant gene but is unaffected by this form of hearing loss. Another 20 to 25 percent of cases of nonsyndromic hearing loss and / or hearing loss are autosomal dominant, meaning that one copy of the modified gene in each cell is sufficient to cause hearing loss and / or hearing loss. People with autosomal dominant hearing loss and / or hearing loss most often inherit an altered copy of the gene from a parent who has hearing loss and / or hearing loss. One to two percent of cases of hearing loss and / or hearing loss exhibit an X-linked inheritance pattern, meaning that the mutant gene responsible for the condition is located on the X chromosome (one of the two sex chromosomes). Males with X-linked nonsyndromic hearing loss and / or hearing loss tend to develop hearing loss earlier and more severely than females who inherit copies of the same gene mutation. X-linked inheritance is characterized by the inability of fathers to pass the X-linked trait to their sons. Mitochondrial nonsyndromic hearing loss, resulting from changes to mitochondrial DNA, occurs in less than 1 percent of cases in the United States. The altered mitochondrial DNA is passed from mother to all of her sons and daughters.This type of hearing loss is not inherited from the father. The causes of syndromic and non-syndromic hearing loss and / or hearing loss are complex. Researchers have identified more than 30 genes that, when altered, are associated with syndromic and / or non-syndromic hearing loss and / or hearing loss. However, some of these genes have not been fully characterized. Different mutations in the same gene may be associated with different types of hearing loss and / or hearing loss, and some genes are associated with both syndromic and non-syndromic hearing loss and / or hearing loss.

[0208] In some embodiments, hearing loss and / or hearing loss can be conductive (originating from the ear canal or middle ear), sensorineural (originating from the inner ear or auditory nerve), or mixed. In some embodiments, non-syndromic hearing loss and / or hearing loss is associated with permanent hearing loss caused by damage to structures in the inner ear (sensorineural hearing loss). In some embodiments, sensorineural hearing loss can be due to poor hair cell function. In some embodiments, sensorineural hearing impairment involves the eighth cranial nerve (vestibulocochlear nerve) or the auditory portion of the brain. In some such embodiments, only the auditory center of the brain is affected. In such situations, cortical hearing loss can occur, in which sounds can be heard at normal thresholds, but the perceived sound quality is so poor that speech cannot be understood. Hearing loss resulting from changes in the middle ear is called conductive hearing loss. Some forms of non-syndromic hearing loss and / or hearing loss involve changes in both the inner and middle ear, called mixed hearing loss. Hearing loss and / or hearing loss that is present before a child learns to speak can be classified as prelingual (anterior lingual) or congenital. Hearing loss and / or hearing loss that occurs after the development of language can be classified as postlingual (posterior lingual). Most autosomal recessive loci associated with syndromic or nonsyndromic hearing loss cause severe to profound hearing loss in the anterior lingual.

[0209] As known to those skilled in the art, hair cells are sensory receptors in both the auditory and vestibular systems of the vertebrate ear. Hair cells detect motion in the environment, and in mammals, hair cells are located in the cochlea of ​​the ear in the organ of Corti. The mammalian ear is known to have two types of hair cells: inner hair cells and outer hair cells. Outer hair cells can amplify low-level sound frequencies either through mechanical movement of the hair cell bundle or electrically driven movement of the hair cell soma. Inner hair cells convert vibrations in the cochlear fluid into electrical signals that the auditory nerve transmits to the brain. In some aspects, hair cells can be abnormal at birth or damaged during an individual's lifetime. In some aspects, outer hair cells can regenerate. In some aspects, inner hair cells cannot regenerate after disease or injury. In some aspects, sensorineural hearing loss is due to abnormalities in hair cells.

[0210] As known to those skilled in the art, hair cells do not occur alone, but rather their function is supported by a wide variety of cells that can be collectively referred to as supporting cells. Supporting cells can perform numerous functions and include numerous cell types, including, but not limited to, inner phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of corikers (KOs), greater ridge epithelial cells (GERs) (including lateral greater ridge cells (LGERs)), and OC90+ cells (OC90s), fibroblasts, and other cells of the lateral wall.

[0211] In some aspects, sensorineural hearing loss is due to abnormalities in supporting cells. In some aspects, supporting cells may be abnormal at birth or damaged during an individual's lifetime. In some aspects, supporting cells may be capable of regenerating. In some aspects, certain supporting cells may not be capable of regenerating.

[0212] Polypeptides Certain aspects of the present disclosure relate to polynucleotides encoding polypeptides. The polynucleotides can encode polypeptides that can be expressed in cells (e.g., inner ear cells). The polynucleotides can encode full-length polypeptides or functional fragments thereof.

[0213] Exemplary polypeptides encoded by the polynucleotides include, but are not limited to, transmembrane proteins, enzymes, growth factors, cytokines, receptors, receptor ligands, hormones, membrane proteins, membrane-associated proteins, antigens, and antibodies.

[0214] Exemplary polynucleotides encoding polypeptides include ATPase plasma membrane Ca2+ transporter 2 (ATP2B2), cholinergic receptor nicotinic alpha 9 subunit (CHRNA9), cadherin 23 (CDH23), coiled-coil glutamic acid-rich protein 2 (CCER2), cularin 1 (CLRN1), cularin 2 (CLRN2), cochlin (COCH or DFNA9), dystrotelin (DYTN), epidermal growth factor receptor pathway substrate 8 (EPS8), EPS8-like 2 (EPS8L2), espin (ESPN), espin-like (ESPNL), gap junction protein beta 2 (GJB2), gap junction protein beta 6 (GJB6), gap junction protein beta 3 (GJB7), and the like. These include, but are not limited to, GJB3), gasdermin E protein (GSDME or DFNA5), insulinoma-associated 1 (INSM1), Ikaros family zinc finger 2 (IKZF2), LIM homeobox protein 3 (LHX3), myosin 7A (MYO7A), myosin 11 (MYO3A), normal cystine knot growth factor receptor (NDP), protocadherin 15 (PCDH15), protein tyrosine phosphatase, receptor type Q (PTPRQ), stereocillin (STRC), protein network component harmonin (USH1C), ashurin (USH2A), and spectrin repeat-containing nuclear envelope family member 4 (SYNE4).

[0215] In some embodiments, the polynucleotide can comprise a GJB2 gene. In some embodiments, the polynucleotide can comprise a nucleic acid encoding a connexin 26 polypeptide. In some embodiments, the nucleic acid comprises a coding sequence for a connexin 26 polypeptide.

[0216] In some embodiments, the polynucleotide or nucleic acid comprises the gap junction beta-2 (GJB2) gene. The GJB2 gene is highly conserved across mammalian species and encodes connexin 26 (Cx26), also known as the gap junction beta-2 (GJB2) protein. Connexin 26 is a member of the gap junction protein family, also known as the connexin family. Gap junction proteins are specialized proteins involved in intracellular communication. Mutations in the human GJB2 gene are associated with hearing loss and hearing loss (Amorini et al., Ann. Hum. Genet. 79(5):341-349, 2015; Qing et al., Genet. Test Mol. Biomarkers 19(1):52-58, 2015).

[0217] The human GJB2 gene is located on chromosome 13q12. It contains two transcript isoforms that initiate from alternative transcription start sites, both of which contain two exons and a single intron encompassing a total of approximately 5 kilobases (kb) (approximately 5,469 or 4,675 nucleotides, respectively) (NCBI gene ID 2706, NCBI reference sequence: NG_008358.1). Both human GJB2 mRNA isoforms contain a second exon in exon 2 that completely encodes full-length connexin 26. This coding sequence is approximately 681 nucleotides long and encodes a 226-amino acid long connexin 26.

[0218] Connexin 26 monomers contain four transmembrane helices connected by two extracellular loops and one shorter intracellular loop, with the N- and C-termini located on the cytoplasmic side of the plasma membrane. Gap junctions between cells can form homomeric and / or heteromeric channels. Connexin 26 has been shown to form functional homomeric channels as well as functional heteromeric channels with at least connexin 30, connexin 32, connexin 46, and connexin 50. In some embodiments, GJB2 gene-associated sensorineural hearing loss (e.g., nonsyndromic or syndromic) may be due to compound heterozygous mutations in GJB2 and alternative connexin protein-encoding genes. Gap junctions formed with connexin 26 transport potassium ions and certain other small molecules across cells. Connexin 26 helps maintain precise levels of intracellular potassium ions and is necessary for the maturation of certain cells in the cochlea.

[0219] The human GJB2 gene is expressed in many tissues and is known to play important cell homeostatic roles in the epidermis and inner ear. Within the inner ear, connexin 26 is synthesized by all supporting cell types within the organ of Corti, including inner phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / external sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of Corti cells (KOs), greater epithelial ridge cells (GERs) (including greater epithelial ridge cells (LGERs)), OC90+ cells (OC90s), root cells, fibrocytes, fibroblasts, basal and intermediate cells from the stria vascularis, and other cells of the lateral wall. Furthermore, connexin 26 is known to be present in mesenchymal cells in the lateral wall and in type 1 neurons in the spiral ganglion.

[0220] The human GJB2 gene has a defined 128-bp basal / minimal promoter immediately upstream of the canonical first exon in the most abundant isoform. This sequence contains a TATA box and two GC boxes, which are known to be bound by the Sp1 and Sp3 TFs.

[0221] There are over 200 distinct mutations in GJB2, which may be pathogenic to some degree, and various mutations in the GJB2 gene are associated with hearing loss (e.g., nonsyndromic or syndromic sensorineural hearing loss). For example, the c.35delG allele was found in 65.5% of patients from Eastern Sicily (Amorini et al., Ann. Hum. Genet. 79(5):341-349, 2015). Further exemplary mutations in the GJB2 gene detected in subjects with nonsyndromic sensorineural hearing loss or syndromic sensorineural hearing loss, and methods for sequencing nucleic acids encoding GJB2, are described, for example, in Snoeckx et al., Am.J.Hum.Genet 77:945-957, 2005; Welch et al., Am.J.Med.Genet A 143:1567-1573, 2007; Zelante et al., Hum.Mol.Genet.6:1605-1609, 1997; and Tsukada et al., Annals of Otology, Rhinology & Laryngology. 2015, Vol. 124(5S)61S-76S, each of which is incorporated herein by reference in its entirety. Methods for detecting mutations in genes are well known in the art. Non-limiting examples of such techniques include real-time polymerase chain reaction (RT-PCR), PCR, Sanger sequencing, next-generation sequencing, Southern blotting, and Northern blotting. Several disease states associated with sensorineural hearing loss, with either nonsyndromic or syndromic manifestations, have been associated with specific mutations in the human GJB2 gene (see Nickel & Forge, Curr Opin Otolaryngol Head Neck Surg. 2008 Oct;16(5):452-7, the entire contents of which are incorporated herein by reference). Human GBJ2 gene mutations that cause syndromic or nonsyndromic hearing loss range from large deletions that remove either the entire GJB2 or the GJB2 gene regulatory region to hundreds of smaller changes, including nonsense, missense, indels (which cause phase shifts), and splice site point mutations.

[0222] In some embodiments, GJB2 gene mutations, such as Gly59Ser and Asn52Lys, are associated with Bart-Pumphrey syndrome, a syndrome characterized by thickened skin, warty growths, and generally congenital, moderate to severe sensorineural hearing loss. In other embodiments, GJB2 gene mutations, such as Aspn50Asn, are associated with histolytic ichthyosis with hearing loss and keratitis-ichthyosis-deafness syndrome. These syndromes are associated with dry, scaly skin, generally congenital, severe sensorineural hearing loss, and keratitis-ichthyosis-deafness syndrome, further inflammation of the cornea.

[0223] In some embodiments, GJB2 gene missense mutations are associated with palmar keratoderma with hearing loss. This syndrome is associated with thickened skin on the palms and soles of the feet, as well as mild to severe sensorineural hearing loss that begins in early childhood and worsens over time, and affected individuals may have particular difficulty hearing high-pitched sounds. Meanwhile, in other embodiments, GJB2 gene missense mutations are associated with Vohwinkel syndrome, a syndrome associated with skin abnormalities (e.g., thick bands of fibrous tissue around the fingers and toes that can block circulation to the fingers and lead to spontaneous amputation) and sensorineural hearing loss.

[0224] In some embodiments, GJB2 gene mutations are associated with non-syndromic hearing loss, which can be inherited in either a dominant (e.g., DFNA3) or recessive (DFNB1) manner. In some embodiments, loss-of-function GJB2 gene mutations are associated with non-syndromic DFNB1, which is inherited in an autosomal recessive manner and manifests as mild to profound hearing loss that is generally anterior and does not become more severe over time. DFNB1 is estimated to be present in approximately 14 per 100,000 live births in the United States and EU5. It is postulated that an early, but not necessarily congenital, onset of DFNB1 hearing impairment can be followed by rapidly progressive hearing loss. Generally, treatment options for DFNB1 patients include education, hearing aids, and cochlear implants. Patients generally have no further symptoms and live a normal lifespan. DFNB1 is estimated to account for approximately 50% of congenital severe-to-profound autosomal recessive non-syndromic hearing loss in many developed countries (eg, the United States, France, the United Kingdom, and Australia).

[0225] In some embodiments, sensorineural hearing loss due to GJB2 gene mutations is inherited in an autosomal dominant manner as non-syndromic DFNA3. These mutations are generally dominant-negative missense mutations that prevent the formation of necessary functional gap junctions. This disease state presents with hearing loss that can range from mild to profound, either anterior or posterior, which generally becomes more severe over time.

[0226] Among other things, the present disclosure provides polynucleotides, such as polynucleotides comprising the GJB2 gene or characteristic portions thereof, as well as compositions comprising such polynucleotides, and methods utilizing such polynucleotides and / or compositions.

[0227] In some embodiments, the polynucleotide comprising the GJB2 gene or a characteristic portion thereof can be DNA or RNA. In some embodiments, the DNA can be genomic DNA or cDNA. In some embodiments, the RNA can be mRNA. In some embodiments, the polynucleotide comprises exons and / or introns of the GJB2 gene.

[0228] In some embodiments, a gene product is expressed from a polynucleotide comprising the GJB2 gene or a characteristic portion thereof. In some embodiments, expression of such a polynucleotide can utilize one or more control elements (e.g., a promoter, an enhancer, a splice site, a polyadenylation site, a translation initiation site, etc.). Thus, in some embodiments, the polynucleotides provided herein can comprise one or more control elements.

[0229] In some embodiments, the GJB2 gene is a mammalian GJB2 gene. In some embodiments, the GJB2 gene is a mouse GJB2 gene. In some embodiments, the GJB2 gene is a primate GJB2 gene. In some embodiments, the GJB2 gene is a human GJB2 gene. In some embodiments, the GJB2 gene is codon-optimized. An exemplary human GJB2-encoding cDNA sequence is or includes SEQ ID NO: 117 or SEQ ID NO: 118. An exemplary human GJB2 splice cDNA sequence with an untranslated region is or includes SEQ ID NO: 119. An exemplary human GJB2 splice cDNA sequence with an alternative transcription start site with an untranslated region is or includes SEQ ID NO: 120. An exemplary human GJB2 genomic DNA sequence can be found in SEQ ID NO: 121. Exemplary codon-optimized GJB2 DNA sequences can be found in SEQ ID NOs: 123-126. An exemplary human GJB2 cDNA coding sequence (SEQ ID NO: 117). ATGGATTGGGGCACGCTGCAGACGATCCTGGGGGGTGTGAACAAACACTCCACCAGCATTGGAAAGATCTGGCTCACCGTCCTCTTCATTTTTCGCATTATGATCCTCGTTGTGGCTGCAAAGGAGGTGTGGGGAGATGAGCAGGCCGACTTTGTCTGCAACACCCTGCAGCCAGGCTGCAAGAACGTGTGCTACGATCACTACTTCCCCATCTCCCACATCCGGCTATGGGCCCTGCAGCTGATCTTCGTGTCCACGCCAGCGCTCCTAGTGGCCATGCACGTGGCCTACCGGAGACATGAGAAGAAGAGGAAGTTCATCAAGGGGGAGATAAAGAGTGAATTTAAGGACATCGAGGAGATCAAAACCCAGAAGGTCCGCATCGAAGGCTCCCTGTGGTGGACCTACACAAGCAGCATCTTCTTCCGGGTCATCTTCGAAGCCGCCTTCATGTACGTCTTCTATGTCATGTACGACGGCTTCTCCATGCAGCGGCTGGTGAAGTGCAACGCCTGGCCTTGTCCCAACACTGTGGACTGCTTTGTGTCCCGGCCCACGGAGAAGACTGTCTTCACAGTGTTCATGATTGCAGTGTCTGGAATTTGCATCCTGCTGAATGTCACTGAATTGTGTTATTTGCTAATTAGATATTGTTCTGGGAAGTCAAAAAAGCCAGTT Exemplary human GJB2 cDNA coding sequence (SEQ ID NO: 118). ATGGATTGGGGCACGCTGCAGACGATCCTGGGGGGTGTGAACAAACACTCCACCAGCATTGGAAAGATCTGGCTCACCGTCCTCTTCATTTTTCGCATTATGATCCTCGTTGTGGCTGCAAAGGAGGTGGGGAGATGAGCAGGCCGACTTTGTCTGCAACACCCTGCA GCCAGGCTGCAAGAACGTGTGCTACGATCACTACTTCCCCATCTCCCACATCCGGCTATGGGCCCTGCAGCTGATCTTCGTGTCCACGCCAGCGCTCCTAGTGGCCATGCACGTGGCCTACCGGAGACATGAGAAGAAGAGGAAGTTCATCAAGGGGGAGATAAAGAGTG AATTTAAGGACATCGAGGAGATCAAAACCCAGAAGGTCCGCATCGAAGGCTCCCTGTGGTGGACCTACACAAGCAGCATCTTCTTCCGGGTCATCTTCGAAGCCGCCTTCATGTACGTCTTCTATGTCATGTACGACGGCTTCTCCATGCAGCGGCTGGTGAAGTGCAAC GCCTGGCCTTGTCCCAACACTGTGGACTGCTTTGTGTCCCGGCCCACGGAGAAGACTGTCTTCACAGTGTTCATGATTGCAGTGTCTGGAATTTGCATCCTGCTGAATGTCACTGAATTGTGTTATTTGCTAATTAGATATTGTTCTGGGAAGTCAAAAAAGCCAGTTTAA An exemplary spliced ​​human GJB2 isoform 1 cDNA sequence including untranslated regions (SEQ ID NO: 119). An exemplary spliced ​​human GJB2 isoform X1 cDNA sequence including untranslated regions (SEQ ID NO: 120). An exemplary human GJB2 genomic DNA sequence (SEQ ID NO: 121). TGAGCTTTGTCTACTTCAAAAGTTTGTTTGCTTACCCCTTCAGCCTCCAATTTTTTTAAGTGAAAATATAGCTAATAACATGTGAAAAGAATAGAAGCTAAGGTTTAGATAAATATTGAGCAGATCTATAGGAAGATTGAACCTGAATATTGCCATTATGCTTGACATGGTTTCC AAAAAATGGTACTCCACATATTTCAGTGAGGGTAAGTATTTTCCTGTTGTCAAGAATAGCATTGTAAAAGCATTTTGTAATAATAAAGAATAGCTTTAATGATATGCTTGTAACTAAAATAATTTTGTAATGTATCAAATACATTTAAAACATTAAAATATAATCTCTATAATAA An exemplary extended human GJB2 genomic DNA sequence (SEQ ID NO: 122) containing specific regulatory regions.

[0230] In some embodiments, the GJB2 gene is codon-optimized. In some embodiments, the codon-optimized GJB2 gene is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 123-126. In some embodiments, the codon-optimized GJB2 gene has the sequence of any one of SEQ ID NOs: 123-126. An exemplary codon-optimized human GJB2 DNA sequence (SEQ ID NO: 123). ATGGACTGGGGCACCCTGCAGACTATCCTGGGGGGCGTCAATAAGCATTCAACTAGCATCGGAAAGATTTGGCTGACTGTCCTGTTTATCTTTCGGATCATGATCCTGGTGGTGGCAGCAAAGGAAGTGTGGGGCGACGAGCAGGCCGATTTCGTGTGCAACACACTGCAGCCAGGCTGCAAGAACGTGTGCTACGACCACTATTTTCCCATCTCTCACATCAGGCTGTGGGCCCTGCAGCTGATCTTCGTGAGCACCCCTGCCCTGCTGGTGGCAATGCACGTGGCCTATCGGAGACACGAGAAGAAGCGCAAGTTTATCAAGGGCGAGATCAAGAGCGAGTTCAAGGATATCGAGGAGATCAAGACACAGAAGGTGAGGATCGAGGGCTCCCTGTGGTGGACCTACACAAGCTCCATCTTCTTTCGCGTGATCTTCGAGGCCGCCTTTATGTACGTGTTCTATGTGATGTACGACGGCTTTTCTATGCAGCGGCTGGTGAAGTGCAACGCCTGGCCCTGTCCTAATACAGTGGATTGTTTCGTGTCCAGACCCACCGAGAAGACAGTGTTCACCGTGTTTATGATCGCCGTGTCTGGCATCTGCATCCTGCTGAACGTGACCGAGCTGTGCTATCTGCTGATCCGGTACTGTAGTGGAAAGAGCAAAAAACCCGTG Exemplary codon-optimized human GJB2 DNA sequence (SEQ ID NO: 124). ATGGACTGGGGAACATTGCAAACTATTTTGGGAGGAGTCAACAAGCATTCAACTAGCATCGGGAAGATCTGGCTGACCGTGCTGTTCATCTTTCGCATCATGATTCTCGTGGTGGCCGCTAAGGAAGTCTGGGGCGATGAACAGGCCGACTTCGTGTGTAACACGCTGCAGCCCGGTTGCAAAAACGTCTGCTACGATCACTACTTCCCCATCTCACACATTAGACTGTGGGCGCTGCAGCTGATTTTCGTGTCCACCCCGGCACTTCTTGTGGCGATGCACGTGGCCTACCGGCGGCACGAGAAGAAAAGGAAGTTCATTAAGGGCGAAATCAAGTCCGAGTTCAAGGACATCGAAGAAATCAAGACCCAGAAGGTCCGCATTGAGGGCTCCCTCTGGTGGACCTACACCTCGTCCATCTTCTTCCGGGTCATATTCGAGGCCGCCTTTATGTACGTGTTTTACGTGATGTACGACGGTTTCAGCATGCAAAGACTCGTCAAGTGCAACGCTTGGCCTTGCCCCAATACCGTGGATTGCTTCGTGTCCCGCCCGACCGAGAAAACTGTGTTCACTGTGTTCATGATCGCCGTGTCCGGCATCTGCATCCTGCTGAACGTGACCGAGCTGTGCTATCTCCTGATCCGGTACTGTAGCGGAAAGTCGAAGAAGCCTGTG Exemplary codon-optimized human GJB2 DNA sequence (SEQ ID NO: 125). ATGGATTGGGGGACGCTCCAGACTATACTTGGCGGGTAAACAAACATTCCACCTCAATTGGCAAAATCTGGCTCACAGTCCTCTTCATCTTCAGAATAATGATACTCGTGTTGCCGCTAAAGAAGTTTGGGGTGACGAGCAAGCCGATTTCGTCTGTAACACCCTCC AACCAGGTTGCAAAAATGTCTGTTACGATCACTACTTTCCTATTAGCCATATTAGACTCTGGGCCCTGCAACTTATCTTCGTTTCCACTCCTGCTCTGCTCGTCGCTATGCACGTTGCCTATCGCCGCCATGAAAAAAAACGGAAATTCATTAAGGGAGAGATTAAGAGT GAATTCAAGGATATTGAAGAGATTAAAACGCAAAAAGTTAGAATTGAGGGATCACTGTGGTGGACTTATACCAGTAGCATCTTTTTTAGGGTCATTTTCGAAGCTGCTTTCATGTATGTTTTCTATGTAATGTACGACGGTTTCTCCATGCAACGCTTGGTTAAATGTA ACGCCTGGCCATGCCCTAATACGGTTGATTGCTTTGTCTCCCGCCCTACTGAAAAGACAGTGTTTACCGTTTTCATGATCGCCGTAAGTGGAATTTGTATCCTTCTTAACGTGACCGAGTTGTGCTATCTCCTTATTCGCTACTGTTCAGGAAAAAGTAAAAAACCAGTA An exemplary codon-optimized human GJB2 DNA sequence (SEQ ID NO: 126). ATGGACTGGGGCACGCTGCAGACTATCCTGGGGGGTGTCAACAAGCATTCAACTAGCATCGGAAAGATCTGGCTGACCGTCCTGTTCATCTTTCGCATCATGATCCTCGTGGTGGCCGCTAAGGAAGTGTGGGGCGACGAGCAGGCCGATTTCGTGTGTAACACCCTGCAGCCAGGTTGCAAAAACGTCTGCTACGATCACTACTTTCCCATCTCCCACATTAGACTGTGGGCCCTGCAGCTGATCTTCGTGTCCACCCCTGCGCTGCTAGTGGCCATGCACGTGGCCTATCGGCGACACGAGAAGAAACGGAAGTTCATTAAGGGCGAGATCAAGAGCGAGTTCAAGGATATCGAAGAGATCAAGACCCAGAAGGTCCGCATTGAGGGCTCCCTGTGGTGGACCTACACCAGCTCCATCTTCTTTCGGGTCATCTTCGAGGCCGCCTTTATGTACGTGTTCTATGTGATGTACGACGGTTTCTCCATGCAACGGCTGGTGAAGTGCAACGCCTGGCCTTGCCCTAATACTGTGGATTGCTTCGTGTCCCGCCCCACCGAGAAGACAGTGTTCACCGTGTTCATGATCGCCGTGTCTGGCATCTGCATCCTGCTGAACGTGACCGAGCTGTGCTATCTCCTGATCCGGTACTGTAGTGGAAAGTCAAAAAAACCAGTGTAA

[0231] The present disclosure recognizes that certain changes to a polynucleotide sequence do not affect its expression or the protein encoded by the polynucleotide. In some embodiments, a polynucleotide comprises a GJB2 gene having one or more silent mutations. In some embodiments, the present disclosure provides polynucleotides comprising a GJB2 gene having one or more silent mutations, e.g., a GJB2 gene having a sequence different from SEQ ID NOs: 117-126, but encoding the same amino acid sequence as a functional GJB2 gene. In some embodiments, the present disclosure provides polynucleotides comprising a GJB2 gene having a sequence different from SEQ ID NOs: 117-126, encoding an amino acid sequence containing one or more mutations (e.g., a different amino acid sequence when compared to the amino acid sequence produced from a functional GJB2 gene), wherein the one or more mutations are conservative amino acid substitutions.

[0232] In some aspects, the present disclosure provides polynucleotides comprising a GJB2 gene having a sequence different from SEQ ID NOs: 117-126 that encodes an amino acid sequence containing one or more mutations (e.g., a different amino acid sequence when compared to the amino acid sequence produced from a functional GJB2 gene), wherein the one or more mutations are not within a distinctive portion of the GJB2 gene or the encoded connexin 26 protein. In some aspects, polynucleotides according to the present disclosure comprise a GJB2 gene that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NOs: 117-126. In some aspects, polynucleotides according to the present disclosure comprise a GJB2 gene that is identical to the sequence of SEQ ID NOs: 117-126. As can be understood in the art, SEQ ID NOs: 117-126 can be optimized (e.g., codon optimized) to achieve increased or optimized expression in an animal, e.g., a mammal, e.g., a human.

[0233] In particular, the present disclosure provides polypeptides encoded by the GJB2 gene or characteristic portions thereof. In some embodiments, the GJB2 gene is a mammalian GJB2 gene. In some embodiments, the GJB2 gene is a mouse GJB2 gene. In some embodiments, the GJB2 gene is a primate GJB2 gene. In some embodiments, the GJB2 gene is a human GJB2 gene.

[0234] In some embodiments, the polypeptide comprises a connexin 26 protein or a characteristic portion thereof. In some embodiments, the connexin 26 protein or a characteristic portion thereof is a mammalian connexin 26 protein or a characteristic portion thereof, such as a primate connexin 26 protein or a characteristic portion thereof. In some embodiments, the connexin 26 protein or a characteristic portion thereof is a human connexin 26 protein or a characteristic portion thereof.

[0235] In some embodiments, the polypeptides provided herein comprise a post-translational modification. In some embodiments, the connexin 26 proteins or characteristic portions thereof provided herein comprise a post-translational modification. In some embodiments, the post-translational modification can include, but is not limited to, glycosylation (e.g., N-linked glycosylation, O-linked glycosylation), phosphorylation, acetylation, amidation, hydroxylation, methylation, ubiquitination, sulfation, and / or combinations thereof. An exemplary human connexin 26 protein sequence is or comprises the sequence of SEQ ID NO: 127. Exemplary Human Connexin 26 Protein Sequence (SEQ ID NO: 127) MDWGTLQTILGGVNKHSTSIGKIWLTVLFIFRIMILVVAAKEVWGDEQADFVCNTLQPGCKNVCYDHYFPISHIRLWALQLIFVSTPALLVAMHVAYRRHEKKRKFIKGEIKS EFKDIEEIKTQKVRIEEGSLWWTYTSSIFFRVIFEAAFMYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKTVFTVFMIAVSGICILLNVTELCYLLIRYCSGKSKKPV

[0236] The present disclosure recognizes that certain mutations in the amino acid sequence of a polypeptide described herein (e.g., comprising connexin 26 or a characteristic portion thereof) do not affect the expression, folding, or activity of the polypeptide. In some embodiments, a polypeptide (e.g., comprising connexin 26 or a characteristic portion thereof) comprises one or more mutations, wherein the one or more mutations are conservative amino acid substitutions. In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 or a characteristic portion thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 127. In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 or a characteristic portion thereof that is identical to the sequence of SEQ ID NO: 127. In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 or a characteristic portion thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 127. In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 protein or a characteristic portion thereof that is identical to the sequence of SEQ ID NO: 127.

[0237] In some embodiments, the polypeptide is a therapeutic polypeptide (e.g., a connexin 26 polypeptide). In some embodiments, the polypeptide is a support cell polypeptide (e.g., a connexin 26 polypeptide). In some embodiments, the polypeptide is a reporter polypeptide.

[0238] Feeder Cell Polypeptides Certain aspects of the present disclosure relate to polynucleotides encoding supporting cell polypeptides (e.g., connexin 26 polypeptides). The polynucleotides can encode polypeptides that can be expressed in cells (e.g., inner ear cells). In some aspects, the supporting cell polypeptide (e.g., connexin 26 polypeptide) is a polypeptide that is endogenously expressed in supporting cells of the inner ear. In some embodiments, the inner ear supporting cells are selected from one or more of medial phalangeal / border cells (IPhCs), inner pillar cells (IPCs), outer pillar cells (OPCs), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSCs), interdental cells (Idc), inner sulcus cells (ISCs), organ of corikers cells (KOs), greater ridge epithelial cells (GERs) (including lateral greater ridge epithelial cells (LGERs)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall. The polynucleotide can encode a full-length polypeptide or a functional fragment thereof.

[0239] Exemplary feeder cell polypeptides encoded by the polynucleotides include, but are not limited to, transmembrane proteins, enzymes, growth factors, cytokines, receptors, receptor ligands, hormones, membrane proteins, membrane-associated proteins, antigens, and antibodies.

[0240] Exemplary feeder cell polynucleotides encoding polypeptides include ATPase plasma membrane Ca2+ transporter 2 (ATP2B2), cholinergic receptor nicotinic alpha 9 subunit (CHRNA9), cadherin 23 (CDH23), coiled-coil glutamic acid-rich protein 2 (CCER2), cularin 1 (CLRN1), cularin 2 (CLRN2), cochlin (COCH or DFNA9), dystrotelin (DYTN), epidermal growth factor receptor pathway substrate 8 (EPS8), EPS8-like 2 (EPS8L2), espin (ESPN), espin-like (ESPNL), gap junction protein beta 2 (GJB2), gap junction protein beta 6 (GJB6), gap junction protein beta Examples of suitable polynucleotides include, but are not limited to, gap junction protein beta 2 (GJB3), gasdermin E protein (GSDME or DFNA5), insulinoma-associated 1 (INSM1), Ikaros family zinc finger 2 (IKZF2), LIM homeobox protein 3 (LHX3), myosin 7A (MYO7A), myosin 11 (MYO3A), normal cystine-knot growth factor receptor (NDP), protocadherin 15 (PCDH15), protein tyrosine phosphatase, receptor type Q (PTPRQ), stereocillin (STRC), protein network component harmonin (USH1C), ashurin (USH2A), and spectrin repeat-containing nuclear envelope family member 4 (SYNE4). In some embodiments, the polynucleotide comprises a gap junction protein beta 2 (GJB2) gene. In some embodiments, the polynucleotide encodes a gap junction protein beta 2 polypeptide. In some embodiments, the polynucleotide encodes a connexin 26 polypeptide. In some embodiments, the support cell polypeptide is a gap junction protein beta 2 polypeptide. In some embodiments, the support cell polypeptide is a connexin 26 polypeptide.

[0241] In some embodiments, a polynucleotide according to the present disclosure comprises a GJB2 gene that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NOs: 117-126. In some embodiments, a polynucleotide according to the present disclosure comprises a GJB2 gene that is identical to the sequence of SEQ ID NOs: 117-126. As can be understood in the art, SEQ ID NOs: 117-126 can be optimized (e.g., codon optimized) to achieve increased or optimized expression in an animal, e.g., a mammal, e.g., a human.

[0242] In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 or a characteristic portion thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 127. In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 or a characteristic portion thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 127. In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 protein identical to the sequence of SEQ ID NO: 127 or a characteristic portion thereof.

[0243] Therapeutic Polypeptides

[0244] Certain aspects of the present disclosure relate to polynucleotides that encode a polypeptide (e.g., a therapeutic polypeptide, a connexin 26 polypeptide). The polynucleotide can encode a polypeptide that can be expressed in a cell (e.g., an inner ear cell). The polynucleotide can encode a full-length polypeptide or a functional fragment thereof.

[0245] Exemplary polypeptides encoded by the polynucleotides include, but are not limited to, transmembrane proteins, enzymes, growth factors, cytokines, receptors, receptor ligands, hormones, membrane proteins, membrane-associated proteins, antigens, and antibodies.

[0246] Exemplary polynucleotides encoding therapeutic polypeptides (e.g., connexin 26 polypeptides) include ATPase plasma membrane Ca2+ transporter 2 (ATP2B2), cholinergic receptor nicotinic alpha 9 subunit (CHRNA9), cadherin 23 (CDH23), coiled-coil glutamic acid-rich protein 2 (CCER2), cularin 1 (CLRN1), cularin 2 (CLRN2), cochlin (COCH or DFNA9), dystrotelin (DYTN), epidermal growth factor receptor pathway substrate 8 (EPS8), EPS8-like 2 (EPS8L2), espin (ESPN), espin-like (ESPNL), gap junction protein beta 2 (GJB2), gap junction protein beta 6 (GJB6), gap junction protein beta 6 (GJB7), gap junction protein beta 6 (GJB8), gap junction protein beta 6 (GJB9), gap junction protein beta 6 (GJB9), gap junction protein beta 6 (GJB10), gap junction protein beta 6 (GJB11), gap junction protein beta 6 (GJB12), gap junction protein beta 6 (GJB13), gap junction protein beta 6 (GJB14), gap junction protein beta 6 (GJB15), gap junction protein beta 6 (GJB16), gap junction protein beta 6 (GJB17), gap junction protein beta 6 (GJB18), gap junction protein beta 6 (GJB19 ...1 Examples of suitable polynucleotides include, but are not limited to, fusion protein beta 3 (GJB3), gasdermin E protein (GSDME or DFNA5), insulinoma-related 1 (INSM1), Ikaros family zinc finger 2 (IKZF2), LIM homeobox protein 3 (LHX3), myosin 7A (MYO7A), myosin 11 (MYO3A), normal cystine-knot growth factor receptor (NDP), protocadherin 15 (PCDH15), protein tyrosine phosphatase, receptor type Q (PTPRQ), stereocillin (STRC), protein network component harmonin (USH1C), ashurin (USH2A), and spectrin repeat-containing nuclear envelope family member 4 (SYNE4). In some embodiments, the polynucleotide comprises a gap junction protein beta 2 (GJB2) gene. In some embodiments, the polynucleotide encodes a gap junction protein beta 2 polypeptide. In some embodiments, the polynucleotide encodes a connexin 26 polypeptide. In some embodiments, the therapeutic polypeptide is a gap junction protein beta 2 polypeptide. In some embodiments, the therapeutic polypeptide is a connexin 26 polypeptide.

[0247] In some embodiments, a polynucleotide according to the present disclosure comprises a GJB2 gene that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NOs: 117-126. In some embodiments, a polynucleotide according to the present disclosure comprises a GJB2 gene that is identical to the sequence of SEQ ID NOs: 117-126. As can be understood in the art, SEQ ID NOs: 117-126 can be optimized (e.g., codon optimized) to achieve increased or optimized expression in an animal, e.g., a mammal, e.g., a human.

[0248] In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 or a characteristic portion thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 127. In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 or a characteristic portion thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 127. In some embodiments, a polypeptide according to the present disclosure comprises a connexin 26 protein identical to the sequence of SEQ ID NO: 127 or a characteristic portion thereof.

[0249] construct Among other things, the present disclosure provides that some polynucleotides described herein are polynucleotide constructs. Polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno-associated viral constructs) that incorporate a polynucleotide containing a nucleic acid sequence (e.g., GJB2 gene) encoding a polypeptide (e.g., connexin 26) or a characteristic portion thereof. One of skill in the art would be able to select appropriate constructs and cells for producing any of the polynucleotides described herein. In some embodiments, the construct is a plasmid (i.e., a circular DNA molecule capable of autonomous replication within a cell). In some embodiments, the construct can be a cosmid (e.g., the pWE or sCos series). In some embodiments, the construct is a mammalian or viral vector.

[0250] In some embodiments, the construct is a viral construct. In some embodiments, the viral construct is a lentiviral, retroviral, adenoviral, or adeno-associated viral construct. In some embodiments, the construct is an adeno-associated viral (AAV) construct (see, e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, incorporated herein by reference in its entirety). In some embodiments, the construct is a viral vector. In some embodiments, the construct is a lentiviral, retroviral, adenoviral, or adeno-associated viral vector. In some embodiments, the construct is an AAV vector. In some embodiments, the viral construct is an adenoviral construct. In some embodiments, the viral construct may also be based on or derived from an alphavirus.Alphaviruses include Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, and others. These include Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, salmon pancreatic disease virus, Semliki Forest virus, southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus. Generally, the genomes of such viruses encode nonstructural proteins (e.g., replicons) and structural proteins (e.g., capsid and envelope) that can be translated in the cytoplasm of host cells. Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop viral constructs for delivery of coding sequences. Pseudotyped viruses may be formed by combining alphavirus envelope glycoproteins with retroviral capsids.Examples of alphavirus constructs can be found in U.S. Patent Application Publication Nos. 20150050243, 20090305344, and 20060177819; the constructs and methods for their production are incorporated herein by reference in their entirety.

[0251] The constructs provided herein can vary in size, hi some embodiments, the constructs are plasmids and can comprise an overall length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb. In some embodiments, the construct is a plasmid and can have a total length in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.

[0252] In some embodiments, the construct is a viral construct and can have a total number of nucleotides of up to 10 kb. In some embodiments, the viral construct may be from about 1 kb to about 2 kb, 1 kb to about 3 kb, from about 1 kb to about 4 kb, from about 1 kb to about 5 kb, from about 1 kb to about 6 kb, from about 1 kb to about 7 kb, from about 1 kb to about 8 kb, from about 1 kb to about 9 kb, from about 1 kb to about 10 kb, from about 2 kb to about 3 kb, from about 2 kb to about 4 kb, from about 2 kb to about 5 kb, from about 2 kb to about 6 kb, from about 2 kb to about 7 kb, from about 2 kb to about 8 kb, from about 2 kb to about 9 kb, from about 2 kb to about 10 kb, from about 3 kb to about 4 kb, from about 3 kb to about 5 kb, from about 3 kb to about 6 kb, from about 3 kb to about 7 kb, from about 3 kb to about 8 kb, from about 3 kb to about 9 kb, The total number of nucleotides may be within the range of 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or about 9 kb to about 10 kb.

[0253] In some embodiments, the construct is a lentiviral construct and can have a total number of nucleotides of up to 8 kb. In some examples, the lentiviral construct can be about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, It may have a total number of nucleotides of about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb.

[0254] In some embodiments, the construct is an adeno-associated virus construct and can have a total number of nucleotides of up to 8 kb. In some embodiments, the adeno-associated virus construct can be from about 1 kb to about 2 kb, from about 1 kb to about 3 kb, from about 1 kb to about 4 kb, from about 1 kb to about 5 kb, from about 1 kb to about 6 kb, from about 1 kb to about 7 kb, from about 1 kb to about 8 kb, from about 2 kb to about 3 kb, from about 2 kb to about 4 kb, from about 2 kb to about 5 kb, from about 2 kb to about 6 kb, from about 2 kb to about 7 kb, from about 2 kb to about 8 kb, from about 3 kb to about 4 kb, The total number of nucleotides may be within the range of 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0255] In some embodiments, the construct is an adenoviral construct and can have a total number of nucleotides of up to 8 kb. In some embodiments, the adenoviral construct can be from about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 ...5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb The total number of nucleotides may be within the range of about 5 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0256] Any of the constructs described herein can further comprise regulatory sequences (e.g., regulatory sequences selected from the group of transcription initiation sequences, transcription termination sequences, promoter sequences, enhancer sequences, RNA splicing sequences, polyadenylation (poly(A)) sequences, Kozak consensus sequences, and / or additional untranslated regions that may harbor pre- or post-transcriptional regulatory and / or control elements). In some aspects, the promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of regulatory sequences are described herein.

[0257] In some embodiments, the construct comprises a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter that selectively expresses the polynucleotide in inner ear supporting cells. In some embodiments, the construct comprises a 5' ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5' UTR, a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide), a 3' UTR, a polyA, and a 3' ITR. In some embodiments, the construct comprises a 5' ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5' UTR, a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide), a tag, a 3' UTR, a polyA, and a 3' ITR. In some embodiments, the construct comprises a 5' ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5' UTR, a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide), a tag, a 3' UTR, a microRNA regulatory target site, a polyA, and a 3' ITR.

[0258] In some embodiments, the construct comprises a polynucleotide encoding a polypeptide operably linked to a promoter that selectively expresses the polynucleotide in inner ear supporting cells. In some embodiments, the construct comprises a 5'ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5'UTR, a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide), a 3'UTR, a polyA, and the 3'ITR. In some embodiments, the construct comprises a 5'ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5'UTR, a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide), a tag, a 3'UTR, a polyA, and the 3'ITR. In some embodiments, the construct comprises a 5'ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5'UTR, a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide), a tag, a 3'UTR, a microRNA regulatory target site, a polyA, and the 3'ITR.

[0259] In some embodiments, the construct comprises a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and the construct comprises a miRNA regulatory target site for a microRNA expressed in inner ear cells (e.g., hair cells). In some embodiments, the construct comprises a 5' ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5' UTR, a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide), a 3' UTR, a microRNA regulatory target site, a polyA, and the 3' ITR. In some embodiments, the construct comprises a 5' ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5' UTR, a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide), a tag, a 3' UTR, a microRNA regulatory target site, a polyA, and the 3' ITR. In some embodiments, the construct comprises a 5'ITR, a constitutive promoter, a 5'UTR, a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide), a 3'UTR, a microRNA regulatory target site, a polyA, and the 3'ITR. In some embodiments, the construct comprises a 5'ITR, a constitutive promoter, a 5'UTR, a polynucleotide encoding a therapeutic polypeptide (e.g., a connexin 26 polypeptide), a tag, a 3'UTR, a microRNA regulatory target site, a polyA, and the 3'ITR.

[0260] In some embodiments, the construct comprises a polynucleotide encoding a polypeptide operably linked to a promoter, wherein the construct comprises a miRNA regulatory target site for a microRNA expressed in inner ear cells (e.g., hair cells). In some embodiments, the construct comprises a 5'ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5'UTR, a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide), a 3'UTR, a microRNA regulatory target site, a polyA, and the 3'ITR. In some embodiments, the construct comprises a 5'ITR, a promoter that selectively expresses the polynucleotide in inner ear supporting cells, a 5'UTR, a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide), a tag, a 3'UTR, a microRNA regulatory target site, a polyA, and the 3'ITR. In some embodiments, the construct comprises a 5'ITR, a constitutive promoter, a 5'UTR, a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide), a 3'UTR, a microRNA regulatory target site, a polyA, and the 3'ITR. In some embodiments, the construct comprises a 5'ITR, a constitutive promoter, a 5'UTR, a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide), a tag, a 3'UTR, a microRNA regulatory target site, polyA, and a 3'ITR.

[0261] In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter that expresses the polynucleotide in an inner ear supporting cell, and (iii) a 3' ITR, wherein the promoter is heterologous to the polynucleotide.

[0262] In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 16, 28, 40, 57, or 90-99.

[0263] In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 40. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 90. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 96. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 99.

[0264] In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 16. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 28. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 57. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 91.In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 92. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 93. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 94. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 95.In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 97. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 98.

[0265] In some embodiments, the construct further comprises a minimal GJB2 promoter. In some embodiments, the minimal GJB2 promoter comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:86.

[0266] In some aspects, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a minimal GJB2 promoter comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 40, and a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 86. In some embodiments, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a minimal GJB2 promoter comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 90, and a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 86.In some aspects, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 96, and a minimal GJB2 promoter comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 86. In some aspects, the construct comprises (i) a 5' inverted terminal repeat (ITR), (ii) a polynucleotide encoding a polypeptide (e.g., a connexin 26 polypeptide) operably linked to a promoter, and (iii) a 3' ITR, wherein the promoter comprises a minimal GJB2 promoter comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, o...

Claims

1. A polynucleotide comprising a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 40, 90, 96 or 99.

2. A construct comprising the polynucleotide of claim 1 and a nucleic acid sequence comprising a coding sequence for a connexin 26 polypeptide or a functional fragment thereof.

3. The polynucleotide of claim 1 or the construct of claim 2, wherein the polynucleotide is a promoter and is operably linked to a coding sequence.

4. 4. The polynucleotide or construct of claim 3, wherein the polynucleotide is capable of directing transcription of the coding sequence in inner ear supporting cells.

5. 4. The polynucleotide or construct of claim 3, wherein the promoter comprises a nucleic acid sequence having at least 95% identity to a sequence selected from one or more of SEQ ID NOs: 40, 90, 96 or 99.

6. 5. The polynucleotide or construct of claim 4, wherein the inner ear supporting cells are selected from one or more of medial phalangeal / border cells (IPhC), inner pillar cells (IPC), outer pillar cells (OPC), Deiters cell rows 1 and 2 (DC1 / 2), Deiters cell row 3 (DC3), Hensen cells (Hec), Claudius cells / outer sulcus cells (CC / OSC), interdental cells (Idc), inner sulcus cells (ISC), organ of corikers cells (KO), greater ridge epithelial ridge cells (GER) (including greater ridge epithelial ridge cells (LGER)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall.

7. The construct of claim 2, further comprising a minimal GJB2 promoter operably linked to the coding sequence of the connexin 26 polypeptide or a functional fragment thereof.

8. 3. The polynucleotide of claim 1 or the construct of claim 2, comprising a GJB2 nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 117-126.

9. 1. A viral vector construct comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a coding sequence for a connexin 26 polypeptide or a functional fragment thereof, operably linked to a promoter capable of directing transcription of the coding sequence in an inner ear supporting cell; and (iii) a 3' ITR, wherein the promoter is heterologous to the coding sequence, and the promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 40, 90, 96 or 99.

10. 10. The polynucleotide of claim 1, the construct of claim 2, or the viral vector construct of claim 9, further comprising a 5' untranslated region (UTR) and a 3' UTR.

11. 11. The polynucleotide, construct, or viral vector construct of claim 10, wherein the 5'UTR comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to any one of SEQ ID NOs: 20, 21 or 66, and the 3'UTR comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to any one of SEQ ID NOs: 22, 67, 68, or 69.

12. The polynucleotide, construct, or viral vector construct of claim 11, further comprising a polyA tail.

13. 10. The polynucleotide, construct, or viral vector construct of claim 9, wherein the 5' ITR and the 3' ITR are AAV ITRs from a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV Anc80 ITRs.

14. a. the 5' ITR comprises the nucleic acid sequence of SEQ ID NO:8 and the 3' ITR comprises the nucleic acid sequence of SEQ ID NO:9; or b. The polynucleotide, construct, or viral vector construct of claim 9 or 13, wherein the 5' ITR comprises the nucleic acid sequence of SEQ ID NO:52 and the 3' ITR comprises the nucleic acid sequence of SEQ ID NO:

53.

15. 10. A viral vector comprising the polynucleotide of claim 1, the construct of claim 2, or the viral vector construct of claim 9, optionally wherein the viral vector is selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, or a lentivirus vector.

16. 10. An AAV particle comprising the polynucleotide of claim 1, the construct of claim 2, or the viral vector construct of claim 9.

17. 17. The AAV particle of claim 16, comprising an AAV capsid, wherein the AAV capsid is or is derived from an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-rh8, AAV-rhlO, AAV-rh39, AAV-rh43, or AAV Anc80 serotype capsid.

18. The AAV particle of claim 17, wherein the AAV capsid is an AAV Anc80 capsid.

19. A viral vector construct comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a polynucleotide encoding a polypeptide operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter capable of driving transcription of the polynucleotide in an inner ear supporting cell; and (iii) a 3' ITR, wherein the inner ear supporting cell-selective promoter is heterologous to the polynucleotide.

20. 20. The viral vector construct of claim 19, comprising: (i) a 5' inverted terminal repeat (ITR); (ii) a polynucleotide encoding a polypeptide operably linked to an inner ear supporting cell-selective promoter and a minimal GJB2 promoter; and (iii) a 3' ITR, wherein the inner ear supporting cell-selective promoter comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 40, 90, 96 or 99.

21. 20. The construct of claim 2, or the viral vector construct of claim 9 or 19, further comprising a miRNA regulatory target site (miRTS) for a microRNA expressed in an inner ear cell, said microRNA being one or more of miR-194, miR-140, miR-18a, miR-99a, miR-30b, miR-15a, miR182, or miR-183.

22. A composition comprising the polynucleotide of claim 1, the construct of claim 2, or the viral vector construct of claim 9 or 19, wherein the composition is a pharmaceutical composition further comprising a pharma- ceutical acceptable carrier.

23. 23. The composition of claim 22, wherein the pharmaceutical composition is a synthetic perilymphatic solution.

24. 20. An ex vivo cell comprising a polynucleotide according to claim 1, a construct according to claim 2, or a viral vector construct according to claim 9 or 19.

25. 25. The ex vivo cell of claim 24, wherein the ex vivo cell is an inner ear supporting cell.

26. Ex vivo cells a. a polynucleotide according to claim 1, a construct according to claim 2, a viral vector construct according to claim 9 or 19; and b. A method comprising transducing with one or more helper plasmids collectively comprising an AAV Rep gene, an AAV Cap gene, an AAV VA gene, an AAV E2a gene, and an AAV E4 gene.

27. A polynucleotide as described in claim 1, a construct as described in claim 2, or a viral vector construct as described in claim 9 or 19 for use in increasing expression of the polypeptide in inner ear supporting cells.

28. 20. A polynucleotide according to claim 1, a construct according to claim 2, a viral vector construct according to claim 9 or 19 for use in the treatment of hearing loss in a subject suffering from or at risk of hearing loss.

29. 20. A kit comprising a polynucleotide according to claim 1, a construct according to claim 2, or a viral vector construct according to claim 9 or 19.

30. 30. The kit of claim 29, further comprising a device, said device being a device as depicted in any one of Figures 5-8.

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