Gene Therapy Delivery Compositions and Methods for Treating Hearing Loss

Abstract

The present disclosure provides constructs that include a coding sequence operably linked to a promoter that expresses a polynucleotide in an outer hair cell, the coding sequence encoding a polypeptide (e.g., a heterologous polypeptide). Exemplary constructs include AAV constructs. Also provided are methods of using the disclosed constructs for the treatment of hearing loss and / or hearing loss.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 251,017, filed September 30, 2021, the entire contents of which are incorporated herein by reference.

[0002] Reference to an Electronically Submitted Sequence Listing The contents of the electronically submitted sequence listing (Name: 4833_013PC01_SeqListing_ST26.xml, Size: 164,451 bytes, and Creation Date: September 29, 2022) are incorporated by reference in their entirety into this specification.

[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 structures in the inner ear (sensorineural hearing loss), but some forms may involve changes in the middle ear (conductive hearing loss). The majority of sensorineural hearing loss in humans is caused by abnormalities in 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 life (e.g., as a result of noise trauma or infection).

[0004] Treatments for hearing loss currently include hearing amplification for mild to severe loss and cochlear implants for severe to profound loss (Kral and O'Donoghue, 2010, N. Engl. J. Med. 363:1438-1450). There is a need for improved treatment options for nonsyndromic hearing loss and other forms of hearing loss. Summary of the Invention

[0005] Certain aspects of the present disclosure relate to constructs comprising a polynucleotide encoding a polypeptide operably linked to a promoter that causes expression of the polynucleotide in an outer hair cell, the promoter being selected from one or more of an oncomodulin (OCM) promoter, a prestin promoter, a cholinergic receptor nicotinic alpha 10 (CHRNA10) promoter, a dynamin 3 (DNM3) promoter, a mucin 14 (MUC15) promoter, a phospholipase D (PLDB1) promoter, a RAR-related orphan receptor B (RORB) promoter, a striatin interacting protein 2 (STRIP2) promoter, an aquaporin 11 (AQP11) promoter, a voltage-gated potassium channel subfamily Q member 4 (KCNQ4) promoter, a LBH promoter, a stereocillin (STRC) promoter, a tubulin alpha 8 (TUBA8) promoter, or a combination thereof, an oncomodulin (OCM) promoter.

[0006] Certain aspects of the present disclosure relate to a construct comprising a polynucleotide encoding a polypeptide operably linked to a promoter that causes expression of the polynucleotide in an outer hair cell, the promoter being heterologous to the polynucleotide.

[0007] 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 any one of SEQ ID NOs:1-15.

[0008] Certain aspects of the present disclosure relate to constructs comprising a polynucleotide encoding a polypeptide operably linked to a promoter that causes expression of the polynucleotide in an outer hair cell, the 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:1-15.

[0009] In some embodiments, the prestin 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:3 or SEQ ID NO:15.

[0010] In some embodiments, the oncomodulin (OCM) 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:1 or SEQ ID NO:2.

[0011] In some aspects, the promoter is heterologous to the polynucleotide.

[0012] In some aspects, the polypeptide is an outer hair cell polypeptide, a therapeutic polypeptide, or a reporter polypeptide.

[0013] In some aspects, the polynucleotide encoding the outer hair cell polypeptide is selected from the group consisting of actin gamma 1 (ACTG1), adenylate cyclase type 1 (ADCY1), calcium binding protein 2 (CABP2), coiled-coil domain containing 50 (CCDC50), cadherin-associated 23 (CDH23), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), chromodomain helicase DNA binding protein 7 (CHD7), calcium and integrin binding family member 2 (CIB2), clonidine kinase 1 (CLK1), clonidine kinase 2 (CLK2), clonidine kinase 1 (CLK3), clonidine kinase 2 (CLK4), clonidine kinase 1 (CLK5), clonidine kinase 2 (CLK6), clonidine kinase 1 (CLK7), clonidine kinase 2 (CLK8), clonidine kinase 1 (CLK9), clonidine kinase 1 (CLK10), clonidine kinase 1 (CLK11), clonidine kinase 1 (CLK12), clonidine kinase 1 (CLK13), clonidine kinase 1 (CLK14), clonidine kinase 1 (CLK25), clonidine kinase 1 (CLK15), clonidine kinase 1 (CLK16), clonidine kinase 1 (CLK16), clonidine kinase 1 (CLK17), clonidine kinase 1 (CLK26), clonidine kinase 1 (CLK18), clonidine kinase 1 (CLK19), clonidine kinase 1 (CLK27), clonidine kinase 1 (CLK28), clonidine kinase 1 (CLK30), clonidine kinase 1 (CLK31), clonidine kinase 1 (CLK41), clonidine kinase 1 (CLK52), clonidine kinase 1 (CLK19), clonidine kinase 1 (CLK19), clonidine kinase 1 (CLK25), clonidine kinase din14 (CLDN14), intracellular chloride channel 5 (CLIC5), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), clarrin1 (CLRN1), pejvakin (DFNB59), endothelin 3 (EDN3), ELMO domain-containing protein 3 (ELMOD3), epidermal growth factor receptor kinase substrate 8 (EPS8), espin (ESPN), estrogen-related receptor beta (ESRRB), eyes absent (eyes absent) homolog 1 (EYA1), GIPC PDZ domain-containing family, member 3 (GIPC3), G protein-coupled receptor 98 (GPR98), G protein signaling modulator 2 (GPSM2), glutaredoxin, cysteine-rich 1 (GRXCR1), glutaredoxin, cysteine-rich 2 (GRXCR2), immunoglobulin-like domain-containing receptor 1 (ILDR1), lysyl-tRNA synthetase (KARS), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), lipoma HMGIC fusion partner-like 5 (LHFPL5), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT1 COMT2), tricellulin (MARVELD2), microRNA96 (MIR96), methionine sulfoxide reductase B3 (MSRB3), nonmuscle myosin heavy chain 9 (MYH9), nonmuscle myosin heavy chain 14 (MYH14), atypical myosin IIIA (MYO3A), atypical myosin VI (MYO6), atypical myosin VIIA (MYO7A), atypical myosin XVA (MYO15A), otoferlin (OTOF), otogelin-like protein (OTOGL), purinergic receptor (recetpro) P2X, ligand-gated ion channel, 2 (P2RX2), protocadherin 15 (PCDH15), PDZ domain-containing 7 (PDZD7), polyribonucleotide nucleotidyltransferase (P2RX2) transferase 1, mitochondrial (PNPT1), POU domain, class 4, transcription factor 3 (POU4F3), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), protein tyrosine phosphatase, receptor type Q (PTPRQ), radixin (RDX), scaffold-containing ankyrin repeats and SAM domain (SANS), serpin peptidase inhibitor, clade B, member 6 (SERPINB6), SIX homeobox 1 (SIX1), SIX homeobox 5 (SIX5), prestin (SLC26A5), second mitochondrial-derived caspase activator (SMAC / DIABLO), small muscle (small muscle protein, x-linked (SMPX), stereocillin (STRC), nesprin-4 (SYNE4), TBC1 domain family, member 24 (TBC / D24), tight junction protein XO 2 (TJP2), transmembrane channel-like protein 1 (TMC1), transmembrane inner ear expressed protein (TMIE), transmembrane protease, serine 3 (TMPRSS3), taperin (TPRN), TRIO and F-actin binding protein (TRIOBP), thrombospondin-type laminin G domain and EAR repeats (TSPEAR), harmonin (USH1C), usherin (USH2A), wolframin (WFS1), and whirlin (WHRN), or any combination thereof.

[0014] Certain aspects of the present disclosure relate to methods of using the constructs, vectors, viral particles (e.g., AAV), cells, compositions, and pharmaceutical compositions disclosed herein for expressing polypeptides in outer hair cells.

[0015] Certain aspects of the present disclosure relate to methods of using the constructs, vectors, viral particles (e.g., AAV), cells, compositions, and pharmaceutical compositions disclosed herein to increase expression of a polypeptide in an outer hair cell, hi some aspects, the increased expression is relative to endogenous polypeptide expression in the outer hair cell.

[0016] Certain aspects of the present disclosure relate to methods of using the constructs, vectors, viral particles (e.g., AAV), cells, compositions, and pharmaceutical compositions disclosed herein to treat hearing loss. [Brief description of the drawings]

[0017] [Figure 1A] Illustrates in vitro expression of KCNQ4 protein from constructs containing outer hair cell promoters. Shown are KCNQ4-FLAG protein levels ("KCNQ4-FLAG") in HEK293 cells transfected with 500 ng of exemplary plasmids containing constructs driven by prestin, oncomodulin, CMV, or CAG promoters (red band, white box). GAPDH is shown as a loading control (green). [Figure 1B] Illustrates in vitro expression of KCNQ4 protein from constructs containing outer hair cell promoters. KCNQ4-FLAG protein levels are shown in HEK293 cells transfected with 400 ng of exemplary plasmids containing constructs driven by the DNM3, STRIP2, MUC15, LBD1, RORB, CHRNA10, prestin, oncomodulin, or CMV promoters (red band, white box). GAPDH is shown as a loading control (green). [Figure 1C]Illustrates in vitro expression of KCNQ4 protein from constructs containing outer hair cell promoters. KCNQ4-FLAG protein levels are shown in HEK293 cells transfected with 400 ng of exemplary plasmids containing constructs driven by the AQP11, KCNQ4, LBH, TUBA8, STRC, prestin, oncomodulin, or CMV promoters (red bands, white boxes). GAPDH is shown as a loading control (green). [Diagram 2] 1 illustrates a perspective view of a device for delivering fluid to the inner ear, according to an aspect of the present disclosure. [Diagram 3] 1 illustrates a side view of a curved needle subassembly according to an aspect of the present disclosure. [Figure 4] 1 illustrates a perspective view of a device for delivering fluid to the inner ear, according to an aspect of the present disclosure. [Diagram 5] 1 illustrates a perspective view of a curved needle subassembly coupled to a distal end of a device, according to an aspect of the present disclosure. [Figure 6] 1A-C illustrate in vivo expression of a rAAV construct encoding the KCNQ4 protein under the control of the prestin promoter. A shows phalloidin staining of F-actin in the cochlea 28 days after administration of rAAV particles containing the construct of SEQ ID NO:26 into the inner ear of a 2-day-old Kcnq4dn / +KI mouse. B shows expression of heterologous KCNQ4 in outer hair cells 28 days after administration of rAAV particles containing the construct of SEQ ID NO:26 into the inner ear of a 2-day-old Kcnq4dn / +KI mouse. C shows a close-up of heterologous KCNQ4 expression in outer hair cells at the 16 kHz frequency position in the cochlea 28 days after administration of rAAV particles containing the construct of SEQ ID NO:26 into the inner ear of a 2-day-old Kcnq4dn / +KI mouse.

[0018] definition The scope of the present disclosure is defined by the claims attached hereto, and is not limited by any particular embodiment described herein. Those skilled in the art will recognize upon reading this specification various modifications that may be equivalent to the embodiments described herein or may otherwise be within the scope of the claims. In general, the terms used herein follow their meanings as understood in the art, unless expressly indicated otherwise. Explicit definitions of certain terms are provided below. The meanings of these and other terms in specific occurrences throughout this specification will be clear to those skilled in the art from the context.

[0019] 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 over another claim element, or the temporal order in which acts of a method are performed, but rather is merely used as a label to distinguish one claim element having a particular name from another element having the same name (except for the use of ordinal terms) to distinguish among the claim elements.

[0020] As used herein, the articles "a" and "an" 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 considered to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. In some embodiments, exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. In some embodiments, more than one or all of the members of a group are present in, used in, or otherwise relevant to a given product or process. It is to 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 that are introduced into another claim that is dependent on the same base claim (or any other claim, if relevant), unless otherwise indicated or unless it is obvious to one of ordinary skill in the art that a contradiction or inconsistency would result. It is to be understood that when elements are presented as a list (e.g., in a Markush group or similar format), each subgroup of elements is also disclosed, and any element(s) may be removed from the group. In general, when an aspect or aspect is referred to as "comprising" certain elements, features, etc., it is to be understood that a particular aspect or aspect "consists of" or "consists essentially of" such elements, features, etc. For purposes of brevity, those aspects have not been explicitly and explicitly described herein in all cases. It is also to be understood that any embodiment or aspect may be expressly excluded from the claims, regardless of whether that specific exclusion is enumerated herein.

[0021] Throughout this specification, whenever a polynucleotide or polypeptide is represented by a series of letters (e.g., A, C, G, and T, which in polynucleotides refer to adenosine, cytidine, guanosine, and thymidine, respectively), such polynucleotide or polypeptide will be presented in left to right, 5' to 3' or N-terminal to C-terminal order.

[0022] Administration: As used herein, the term "administration" typically refers to 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 or is included in a composition, and in some embodiments, the agent is produced by metabolism of the composition or one or more components thereof. A person skilled in the art will be aware of various routes that may be utilized for administration to a subject, e.g., a human, in the 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 involve, for example, delivery to the perilymph of the cochlea via injection through the round window membrane or into the scala tympani, injection through the scala media through the endolymph, perilymph and / or endolymph after canalostomy. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some aspects, administration may involve dosing that is intermittent (e.g., multiple doses spaced apart over time) and / or periodic (e.g., individual doses spaced apart by a common period of time) dosing. In some aspects, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0023] Allele: As used herein, the term "allele" refers to one of two or more existing genetic variants at a particular polymorphic locus.

[0024] Amelioration: As used herein, the term "amelioration" refers to the prevention, reduction, or palliative of a subject's condition, or a reversal of the condition. Amelioration may include, but does not require, complete recovery or complete prevention of a disease, disorder, or condition.

[0025] 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, e.g., by formation of one or more peptide bonds. In some aspects, an amino acid is, for example, H 2 The amino acids have the general structure NC(H)(R)-COOH. In some aspects, the amino acids are naturally occurring amino acids. In some aspects, the amino acids are unnatural amino acids, in some aspects, the amino acids are D-amino acids, and in some aspects, the amino acids are L-amino acids. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "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 aspects, amino acids, including carboxy- and / or amino-terminal amino acids in a polypeptide, may contain structural modifications compared to the general structure shown above. For example, in some aspects, amino acids may be modified 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) compared to the general structure. In some aspects, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to one that otherwise contains the same unmodified amino acid, while in some aspects, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to one that otherwise contains the same unmodified amino acid.

[0026] 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 fall within ±10% (greater or less) of a stated reference value, unless otherwise specified or otherwise clear from the context (except when such number exceeds 100% of possible values). For example, in some embodiments, the term "approximately" or "about" may encompass a range of values ​​that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a reference value.

[0027] Associated: As used herein, the term "associated" describes two events or entities as "associated" with each other when the presence, level, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, a gene signature, a metabolite, a microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition when 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 each other when they interact directly or indirectly such that they are in physical proximity to each other and / or remain in physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other, and in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other but are non-covalently associated by means of, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0028] Biologically active: As used herein, the term "biologically active" refers to an observable biological effect or outcome achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, the regulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is assessed by detecting a direct or indirect product produced by the biological pathway or event of interest.

[0029] 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 and / or control protein that binds to the tissue-specific promoter). In some aspects, an inner ear outer hair cell-selective promoter is a promoter that is primarily active in one or more outer hair cells of the inner ear.

[0030] Characteristic portion: As used herein, the term "characteristic portion" in its broadest sense refers to a portion of a substance whose presence (or absence) correlates with the presence (or absence) of a particular characteristic, attribute, or activity of that 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 those that do not share that 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 unique to the protein or polypeptide. In some embodiments, each such contiguous stretch typically contains 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 property with the relevant intact substance, in addition to the sequence and / or structural identity set forth above. In some aspects, the characteristic portion may be biologically active.

[0031] 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 thus can be used by those of skill in the art to define the members of that family.

[0032] 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 being (or not being) a member 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 a first stretch of contiguous monomers and a second stretch of contiguous monomers separated by one or more spacer regions that may or may not vary in length across polymers that share the sequence element.

[0033] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the two or more agents may be administered simultaneously. In some embodiments, the two or more agents may be administered sequentially. In some embodiments, the two or more agents may be administered in overlapping dosing regimens.

[0034] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, situations, sets of conditions, subjects, populations, etc. that may not be identical to one another, but are sufficiently similar to permit a comparison between them, such that a person of ordinary skill in the art would understand that conclusions may be reasonably drawn based on the differences or similarities observed. In some embodiments, a set of equivalent agents, entities, situations, sets of conditions, subjects, populations, etc. is characterized by a number of substantially identical characteristics and one or a few variable characteristics. A person of ordinary skill in the art will understand in context what degree of identity is required for two or more such agents, entities, situations, sets of conditions, subjects, populations, etc. to be considered equivalent in any given situation. For example, one of ordinary skill in the art will understand that sets of agents, entities, circumstances, sets of conditions, subjects, populations, etc. are equivalent to one another if they are characterized by a sufficient number and variety of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, stimuli, agents, entities, circumstances, sets of conditions, subjects, populations, etc. are caused by or indicative of variations in those features that vary.

[0035] Construct: As used herein, the term "construct" refers to a composition comprising a polynucleotide capable of carrying at least one heterologous polynucleotide. In some aspects, the construct can be a plasmid, a transposon, a cosmid, an artificial chromosome (e.g., a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC)) or a viral vector, a capsid, a viral particle, and any Gateway® plasmid. The construct can, for example, contain sufficient cis-acting elements for expression, and 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, a transposon, a cosmid, an artificial chromosome, or a viral vector, a capsid, a viral particle, etc.) that can replicate when associated with the appropriate regulatory elements. Thus, in some aspects, a "construct" can include a cloning and / or expression construct and / or a viral construct (e.g., an adeno-associated virus (AAV) construct, an adenoviral construct, a lentiviral construct, or a retroviral construct).

[0036] Conservative: As used herein, the term "conservative" refers to cases describing conservative amino acid substitutions, including the replacement of one 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 desired functional property 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 substitutions 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 aspects, a conservative amino acid substitution can be the substitution of any naturally occurring residue in a protein with alanine, for example, as used in alanine scanning mutagenesis. In some aspects, 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, which is incorporated herein by reference in its entirety. In some aspects, the substitutions are reasonably conservative substitutions, where the substitutions have non-negative values ​​in the PAM250 log-likelihood matrix.One skilled in the art will appreciate that amino acids that are not conserved between the same proteins from different species should be selected for mutation because changes (e.g., substitutions, additions, deletions, etc.) of these amino acids are unlikely to have an effect on the function of the protein. Amino acids that are conserved between the same proteins from different species should not be altered (e.g., deletions, additions, substitutions, etc.) because these mutations are likely to result in changes in the function of the protein. Exemplary conservative amino acid substitutions are shown in Table 1. [Table 1]

[0037] Control: As used herein, the term "control" refers to the art-understood meaning of "control" being a standard against which results are compared. Typically, controls are used to enhance the integrity of an experiment by isolating such variables to make conclusions about the variables. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparison. For example, in one experiment, the "test" (i.e., the variable being tested) is applied. In a second experiment, i.e., the "control," the variable being tested is not applied. 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 stored record. In some embodiments, a control is a positive control. In some embodiments, a control is a negative control.

[0038] Determine, measure, evaluate, assess, assay, and analyze: As used herein, the terms "determine," "measure," "assess," "assess," "assay," and "analyze" may be used interchangeably to refer to any form of measurement and may include determining whether an element is present or not. These terms include both quantitative and / or qualitative determinations. Assaying may be relative or absolute. For example, in some embodiments, "assaying for the presence of" may be determining the amount of something present and / or determining whether it is present or absent.

[0039] 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.

[0040] Engineered: In general, as used herein, the term "engineered" refers to an aspect that has been artificially manipulated. For example, a cell or organism is considered to be "engineered" if it has been manipulated such that its genetic information is altered (e.g., new genetic material not previously present is introduced, for example, by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is modified or removed, for example, by substitution or deletion mutations, or by mating protocols). As is customary and understood by those skilled in the art, the progeny of an engineered polynucleotide or cell is typically still referred to as "engineered", even if the actual manipulation was performed on the previous entity.

[0041] Excipient: As used herein, the term "excipient" refers to an inert (e.g., non-therapeutic) agent that can be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. In some aspects, suitable pharmaceutical excipients can 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.

[0042] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product (e.g., a transcription product, e.g., mRNA, e.g., a polypeptide, etc.) from the nucleic acid sequence. In some aspects, the gene product can be a transcription product. In some aspects, the gene product can be a polypeptide. In some aspects, expression of a nucleic acid sequence involves 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.

[0043] Flanked: As used herein, the term "flanked" refers to a position relative to the ends of a reference item. For example, with respect to a reference nucleic acid sequence(s), "flanked" refers to having sequences upstream and downstream of the reference nucleic acid sequence(s). In some aspects, a flanked referent nucleic acid sequence has a first sequence or series of nucleotide residues located adjacent to the 5' end of the referent nucleic acid and a second sequence or series of nucleotide residues located adjacent to the 3' end of the referent nucleic acid. In some aspects, the upstream and / or downstream flanking sequences are immediately adjacent to the referent nucleic acid sequence. In some aspects, there is an intervening nucleic acid between the upstream and / or downstream flanking sequences and the referent nucleic acid sequence.

[0044] Functional: As used herein, the term "functional" describes something that exists in a form that exhibits a property and / or activity that characterizes it. For example, in some embodiments, a "functional" biomolecule is a biomolecule that exists in a form that exhibits a property and / or activity that characterizes it. 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 biomolecule may have one function, two functions (i.e., bifunctional), or many functions (i.e., multifunctional).

[0045] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that codes for a gene product (e.g., an RNA product, e.g., a polypeptide product). In some aspects, a gene includes a coding sequence (i.e., a sequence that codes for a particular product). In some aspects, a gene includes a non-coding sequence. In some particular aspects, a gene may include both coding (e.g., exon) and non-coding (e.g., intron) sequences. In some aspects, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that may, for example, control or affect 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 codes for a polypeptide or a fragment thereof. As will be clear to one of skill in the art from the context, the term may optionally encompass regulatory sequences. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but rather to clarify that the term as used herein most often refers to a polypeptide-encoding nucleic acid. In some aspects, 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 the wild-type gene, or may not function at all. In some aspects, a gene may encode a transcript, which in some aspects may be toxic above a threshold level. In some aspects, a gene may encode a polypeptide, but the polypeptide may not be functional and / or may be toxic above a threshold level.

[0046] Hair Cell: As used herein, the term "hair cell" or "inner ear hair cell" refers to the sensory receptors of both the auditory and vestibular systems in the ear of all vertebrates. The term "hair cell" or "inner ear hair cell" refers to the inner hair cell and / or the outer hair cell. The term "inner hair cell" or "inner ear inner hair cell" refers to the cell of the inner ear that converts sound vibrations from the fluid in the cochlea into an electrical signal that is then transmitted to the brain via the auditory nerve. The term "outer hair cell" or "inner ear outer hair cell" refers to the cell of the inner ear that mechanically amplifies low-level sounds that enter the fluid of the cochlea.

[0047] Hearing loss: As used herein, the term "hearing loss" may be used for partial or complete lack of hearing ability in a living organism. In some aspects, hearing loss may be acquired. In some aspects, hearing loss may be hereditary. In some aspects, hearing loss may be genetic. In some aspects, 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 aspects, hearing loss may be due to one or more known genetic causes and / or syndromes. In some aspects, hearing loss may be of unknown etiology. In some aspects, hearing loss may or may not be alleviated by the use of hearing aids or other treatments.

[0048] 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 protein may be derived from a different gene than the gene encoding the protein.

[0049] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered "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 percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison (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 sequence 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, and the nucleotides at corresponding positions are then 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 (i.e., first and second) molecules 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 need to be introduced to obtain 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 with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0050] Improve, increase, enhance, inhibit, or reduce: As used herein, the terms "improve," "increase," "enhance," "inhibit," "reduce," or their grammatical equivalents, refer to a value compared to a baseline or other reference measurement. In some embodiments, the value is a statistically significant difference 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., a single individual) under otherwise equivalent conditions except for the presence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate equivalent 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 manner in the presence of the relevant agent or treatment. In some embodiments, a suitable reference is a negative reference, and in some embodiments, a suitable reference is a positive reference.

[0051] Knockdown: As used herein, the term "knockdown" refers to a reduction in the expression of one or more gene products. In some embodiments, an inhibitory nucleic acid achieves the knockdown. In some embodiments, a genome editing system described herein achieves the knockdown.

[0052] 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.

[0053] 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 aspects, 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 aspects, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some aspects, "nucleic acid" refers to an oligonucleotide chain that includes individual nucleic acid residues. In some aspects, "nucleic acid" is or includes RNA, and in some aspects, "nucleic acid" is or includes DNA. In some aspects, a nucleic acid is, includes, or consists of one or more naturally occurring nucleic acid residues. In some aspects, a nucleic acid is, includes, or consists of one or more nucleic acid analogs. In some aspects, 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 naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some aspects, 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 aspects, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugars in natural nucleic acids. In some aspects, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some aspects, the nucleic acid comprises one or more introns. In some aspects, 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), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acids are 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 in length, or more. In some embodiments, the nucleic acid is partially or completely single-stranded, and 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 or is complementary to a sequence that encodes a polypeptide.In some embodiments, the nucleic acid has an enzymatic activity.

[0054] Operably linked: As used herein, refers to a juxtaposition in which the described components are in a relationship that allows them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a manner that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some aspects, an "operably linked" control element is contiguous (e.g., covalently linked) with the coding element of interest, and in some aspects, the control element acts in trans or otherwise remote from the functional element of interest. In some aspects, "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 to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In some aspects, for example, a functional linkage may include transcriptional control. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, for example, where necessary to join two protein coding regions, in the same reading frame.

[0055] 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 pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predefined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including, for example, one adapted for administration, e.g., an injectable formulation, e.g., an aqueous or non-aqueous solution or suspension, or a drop designed to be administered into the ear canal. In some embodiments, the pharmaceutical composition may be formulated for administration via injection, either directly into a particular organ or compartment, e.g., the ear, or systemically, e.g., intravenously. In some embodiments, the formulation may be or include a drench (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste, capsule, powder, etc. In some embodiments, the active agent may be or include an isolated, purified, or pure compound.

[0056] Pharmaceutically acceptable: As used herein, the term "pharmaceutical acceptable," which may be used in reference to a carrier, diluent, or excipient used, for example, to formulate a pharmaceutical composition disclosed herein, means that the carrier, diluent, or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0057] Pharmaceutically acceptable carrier: As used herein, the term "pharmacologically acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting 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 pharma- ceutically 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 buffers; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0058] Polyadenylation: As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety, or modified variants thereof, 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 stretch of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) that is added to the pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, a poly(A) tail can be added to transcripts that contain a specific sequence, a polyadenylation signal or "poly(A) sequence." The poly(A) tail and its associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation can also affect transcription termination, nuclear export of mRNA, and translation. Typically, polyadenylation occurs in the nucleus immediately following transcription of DNA into RNA, but can additionally occur later in the cytoplasm. After transcription is terminated, 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 induces endonuclease cleavage of mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0059] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (eg, amino acids) typically joined by peptide bonds.

[0060] In some aspects, a polypeptide has an amino acid sequence that occurs in nature. In some aspects, a polypeptide has an amino acid sequence that does not occur in nature. In some aspects, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced by human action. In some aspects, a polypeptide can include or consist of natural amino acids, unnatural amino acids, or both. In some aspects, a polypeptide can include one or more pendant groups or other modifications that modify or are attached to one or more amino acid side chains, for example, at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof. In some aspects, such pendant groups or modifications can be acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some aspects, a polypeptide can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. In some aspects, useful modifications can be or include, for example, terminal acetylation, amidation, methylation, etc. In some aspects, a protein may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to 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.

[0061] Polynucleotide: As used herein, the term "polynucleotide" refers to any polymeric chain of nucleic acid. In some embodiments, a polynucleotide is or comprises RNA, and in some embodiments, a polynucleotide is or comprises DNA. In some embodiments, a polynucleotide is or comprises or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a polynucleotide is or comprises or consists of one or more nucleic acid analogs. In some embodiments, a polynucleotide analog differs from a nucleic acid in that it does 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 or comprises or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some aspects, 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, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some aspects, a polynucleotide comprises one or more modified sugars compared to the sugars in natural nucleic acids (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), in some aspects, a polynucleotide has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein.In some aspects, the polynucleotide comprises one or more introns. In some aspects, 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), replication in a recombinant cell or system, and chemical synthesis. In some aspects, a 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 in length, or more. In some embodiments, the polynucleotide is partially or completely single-stranded, and in some embodiments, the polynucleotide is partially or completely double-stranded.In some embodiments, the polynucleotide has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complementary sequence of the sequence that encodes it.In some embodiments, the polynucleotide has enzymatic activity.

[0062] Promoter: As used herein, the term "promoter" refers to a nucleic acid sequence that functions to control one or more coding sequences (e.g., genes or transgenes that encode a polypeptide) located upstream in the direction of transcription of the transcription start site of the coding sequence. In some aspects, a promoter is structurally identified by the presence of a binding site for a DNA-dependent RNA polymerase, a transcription start site, or other DNA sequences (e.g., transcription factor binding sites, repressor and / or activator protein binding sites, or other nucleotide sequences that act directly or indirectly to regulate the amount of transcription from the promoter).

[0063] 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 appreciate 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 appreciate that a protein may sometimes comprise more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means.

[0064] Recombinant: As used herein, the term "recombinant" refers to a polypeptide that is 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 gene(s) or genetic component(s) that encodes and / or directs the expression of a polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. It is intended to refer to a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that has been transgenic or otherwise engineered to express it; and / or a polypeptide prepared, expressed, produced, or isolated by splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or any other means that involves the otherwise generation of nucleic acids that encode and / or direct the expression of a polypeptide or one or more of its component(s), portion(s), element(s), or domain(s). In some aspects, one or more of such selected sequence elements are found in nature. In some aspects, one or more of such selected sequence elements are designed in silico. In some aspects, one or more of 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, in the germline (e.g., human, mouse, etc.) of a source organism of interest.

[0065] 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 simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those of skill in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those being assessed. Those of skill in the art will understand when there is sufficient similarity to justify the reliability of and / or comparison to a particular possible reference or control. In some embodiments, the reference is a negative control reference, and 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.

[0066] Regulatory element: As used herein, the term "regulatory element" or "regulatory sequence" refers to a non-coding region of DNA that in some way regulates the expression of one or more specific genes. In some aspects, such genes are juxtaposed or "near" a given regulatory element. In some aspects, such genes are located at a significant distance from a given regulatory element. In some aspects, a regulatory element impairs or enhances the transcription of one or more genes. In some aspects, a regulatory element may be located in cis with respect to the gene being regulated. In some aspects, a regulatory element may be located in trans with respect to the gene being regulated. For example, in some aspects, 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 aspects, this sequence may be an enhancer sequence and other regulatory elements that regulate the expression of the gene product.

[0067] 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 can 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 biological fluid. In some aspects, 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, chyme, ejaculate, endolymph, exudate, stool, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some aspects, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or cellular fluid. In some aspects, the biological fluid may be or include plant exudates. In some aspects, 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, washing or lavage (e.g., bronchoalveolar epithelium, breast duct, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some aspects, the biological sample is or comprises cells obtained from an individual. In some aspects, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some aspects, 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 thereof and / or by adding one or more agents thereto). For example, filtration using a semipermeable membrane.Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques, such as amplification or reverse transcription of nucleic acids, isolation and / or purification of certain components, etc.

[0068] Selective expression: As used herein, the term "selective expression" or "selectively expressing" refers to the preferential expression of a gene or polypeptide of interest in a particular specific cell type (e.g., inner ear cells, e.g., inner ear outer hair cells).

[0069] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments prenatal human forms). In some embodiments, the subject suffers from a relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is one who has 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 to whom diagnosis and / or therapy is administered.

[0070] Substantially: As used herein, the term "substantially" refers to a qualitative state of exhibiting a complete or near complete extent or degree of a desired property or characteristic. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, reach perfection and / or proceed to perfection, or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of perfection that is inherently present in many biological and chemical phenomena.

[0071] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of therapy that partially or completely alleviates, improves, eliminates, reverses, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some aspects, such treatment may be of subjects who do not exhibit symptoms of the relevant disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be of subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. In some aspects, treatment may be of subjects who have been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some aspects, treatment may be of a subject known to have one or more predisposition factors that are statistically correlated with an increased risk of developing a given disease, disorder, and / or condition.

[0072] 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, a reference version is typically selected, and the variant differs relative to the reference version. In some embodiments, a variant may have the same or different (e.g., increased or decreased) level of activity or functionality as a wild-type sequence. For example, in some embodiments, a variant may have improved functionality compared to a wild-type sequence, e.g., when 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 a reduced or eliminated activity or functionality that results in a negative outcome, or an altered activity (e.g., increased electrical activity that results in chronic depolarization leading to cell death). Such variants are referred to herein as loss-of-function variants. In some aspects, a gain-of-function variant is a codon-optimized sequence that encodes a transcript or polypeptide that may have improved properties (e.g., reduced susceptibility to degradation, e.g., reduced susceptibility 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 PREFERRED EMBODIMENTS

[0073] The present disclosure relates to constructs comprising polynucleotides encoding polypeptides that are designed for selective expression of a transgene, e.g., preferential expression in outer hair cells, and compositions comprising the same.

[0074] Hearing loss In general, the ear can be described as including the outer ear, middle ear, inner ear, auditory (hearing) 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.

[0075] 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, an illusory noise in the auditory system (ringing, buzzing, chirping, humming, or beating), also have a reduced sensitivity / tolerance to certain sound frequencies and volume ranges, known as hyperacusis (also spelled hyperacousis). Various non-syndromic and syndromic related hearing loss will be known to those skilled in the art (e.g., DFNB1 and DFNA3, or respectively, Bart-Pumphrey syndrome, hystrix-like ichthyosis with deafness (HID), palmoplantar keratitis with deafness, keratitis-ichthyosis-deafness (KID) syndrome, and Vohwinkel syndrome). Environmental causes of hearing impairment or hearing loss may include, for example, certain medications, certain infections before or after birth, and / or exposure to loud noise over time. In some aspects, hearing loss may result from noise, ototoxic drugs, presbycusis, disease, infection, or cancer that affects certain parts of the ear. In some aspects, ischemic injury may cause hearing loss through pathophysiological mechanisms. In some aspects, intrinsic abnormalities such as congenital mutations of 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 may cause or contribute to hearing loss.

[0076] Hearing loss and / or hearing loss is one of the most common sensory defects in humans and can occur for many reasons. In some embodiments, some subjects are born deaf or deaf, while others may gradually lose their hearing over time. Approximately 36 million American adults report some degree of hearing loss, and one in three people over the age of 60 and half of people over the age of 85 experience hearing loss. Approximately 1.5 out of every 1,000 children are born with severe hearing loss, and another 2-3 out of every 1,000 children are born with partial hearing loss (Smith et al., 2005, Lancet 365:879-890, which is incorporated herein by reference in its entirety). More than half of these cases are attributed to a genetic basis (Di Domenico, et al., 2011, J. Cell. Physiol. 226:2494-2499, which is incorporated herein by reference in its entirety).

[0077] Treatments for hearing loss currently consist of hearing amplification for mild to severe loss and cochlear implants for severe to profound loss (Kral and O'Donoghue, 2010, N. Engl. J. Med. 363:1438-1450, which is incorporated herein by reference in its entirety). Recent research in this area has focused on regeneration of cochlear hair cells, which is applicable to the most common forms of hearing loss, including presbycusis, noise pollution, infections, and ototoxicity. There remains a need for effective treatments, such as gene therapy, that can repair and / or reduce the source of the hearing problem (see, e.g., WO2018 / 039375, WO2019 / 165292, and PCT application US2019 / 060328, each of which is incorporated herein by reference in its entirety).

[0078] 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 hair cell dysfunction. In some embodiments, sensorineural hearing impairment involves the eighth cranial nerve (vestibular cochlear nerve) or the auditory part of the brain. In some such embodiments, only the auditory center of the brain is affected. In such situations, cortical deafness may occur, in which sounds may be heard at normal thresholds, but the quality of the sound is not good, so 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 ear and the middle ear, called mixed hearing loss. Hearing loss and / or hearing loss that is present before a child can speak may be classified as prelingual or congenital. Hearing loss and / or hearing loss that occurs after language development may be classified as postlingual. Most autosomal recessive loci associated with syndromic or nonsyndromic hearing loss cause severe-to-profound prelingual hearing loss.

[0079] In some aspects, the hearing loss or deafness may be non-syndromic. In some aspects, the hearing loss or deafness may be syndromic. Non-syndromic hearing loss or deafness is hearing loss that is not associated with other signs and symptoms. In contrast, syndromic hearing loss includes hearing loss that occurs in conjunction with abnormalities in other parts of the body. Most cases of hereditary hearing loss (70 percent to 80 percent) are non-syndromic, with the remaining cases being caused by specific genetic syndromes.

[0080] Non-syndromic hearing loss can have different inheritance patterns and can occur at any age. Types of non-syndromic hearing loss are named according to their inheritance patterns. Autosomal dominant type is designated as DFNA, autosomal recessive type is designated as DFNB, and X-linked type is designated as DFNX. Each type is also numbered in the order in which it is described. For example, DFNA1 is the first autosomal dominant type of non-syndromic hearing loss described. In some embodiments, non-syndromic hearing loss or deafness can have autosomal dominant, autosomal recessive, or X-linked inheritance patterns.

[0081] 75% to 80% of nonsyndromic hearing loss cases are inherited in an autosomal recessive manner, meaning that both copies of the gene in each cell have a mutation. Usually, each parent of an individual with autosomal recessive hearing loss is a carrier of one copy of the mutated gene, but is not affected by this form of hearing loss. In some embodiments, nonsydromic hearing loss or deafness may be inherited in an autosomal recessive manner (Venkatesh, et al., 2015, Med. J. Armed Forces India, 71(4)363-368).

[0082] Another 20 percent to 25 percent of nonsyndromic hearing loss cases are autosomal dominant, meaning that one copy of the altered gene in each cell is sufficient to cause hearing loss. People with autosomal dominant hearing loss most often inherit an altered copy of the gene from a parent who has hearing loss. In some embodiments, nonsydromic hearing loss or deafness can be inherited in an autosomal dominant manner (e.g., DFNA2) (Venkatesh, et al., 2015, Med. J. Armed Forces India, 71(4)363-368).

[0083] One to two percent of hearing loss and deafness cases exhibit an X-linked inheritance pattern, meaning that the mutated gene causing the condition is located on the X chromosome. In some aspects, non-syndromic hearing loss or deafness may be inherited in an X-linked inheritance pattern (Venkatesh, et al., 2015, Med. J. Armed Forces India, 71(4)363-368).

[0084] The causes of nonsyndromic hearing loss are complex. Researchers have identified over 30 genes that, when altered, are associated with nonsyndromic hearing loss, although 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 some genes are associated with both syndromic and nonsyndromic hearing loss (Venkatesh, et al., 2015, Med. J. Armed Forces India, 71(4)363-368).

[0085] For example, genes associated with nonsyndromic hearing loss include, but are not limited to, ATP2B2, ACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5, DFNB31(WHRN), DFNB59, ESPN, EYA4, GJB3, KCNQ4, LHFPL5, MYO15A, MYO6, MYO7A, OTOF, PCDH15, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIOBP, USH1C, and WFS1 (Athena Diagnostics, 2017, "Hearing Loss Advanced Sequencing and CNV Evaluation", 1-3). In some aspects, the non-syndromic hearing loss or hearing loss is associated with a gene selected from ATP2B2, ACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB3, KCNQ4, LHFPL5, MYO15A, MYO6, MYO7A, OTOF, PCDH15, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIOBP, USH1C, and WFS1.

[0086] OTOF-related deafness (DFNB9 nonsyndromic deafness) is characterized by two phenotypes: pre-lingual nonsyndromic deafness and, less frequently, temperature-sensitive nonsyndromic auditory neuropathy (TS-NSAN) (Azaiez, et al., 2008, "OTOF-Related Deafness", GeneReviews, 1-16). Another form of progressive hearing loss is associated with a mutation in the otoferlin gene (e.g., E1700Q mutation) or is not temperature sensitive (Iwasa, et al. 2019, PLoS ONE 14(5):e0215932). In some embodiments, the deafness or hearing loss is otoferlin-related. In some embodiments, the deafness or hearing loss is DFNB9 nonsyndromic deafness. In some embodiments, the deafness or hearing loss is associated with a mutation in the otoferlin gene.

[0087] DFNB59 (Deafness, Autosomal Recessive 59), also known as Pejvakin or PJVK, is a 352 amino acid protein that belongs to the gasdermin family in vertebrates. DFNB59 is encoded by a gene that maps to human chromosome 2q31.2, which is essential for proper function of auditory pathway nerves and outer hair cell function. Mutations in DFNB59 are believed to cause nonsyndromic sensorineural hearing loss, autosomal recessive type 59, a form of sensorineural hearing impairment characterized by an absent or severely abnormal auditory brainstem response, but normal otoacoustic emissions (auditory neuropathy or auditory dyssynchrony). DFNB59 shares significant similarity with DFNA5, indicating that these genes share a common origin (Delmaghani, et al., 2006, Nat. Genet. 38:770-778). In some aspects, the hearing loss or hearing loss is Pejvakin-associated. In some embodiments, the hearing loss or deafness is DFNB59 nonsyndromic deafness. In some embodiments, the hearing loss or deafness is DFNA5 nonsydromic deafness.

[0088] Defects in ion channels are associated with hearing loss, and DFNA2 nonsyndromic hearing loss is inherited as an autosomal dominant mutation in the KCNQ4 gene, which encodes the voltage-gated potassium channel subfamily KQT member 4, also known as the voltage-gated potassium channel subunit Kv7.4. DFNA2 nonsyndromic hearing loss is characterized by symmetric, predominantly high-frequency sensorineural hearing loss (SNHL) that is progressive across all frequencies (Jung, et a., 2019, Exp. Mol. Med. 51:1-12). At younger ages, hearing loss tends to be mild at low frequencies and moderate at high frequencies, while in older adults, hearing loss is moderate at low frequencies and severe to profound at high frequencies. Hearing impairment is often detected during routine hearing evaluations in school-age children, but hearing is likely impaired from birth, especially at high frequencies. Most affected individuals first require hearing aids to aid in sound amplification between the ages of 10 and 40. By age 70, all people with DFNA2 hearing loss have severe-to-profound hearing loss (Smith and Hildebrand, 2008, DFNA2 Nonsydrmoic Hearing Loss, GeneReviews, 1-14).

[0089] Usher syndrome (also known as Hallgren syndrome, Usher-Hallgren syndrome, retinitis pigmentosa-deafness syndrome, and retinal dystrophy-deafness syndrome) is a rare disorder caused by mutations in any one of at least ten genes that results in a combination of hearing loss and gradual visual impairment, and is the leading cause of deaf-blindness. The hearing loss is caused by a defective inner ear, while the vision loss results from retinitis pigmentosa (RP), a degeneration of retinal cells. Usher syndrome has three clinical subtypes, designated as I, II, and III. Subjects with Usher I are born profoundly deaf, begin to lose their vision in the first decade of life, are slow to learn to walk as children due to problems in their vestibular system, and exhibit balance disorders. Subjects with Usher II are not born deaf, but have hearing loss, but do not appear to have any notable problems with balance. Such subjects also begin to lose their vision later (in their teens), and may retain some vision even in middle age. Subjects with Usher syndrome III are not born deaf, but experience a gradual loss of their hearing and vision. Such subjects may or may not have balance disorders (Toms, et al., 2020, Ther. Adv. Ophthalmol., 12:2515841420952194). In some aspects, the hearing loss is symptomatic. In some aspects, the hearing loss is associated with Usher syndrome. In some aspects, the hearing loss or deafness is associated with Usher syndrome I, Usher syndrome II, or Usher syndrome III.

[0090] Mutations in the WFS1 gene cause more than 90 percent of Wolfram syndrome type 1 cases. Wolfram syndrome is a condition that affects many of the body's systems, most often characterized by high blood sugar levels due to a lack of the hormone insulin (diabetes mellitus), and progressive vision loss due to degeneration of the nerves that carry information from the eye to the brain (optic atrophy). However, people with Wolfram syndrome also often have pituitary dysfunction, which results in excessive urine output (diabetes insipidus), hearing loss caused by changes in the inner ear (sensorineural hearing loss), urinary tract problems, reduced amounts of the sex hormone testosterone in men (hypogonadism), or neurological or psychiatric disorders. Approximately 65 percent of people with Wolfram syndrome have sensorineural hearing loss, which can range in severity from hearing loss that begins at birth to mild hearing loss that begins in adolescence and worsens over time. Additionally, approximately 60 percent of people with Wolfram Syndrome develop neurological or psychiatric disorders, most commonly problems with balance and coordination (ataxia), typically beginning in early adulthood (Medlej, et al., 2004, J. Clin. Endocrinol. Metab., 89(4):1656-1661).

[0091] The WFS1 gene encodes a protein called wolframin, which is thought to regulate the amount of calcium in cells. When Wolfram syndrome is caused by a mutation in the WFS1 gene, it is inherited in an autosomal recessive manner, and the wolframin protein is reduced or absent in function. As a result, calcium levels in the cell are not regulated and the endoplasmic reticulum does not work properly. If the endoplasmic reticulum does not have enough functional wolframin, the cell induces its own cell death (apoptosis) (Zmyslowska, et al., 2021, Cell Commun Signal, 19:116). The death of cells in the pancreas, specifically the cells that make insulin (beta cells), causes diabetes mellitus in people with Wolfram syndrome. The gradual loss of cells along the optic nerve eventually results in vision loss in affected individuals. The death of cells in other body systems likely causes the various signs and symptoms of Wolfram syndrome type 1 (Urano, 2016, Curr. Diab. Rep., 16:6). In some embodiments, the hearing loss or deafness is syndromic hearing loss or deafness. In some embodiments, the syndromic hearing loss or deafness is WFS1-associated. In some embodiments, the syndromic hearing loss or deafness is associated with Wolfram syndrome.

[0092] As known to those skilled in the art, hair cells are the sensory receptors of 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. It is known that mammalian ears 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 through electrically driven movement of the hair cell body. The 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 may be abnormal at birth or damaged during the life of an individual.

[0093] Polypeptides Certain aspects of the present disclosure relate to polynucleotides that encode polypeptides. In some aspects, the polynucleotides may encode polypeptides that can be expressed in cells (e.g., inner ear cells). In some aspects, the polynucleotides may encode polypeptides that can be expressed in hair cells. In some aspects, the polynucleotides may encode polypeptides that can be expressed in outer hair cells. In some aspects, the polynucleotides may encode full-length polypeptides or functional fragments thereof.

[0094] 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.

[0095] Exemplary polynucleotides encoding polypeptides include actin gamma 1 (ACTG1), adenylate cyclase type 1 (ADCY1), calcium binding protein 2 (CABP2), coiled-coil domain containing 50 (CCDC50), cadherin-associated 23 (CDH23), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), chromodomain helicase DNA-binding protein 7 (CHD7), calcium and integrin binding family member 2 (CIB2), claudin 14, and the like. (CLDN14), intracellular chloride channel 5 (CLIC5), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), clarin 1 (CLRN1), pedivakin (DFNB59), endothelin 3 (EDN3), ELMO domain-containing protein 3 (ELMOD3), epidermal growth factor receptor kinase substrate 8 (EPS8), espin (ESPN), estrogen-related receptor beta (ESRRB), eyes absent homolog 1 (EYA1), GIPC PDZ domain-containing family, member 3 (GIPC3), G protein-coupled receptor 98 (GPR98), G protein signaling modulator 2 (GPSM2), glutaredoxin, cysteine-rich 1 (GRXCR1), glutaredoxin, cysteine-rich 2 (GRXCR2), immunoglobulin-like domain-containing receptor 1 (ILDR1), lysyl-tRNA synthetase (KARS), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), lipoma HMGIC fusion partner-like 5 (LHFPL5), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT1 COMT2), tricellulin (MARVELD2), microRNA96 (MIR96), methionine sulfoxide reductase B3 (MSRB3), nonmuscle myosin heavy chain 9 (MYH9), nonmuscle myosin heavy chain 14 (MYH14), atypical myosin IIIA (MYO3A), atypical myosin VI (MYO6), atypical myosin VIIA (MYO7A), atypical myosin XVA (MYO15A), otoferlin (OTOF), otogelin-like protein (OTOGL), purinergic receptor (recetpro) P2X, ligand-gated ion channel, 2 (P2RX2), protocadherin 15 (PCDH15), PDZ domain-containing 7 (PDZD7), polyribonucleotide nucleotidyltransferase 1, mitochondrial (PNPT1), POU domain, class 4, transcription factor 3 (POU4F3), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), protein tyrosine phosphatase, receptor type Q (PTPRQ), radixin (RDX), scaffold-containing ankyrin repeats and SAM domain (SANS), serpin peptidase inhibitor, clade B, member 6 (SERPINB6), SIX homeobox 1 (SIX1), SIX homeobox 5 (SIX5), prestin (SLC26A5), second mitochondrial-derived caspase activator (SMAC / DIABLO), small muscle protein, x-linked (SMPX), stereocillin (STRC), nesprin-4 (SYNE4), TBC1 domain family, member 24 (TBC / D24), tight junction protein XO These include, but are not limited to, transmembrane channel-like protein 2 (TJP2), transmembrane inner ear expressed protein (TMIE), transmembrane protease, serine 3 (TMPRSS3), tapellin (TPRN), TRIO and F-actin binding protein (TRIOBP), thrombospondin-type laminin G domain and EAR repeats (TSPEAR), harmonin (USH1C), asshalin (USH2A), wolframin (WFS1), and whirlin (WHRN).

[0096] In some aspects, the polynucleotide encoding the outer hair cell polynucleotide comprises a gene selected from cadherin-related 23 (CDH23), clarin 1 (CLRN1), pedivakin (DFNB59), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), otoferlin (OTOF), protocadherin 15 (PCDH15), POU domain, class 4, transcription factor 3 (POU4F3), prestin (SLC26A5), stereocillin (STRC), transmembrane channel-like protein 1 (TMC1), TRIO and F-actin binding protein (TRIOBP), harmonin (USH1C), usherin (USH2A), wolframin (WFS1), and whirlin (WHRN). In some aspects, the polynucleotide encoding the outer hair cell polynucleotide comprises KQT-like subfamily, member 4 (KCNQ4).

[0097] In some aspects, the encoded polypeptide is a human polypeptide. In some aspects, the encoded polypeptide is a functional fragment of a human polypeptide disclosed herein.

[0098] Exemplary polypeptides are disclosed in Li, Y. et al. Transcriptomes of cochlear inner and outer hair cells from adult mice. Sci. Data. 5:180199 doi:10.1038 / sdata.2018.199 (2018), and Nishio, S. et al. Gene Expression Profiles of the Cochlea and Vestibular Endorgans: Localization and Function of Genes Causing Deafness. Annals Otology, Rhinology & Laryngology 124(55) (2015), which are incorporated by reference in their entireties.

[0099] In some aspects, the polypeptide is an outer hair cell polypeptide. In some aspects, the polypeptide is a therapeutic polypeptide. In some aspects, the polypeptide is a reporter polypeptide.

[0100] Outer Hair Cell Polypeptides Certain aspects of the present disclosure relate to polynucleotides that encode outer hair cell polypeptides. The polynucleotides may encode full-length polypeptides or functional fragments thereof.

[0101] 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.

[0102] Exemplary polynucleotides encoding polypeptides include actin gamma 1 (ACTG1), adenylate cyclase type 1 (ADCY1), calcium binding protein 2 (CABP2), coiled-coil domain containing 50 (CCDC50), cadherin-associated 23 (CDH23), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), chromodomain helicase DNA-binding protein 7 (CHD7), calcium and integrin binding family member 2 (CIB2), claudin 14, and the like. (CLDN14), intracellular chloride channel 5 (CLIC5), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), clarin 1 (CLRN1), pedivakin (DFNB59), endothelin 3 (EDN3), ELMO domain-containing protein 3 (ELMOD3), epidermal growth factor receptor kinase substrate 8 (EPS8), espin (ESPN), estrogen-related receptor beta (ESRRB), eyes absent homolog 1 (EYA1), GIPC PDZ domain-containing family, member 3 (GIPC3), G protein-coupled receptor 98 (GPR98), G protein signaling modulator 2 (GPSM2), glutaredoxin, cysteine-rich 1 (GRXCR1), glutaredoxin, cysteine-rich 2 (GRXCR2), immunoglobulin-like domain-containing receptor 1 (ILDR1), lysyl-tRNA synthetase (KARS), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), lipoma HMGIC fusion partner-like 5 (LHFPL5), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT1 COMT2), tricellulin (MARVELD2), microRNA96 (MIR96), methionine sulfoxide reductase B3 (MSRB3), nonmuscle myosin heavy chain 9 (MYH9), nonmuscle myosin heavy chain 14 (MYH14), atypical myosin IIIA (MYO3A), atypical myosin VI (MYO6), atypical myosin VIIA (MYO7A), atypical myosin XVA (MYO15A), otoferlin (OTOF), otogelin-like protein (OTOGL), purinergic receptor (recetpro) P2X, ligand-gated ion channel, 2 (P2RX2), protocadherin 15 (PCDH15), PDZ domain-containing 7 (PDZD7), polyribonucleotide nucleotidyltransferase 1, mitochondrial (PNPT1), POU domain, class 4, transcription factor 3 (POU4F3), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), protein tyrosine phosphatase, receptor type Q (PTPRQ), radixin (RDX), scaffold-containing ankyrin repeats and SAM domain (SANS), serpin peptidase inhibitor, clade B, member 6 (SERPINB6), SIX homeobox 1 (SIX1), SIX homeobox 5 (SIX5), prestin (SLC26A5), second mitochondrial-derived caspase activator (SMAC / DIABLO), small muscle protein, x-linked (SMPX), stereocillin (STRC), nesprin-4 (SYNE4), TBC1 domain family, member 24 (TBC / D24), tight junction protein XO These include, but are not limited to, transmembrane channel-like protein 2 (TJP2), transmembrane inner ear expressed protein (TMIE), transmembrane protease, serine 3 (TMPRSS3), tapellin (TPRN), TRIO and F-actin binding protein (TRIOBP), thrombospondin-type laminin G domain and EAR repeats (TSPEAR), harmonin (USH1C), asshalin (USH2A), wolframin (WFS1), and whirlin (WHRN).

[0103] In some aspects, the polynucleotide encoding the outer hair cell polynucleotide comprises a gene selected from cadherin-related 23 (CDH23), clarin 1 (CLRN1), pedivakin (DFNB59), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), otoferlin (OTOF), protocadherin 15 (PCDH15), POU domain, class 4, transcription factor 3 (POU4F3), prestin (SLC26A5), stereocillin (STRC), transmembrane channel-like protein 1 (TMC1), TRIO and F-actin binding protein (TRIOBP), harmonin (USH1C), usherin (USH2A), wolframin (WFS1), and whirlin (WHRN). In some aspects, the polynucleotide encoding the outer hair cell polynucleotide comprises KQT-like subfamily, member 4 (KCNQ4).

[0104] In some aspects, the encoded polypeptide is a human polypeptide. In some aspects, the encoded polypeptide is a functional fragment of a human polypeptide disclosed herein.

[0105] Exemplary polypeptides are disclosed in Li, Y. et al. Transcriptomes of cochlear inner and outer hair cells from adult mice. Sci. Data. 5:180199 doi:10.1038 / sdata.2018.199 (2018), and Nishio, S. et al. Gene Expression Profiles of the Cochlea and Vestibular Endorgans: Localization and Function of Genes Causing Deafness. Annals Otology, Rhinology & Laryngology 124(55) (2015), which are incorporated by reference in their entireties.

[0106] Certain aspects of the present disclosure relate to polynucleotides that encode therapeutic polypeptides. The polynucleotides may encode polypeptides that can be expressed in cells (e.g., inner ear cells). The polynucleotides may encode full-length polypeptides or functional fragments thereof.

[0107] 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.

[0108] Exemplary polynucleotides encoding therapeutic polypeptides include actin gamma 1 (ACTG1), adenylate cyclase type 1 (ADCY1), calcium binding protein 2 (CABP2), coiled-coil domain containing 50 (CCDC50), cadherin-associated 23 (CDH23), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), chromodomain helicase DNA binding protein 7 (CHD7), calcium and integrin binding family member 2 (CIB2), claudin 14 (CLDN14), intracellular chloride channel 5 (CLIC5), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), clarrin 1 (CLRN1), pedivakin (DFNB59), endothelin 3 (EDN3), ELMO domain-containing protein 3 (ELMOD3), epidermal growth factor receptor kinase substrate 8 (EPS8), espin (ESPN), estrogen-related receptor beta (ESRRB), eyes absent homolog 1 (EYA1), GIPC PDZ domain-containing family, member 3 (GIPC3), G protein-coupled receptor 98 (GPR98), G protein signaling modulator 2 (GPSM2), glutaredoxin, cysteine-rich 1 (GRXCR1), glutaredoxin, cysteine-rich 2 (GRXCR2), immunoglobulin-like domain-containing receptor 1 (ILDR1), lysyl-tRNA synthetase (KARS), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), lipoma HMGIC fusion partner-like 5 (LHFPL5), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT1 COMT2), tricellulin (MARVELD2), microRNA96 (MIR96), methionine sulfoxide reductase B3 (MSRB3), nonmuscle myosin heavy chain 9 (MYH9), nonmuscle myosin heavy chain 14 (MYH14), atypical myosin IIIA (MYO3A), atypical myosin VI (MYO6), atypical myosin VIIA (MYO7A), atypical myosin XVA (MYO15A), otoferlin (OTOF), otogelin-like protein (OTOGL), purinergic receptor (recetpro) P2X, ligand-gated ion channel, 2 (P2RX2), protocadherin 15 (PCDH15), PDZ domain-containing 7 (PDZD7), polyribonucleotide nucleotidyltransferase 1, mitochondrial (PNPT1), POU domain, class 4, transcription factor 3 (POU4F3), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), protein tyrosine phosphatase, receptor type Q (PTPRQ), radixin (RDX), scaffold-containing ankyrin repeats and SAM domain (SANS), serpin peptidase inhibitor, clade B, member 6 (SERPINB6), SIX homeobox 1 (SIX1), SIX homeobox 5 (SIX5), prestin (SLC26A5), second mitochondrial-derived caspase activator (SMAC / DIABLO), small muscle protein, x-linked (SMPX), stereocillin (STRC), nesprin-4 (SYNE4), TBC1 domain family, member 24 (TBC / D24), tight junction protein XO These include, but are not limited to, transmembrane channel-like protein 2 (TJP2), transmembrane inner ear expressed protein (TMIE), transmembrane protease, serine 3 (TMPRSS3), tapellin (TPRN), TRIO and F-actin binding protein (TRIOBP), thrombospondin-type laminin G domain and EAR repeats (TSPEAR), harmonin (USH1C), asshalin (USH2A), wolframin (WFS1), and whirlin (WHRN).

[0109] Exemplary polypeptides are disclosed in Li, Y. et al. Transcriptomes of cochlear inner and outer hair cells from adult mice. Sci. Data. 5:180199 doi:10.1038 / sdata.2018.199 (2018), and Nishio, S. et al. Gene Expression Profiles of the Cochlea and Vestibular Endorgans: Localization and Function of Genes Causing Deafness. Annals Otology, Rhinology & Laryngology 124(55) (2015), which are incorporated by reference in their entireties.

[0110] Constructs In particular, the present disclosure provides that some polynucleotides described herein are polynucleotide constructs. Polynucleotide constructs according to the present disclosure include all known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viral constructs (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus constructs) that incorporate polynucleotides encoding polypeptides or characteristic portions thereof. Those skilled in the art will be able to select suitable constructs and cells for producing any of the polynucleotides described herein. In some aspects, the construct is a plasmid (i.e., a circular DNA molecule that can replicate autonomously inside a cell). In some aspects, the construct can be a cosmid (e.g., pWE or sCos series). In some aspects, the construct is a mammalian or viral vector.

[0111] In some embodiments, the construct is a viral construct. In some embodiments, the viral construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus construct. In some embodiments, the construct is an adeno-associated virus (AAV) construct (see, e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, which is incorporated herein by reference in its entirety). In some embodiments, the construct is a viral vector. In some embodiments, the construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus vector. In some embodiments, the construct is an AAV vector. In some embodiments, the viral construct is an adenovirus 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 pancreas 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 genome of such viruses encodes nonstructural proteins (e.g., replicons) and structural proteins (e.g., capsid and envelope) that can be translated in the cytoplasm of the host cell. 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 can be formed by combining alphavirus envelope glycoproteins and retroviral capsids.Examples of alphavirus constructs can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819, the constructs and methods for their production being incorporated by reference in their entirety in each of these publications.

[0112] The constructs provided herein can be of different sizes. In some embodiments, the constructs are plasmids and can include a total 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.

[0113] In some embodiments, the construct is a viral construct and can have a total number of nucleotides 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 in the range of about 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.

[0114] 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 have a total number of nucleotides 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 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 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.

[0115] 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 have a total number of nucleotides of up to about 1 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 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.

[0116] 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 have a total number of nucleotides 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 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, The total number of nucleotides may be in the range 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 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0117] Any of the constructs described herein may further comprise a regulatory sequence selected from the group of, for example, 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 house pre- or post-transcriptional regulatory and / or control elements. In some aspects, the promoter may 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.

[0118] In some aspects, the construct comprises a polynucleotide encoding a polypeptide operably linked to a promoter that selectively expresses the polynucleotide in inner ear outer hair cells.

[0119] In some embodiments, the construct comprises a 5'ITR, a promoter that selectively expresses a polynucleotide in the outer hair of the inner ear, a polynucleotide encoding a polypeptide, a polyA, and a 3'ITR. In some embodiments, the construct comprises a 5'ITR, a promoter that selectively expresses a polynucleotide in the outer hair of the inner ear, a 5'UTR, a polynucleotide encoding a polypeptide, a polyA, and a 3'ITR.

[0120] In some aspects, the construct comprises a 5'ITR, a promoter that selectively expresses a polynucleotide in the outer hair of the inner ear, a 5'UTR, a polynucleotide that encodes a polypeptide, a 3'UTR, a polyA, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, a promoter that selectively expresses a polynucleotide in the outer hair of the inner ear, a 5'UTR, a polynucleotide that encodes a polypeptide, a polyA, and a 3'ITR.

[0121] In some aspects, the construct comprises a 5'ITR, a promoter that selectively expresses a polynucleotide in an inner ear outer hair cell, a 5'UTR, a polynucleotide that encodes a polypeptide, a tag, a 3'UTR, a polyA, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, a promoter that selectively expresses a polynucleotide in an inner ear outer hair cell, a 5'UTR, a polynucleotide that encodes a polypeptide, a tag, a polyA, and a 3'ITR.

[0122] In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter that selectively expresses a polynucleotide in the outer hairs of the inner ear, a polynucleotide encoding a polypeptide, a 3'UTR, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter that selectively expresses a polynucleotide in the outer hairs of the inner ear, a 5'UTR, a polynucleotide encoding a polypeptide, a 3'UTR, a polyA, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter that selectively expresses a polynucleotide in the outer hairs of the inner ear, a 5'UTR, a polynucleotide encoding a polypeptide, a polyA, and a 3'ITR.

[0123] In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter that selectively expresses a polynucleotide in an inner ear outer hair cell, a 5'UTR, a polynucleotide that encodes a polypeptide, a tag, a 3'UTR, a polyA, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter that selectively expresses a polynucleotide in an inner ear outer hair cell, a 5'UTR, a polynucleotide that encodes a polypeptide, a tag, a polyA, and a 3'ITR.

[0124] In some aspects, the construct comprises a polynucleotide encoding a polypeptide operably linked to a promoter that causes expression of the polynucleotide in an inner ear outer hair cell.

[0125] In some embodiments, the construct comprises a 5'ITR, a promoter for expressing a polynucleotide in the outer hair of the inner ear, a polynucleotide encoding a polypeptide, a polyA, and a 3'ITR. In some embodiments, the construct comprises a 5'ITR, a promoter for expressing a polynucleotide in the outer hair of the inner ear, a 5'UTR, a polynucleotide encoding a polypeptide, a polyA, and a 3'ITR.

[0126] In some aspects, the construct comprises a 5'ITR, a promoter for expressing a polynucleotide in the outer hair of the inner ear, a 5'UTR, a polynucleotide encoding a polypeptide, a 3'UTR, a polyA, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, a promoter for expressing a polynucleotide in the outer hair of the inner ear, a 5'UTR, a polynucleotide encoding a polypeptide, a polyA, and a 3'ITR.

[0127] In some aspects, the construct comprises a 5'ITR, a promoter for expressing a polynucleotide in an inner ear outer hair cell, a 5'UTR, a polynucleotide encoding a polypeptide, a tag, a 3'UTR, a polyA, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, a promoter for expressing a polynucleotide in an inner ear outer hair cell, a 5'UTR, a polynucleotide encoding a polypeptide, a tag, a polyA, and a 3'ITR.

[0128] In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter for expressing a polynucleotide in the outer hair of the inner ear, a polynucleotide encoding a polypeptide, a 3'UTR, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter for expressing a polynucleotide in the outer hair of the inner ear, a 5'UTR, a polynucleotide encoding a polypeptide, a 3'UTR, a polyA, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter for expressing a polynucleotide in the outer hair of the inner ear, a 5'UTR, a polynucleotide encoding a polypeptide, a polyA, and a 3'ITR.

[0129] In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter that expresses a polynucleotide in an inner ear outer hair cell, a 5'UTR, a polynucleotide that encodes a polypeptide, a tag, a 3'UTR, a polyA, and a 3'ITR. In some aspects, the construct comprises a 5'ITR, an enhancer, a promoter that expresses a polynucleotide in an inner ear outer hair cell, a 5'UTR, a polynucleotide that encodes a polypeptide, a tag, a polyA, and a 3'ITR.

[0130] AAV particles In particular, the present disclosure provides AAV particles comprising constructs and capsids encoding the polypeptides described herein. In some aspects, the AAV particles can be described as having a serotype that is a description of the construct strain and capsid strain. In some aspects, the AAV particles have an AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV Anc80 serotype. In some aspects, the AAV particles have an AAVAnc80 serotype. In some aspects, the AAV particles can be described as AAV2, where the particle has an AAV2 capsid and a construct that includes a characteristic AAV2 inverted terminal repeat (ITR). In some aspects, the AAV particles may be described as pseudotyped, where the capsids and constructs are derived from different AAV strains, e.g., AAV2 / 9 refers to an AAV particle comprising a construct utilizing the AAV2 ITRs and an AAV9 capsid.

[0131] AAV constructs The present disclosure provides a polynucleotide construct comprising a polypeptide or a characteristic portion thereof. In some aspects described herein, the polynucleotide comprising the polypeptide or a characteristic portion thereof can be included in an AAV particle.

[0132] In some embodiments, the polynucleotide construct comprises one or more components derived from or modified from naturally occurring AAV genome construct.In some embodiments, the sequence derived from the AAV construct is AAV1 construct, AAV2 construct, AAV3 construct, AAV4 construct, AAV5 construct, AAV6 construct, AAV7 construct, AAV8 construct, AAV9 construct, AAV2.7m8 construct, AAV8BP2 construct, AAV293 construct or AAV Anc80 construct.In some embodiments, the construct is derived from AAV Anc80 construct.Additional exemplary AAV constructs that can be used herein are known in the art. See, e.g., Kanaan et al., Mol. Ther. Nucleic Acids 8:184-197, 2017; Li et al., Mol. Ther. 16(7):1252-1260, 2008; Adachi et al., Nat. Commun. 5:3075, 2014; Isgrig et al., Nat. Commun. 10(1):427, 2019; and Gao et al., J. Virol. 78(12):6381-6388, 2004, each of which is incorporated by reference in its entirety.

[0133] In some aspects, the provided constructs include a nucleic acid encoding a coding sequence, e.g., a polypeptide, one or more regulatory and / or control sequences, and optionally 5' and 3' AAV-derived inverted terminal repeats (ITRs). In some aspects where 5' and 3' AAV-derived ITRs are utilized, the polynucleotide constructs may be referred to as recombinant AAV (rAAV) constructs. In some aspects, the provided rAAV constructs are packaged into AAV capsids to form AAV particles. In some aspects, the AAV capsid is an Anc80 capsid (e.g., Anc80L65 capsid).

[0134] In some embodiments, the AAV-derived sequences (which are included in the polynucleotide constructs) typically include cis-acting 5' and 3' ITR sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses," ed., P. Tijsser, CRC Press, pp. 155 168, 1990, which is incorporated by reference in its entirety). A typical AAV2-derived ITR sequence is about 145 nucleotides in length. In some embodiments, at least 75% (e.g., at least 80%, at least 85%, at least 90%, or at least 95%) of a typical ITR sequence is incorporated into the constructs provided herein. The ability to modify these ITR sequences is within the skill of one in the art (see, e.g., textbooks such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York, 1989, and K. Fisher et al., J Virol. 70:520 532, 1996, each of which is incorporated by reference in its entirety). In some aspects, any of the coding sequences and / or constructs described herein are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including currently identified AAV types.

[0135] In some aspects, the polynucleotide constructs described by this disclosure and in a manner known in the art (see, e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, which is incorporated herein by reference in its entirety) typically consist of a coding sequence or a portion thereof, at least one and / or regulatory sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). In some aspects, the provided constructs can be packaged into capsids to create AAV particles. The AAV particles can be delivered to selected target cells. In some aspects, the nucleic acid coding sequence is operably linked to regulatory components in a manner that allows for transcription, translation, and / or expression of the coding sequence in cells of the target tissue.

[0136] In some embodiments, the construct is a rAAV construct.In some embodiments, the rAAV construct can comprise at least 500bp, at least 1kb, at least 1.5kb, at least 2kb, at least 2.5kb, at least 3kb, at least 3.5kb, at least 4kb, or at least 4.5kb.In some embodiments, the AAV construct can comprise at most 7.5kb, at most 7kb, at most 6.5kb, at most 6kb, at most 5.5kb, at most 5kb, at most 4.5kb, at most 4kb, at most 3.5kb, at most 3kb, or at most 2.5kb. In some embodiments, the AAV construct may comprise 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 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

[0137] Any of the constructs described herein may further comprise regulatory and / or control sequences, such as control 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 any combination thereof. In some aspects, the promoter may be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of control sequences are described herein.

[0138] Exemplary Construct Components Inverted terminal repeats (ITRs) The AAV-derived sequences of the construct typically include cis-acting 5' and 3' ITRs (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990), which is incorporated herein by reference in its entirety). In general, the ITRs are capable of forming hairpins. The ability to form hairpins may contribute to the self-priming ability of the ITRs, which allows for primase-independent synthesis of the second DNA strand. The ITRs also play a role in the integration of the AAV construct (e.g., a coding sequence, e.g., a polynucleotide encoding a polypeptide) into the genome of the subject's cells. The ITRs may also assist in the efficient encapsidation of the AAV construct within the AAV particle.

[0139] The rAAV particles (e.g., AAV2 / Anc80 particles) of the present disclosure may include rAAV constructs that include coding sequences (e.g., polynucleotides encoding polypeptides) and associated elements flanked by 5' and 3' AAV ITR sequences. In some aspects, the ITRs are or include about 145 nucleic acids. In some aspects, the ITRs are or include about 119 nucleic acids. In some aspects, the ITRs are or include about 130 nucleic acids. In some aspects, all or substantially all of the ITR coding sequences are used. The AAV ITR sequences may be obtained from any known AAV, including currently identified mammalian AAV types. In some aspects, the ITRs are AAV2 ITRs.

[0140] An example of a construct molecule used in this disclosure is a "cis-acting" construct containing a transgene, in which a selected transgene sequence and associated regulatory elements are flanked by 5' or "left" and 3' or "right" AAV ITR sequences. The designations 5' and left refer to the position of the ITR sequence relative to the entire construct, read from left to right in the sense orientation. For example, in some embodiments, the 5' or left ITR is the ITR that is closest to the promoter (as opposed to the polyadenylation sequence) for a given construct when the construct is linearly represented in the sense orientation. At the same time, the designations 3' and right refer to the position of the ITR sequence relative to the entire construct, read from left to right in the sense orientation. For example, in some embodiments, the 3' or right ITR is the ITR that is closest to the polyadenylation sequence (as opposed to the promoter sequence) for a given construct when the construct is linearly represented in the sense orientation. The ITRs provided herein are represented in the order 5' to 3' according to the sense strand. Thus, one skilled in the art will understand that when converted from sense to antisense orientation, a 5' or "left" oriented ITR can also be referred to as a 3' or "right" ITR. Moreover, it is well within the capabilities of one skilled in the art to convert a given sense ITR sequence (e.g., a 5' / left AAV ITR) into an antisense sequence (e.g., a 3' / right ITR sequence). One skilled in the art will know how to modify a given ITR sequence, or its antisense version, for use as either a 5' / left ITR or a 3' / right ITR.

[0141] For example, in some embodiments, an ITR (e.g., a 5'ITR) may have a sequence according to SEQ ID NO: 16. In some embodiments, an ITR (e.g., a 3'ITR) may have a sequence according to SEQ ID NO: 17. In some embodiments, an ITR comprises one or more modifications, e.g., truncations, deletions, substitutions, or insertions, as known in the art. In some embodiments, an ITR comprises fewer than 145 nucleotides, e.g., 119, 127, 130, 134, or 141 nucleotides. For example, in some embodiments, the ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 nucleotides. In some embodiments, the ITR comprises about 119 nucleotides. In some embodiments, the ITR comprises about 130 nucleotides. In some embodiments, the ITR (e.g., the 5'ITR) may have a sequence according to SEQ ID NO: 16. In some embodiments, the ITR (e.g., the 3'ITR) may have a sequence according to SEQ ID NO: 17.

[0142] Non-limiting examples of 5' AAV ITR sequences include SEQ ID NO: 16 or 46. Non-limiting examples of 3' AAV ITR sequences include SEQ ID NO: 17 or 47. In some embodiments, the 5' and 3' AAV ITRs (e.g., SEQ ID NO: 16 and 17, or SEQ ID NO: 46 and 47) flank a portion of a coding sequence, e.g., all or a portion of a polynucleotide encoding a polypeptide. The ability to modify these ITR sequences is within the skill of one in the art (see, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York, 1989, and K. Fisher et al., J Virol. 70:520 532 (1996), each of which is incorporated herein by reference in its entirety). In some embodiments, the 5'ITR 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%, or at least 99%, or 100% identity to SEQ ID NO: 16. In some embodiments, the 5'ITR sequence has the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the 5'ITR 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%, or at least 99%, or 100% identity to SEQ ID NO: 46. In some embodiments, the 5'ITR comprises the nucleic acid sequence of SEQ ID NO: 46.

[0143] In some embodiments, the 3'ITR 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%, or at least 99%, or 100% identity to SEQ ID NO: 17. In some embodiments, the 3'ITR comprises a nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the 3'ITR 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%, or at least 99%, or 100% identity to SEQ ID NO: 47. In some embodiments, the 3'ITR comprises a nucleic acid sequence of SEQ ID NO: 47.

[0144] In some embodiments, the 3' ITR 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%, or at least 99%, or 100% identity to SEQ ID NO: 51. In some embodiments, the 3' ITR comprises the nucleic acid sequence of SEQ ID NO:51. Exemplary 5' AAV ITR (SEQ ID NO:46) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT Exemplary 3' AAV ITR (SEQ ID NO:47) AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA Exemplary 5' AAV ITR (SEQ ID NO: 16) CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT Exemplary 3' AAV ITR (SEQ ID NO:17) AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG Exemplary 5'ITR (SEQ ID NO:51): AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGGGAGTGGCCAA

[0145] promoter In some aspects, the disclosure relates to constructs comprising cell-selective promoters that can be used to regulate (e.g., increase) expression of a polynucleotide encoding a polypeptide in a cell (e.g., an inner ear cell, e.g., an outer hair cell). In some aspects, the increased expression is relative to endogenous polynucleotide expression in the cell.

[0146] In some aspects, a construct (e.g., a rAAV construct) comprises a promoter. The term "promoter" refers to a DNA sequence that is recognized by an enzyme / protein that can promote and / or initiate transcription of an operably linked gene (e.g., a polynucleotide encoding a polypeptide). For example, a promoter typically refers to a nucleotide sequence to which, for example, an RNA polymerase and / or any associated factors can bind and initiate transcription. Thus, in some aspects, a construct (e.g., a rAAV construct) comprises a polynucleotide operably linked to one of the non-limiting examples of promoters described herein.

[0147] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, a cell-selective promoter, or any other type of promoter known in the art. In some embodiments, the promoter is an RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some embodiments, the promoter is an RNA polymerase III promoter, including but not limited to an H1 promoter, a human U6 promoter, a mouse U6 promoter, or a porcine U6 promoter. The promoter is generally one that can promote transcription in inner ear cells. In some embodiments, the promoter is a cochlea-selective promoter or a cochlea-directed promoter. In some embodiments, the promoter is a hair cell-selective promoter or an outer hair cell-selective promoter. In some embodiments, the promoter is an inner ear outer hair cell-selective promoter.

[0148] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a nucleic acid encoding a protein (e.g., a polypeptide), causes transcription of RNA from the nucleic acid in a cell under most or all physiological conditions.

[0149] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al, Cell 41:521-530, 1985, which is incorporated herein by reference in its entirety), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl-alpha promoter (Invitrogen). In some aspects, the promoter is a constitutive promoter. In some aspects, the constitutive promoter is a CAG promoter, a CBA promoter, a CMV promoter, a CMV / CBA enhancer / promoter, or a CB7 promoter.

[0150] In some aspects, the regulatory and / or control sequences confer cell-selective gene expression. In some instances, the cell-selective regulatory and / or control sequences bind cell-selective transcription factors that induce transcription in a cell-selective manner.

[0151] In some aspects, the cell-selective promoter is an ear cell-selective promoter. In some aspects, the cell-selective promoter is an inner ear cell-selective promoter. In some aspects, the promoter is an inner ear outer hair cell-selective promoter.

[0152] In some embodiments, the inner ear outer hair cell selective promoter is selected from one or more of oncomodulin, prestin, CHRNA10, DNM3, MUC15, PLBD1, RORB, STRIP2, AQP11, KCNQ4, LBH, STRC, TUBA8, or any combination thereof.

[0153] In some embodiments, the inner ear outer hair cell selective promoter is an oncomodulin promoter. In some embodiments, the oncomodulin promoter has 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: 1-2. In some embodiments, the oncomodulin promoter has the nucleic acid sequence of any one of SEQ ID NOs: 1-2.

[0154] In some embodiments, the oncomodulin 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 any one of SEQ ID NO: 1. In some embodiments, the oncomodulin promoter is the nucleic acid sequence of SEQ ID NO:1.

[0155] In some embodiments, the oncomodulin 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 any one of SEQ ID NO: 2. In some embodiments, the oncomodulin promoter is the nucleic acid sequence of SEQ ID NO: 2.

[0156] In some embodiments, the oncomodulin promoter is 100-2000, 200-1800, 300-1700, 400-1600, 500-1500, 600-1400, 700-1300, 800-1200, 900-1100, 950-1050, or 1000-1050 nucleotides in length. In some embodiments, the oncomodulin promoter is 1000-1050 nucleotides in length.

[0157] In some embodiments, the oncomodulin promoter is 500-2500, 600-2400, 700-2300, 800-2200, 900-2100, 1000-2000, 1100-1900, 1200-1800, 1300-1700, 1400-1600, or 1450-1500 nucleotides in length. In some embodiments, the oncomodulin promoter is 1450-1500 nucleotides in length. Exemplary Oncomodulin Promoter (SEQ ID NO:1) Exemplary Oncomodulin Promoter (SEQ ID NO:2)

[0158] In some embodiments, the inner ear outer hair cell selective promoter is a prestin promoter. In some embodiments, the prestin promoter has 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 NO: 3 or 15. In some embodiments, the prestin promoter has the nucleic acid sequence of any one of SEQ ID NO: 3 or 15.

[0159] In some embodiments, the inner ear outer hair cell selective promoter is a prestin promoter. In some embodiments, the prestin promoter comprises a nucleic acid sequence that is 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 any one of SEQ ID NO: 3. In some embodiments, the prestin promoter is the nucleic acid sequence of SEQ ID NO: 3.

[0160] In some embodiments, the inner ear outer hair cell selective promoter is a prestin promoter. In some embodiments, the prestin promoter comprises a nucleic acid sequence that is 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 any one of SEQ ID NO: 15. In some embodiments, the prestin promoter is the nucleic acid sequence of SEQ ID NO: 15.

[0161] In some embodiments, the prestin promoter is 500-2500, 600-2400, 700-2300, 800-2200, 900-2100, 1000-2000, 1100-1900, 1200-1800, 1300-1700, 1400-1600, 1450-1550, or 1500-1550 nucleotides in length. In some embodiments, the prestin promoter is 1500-1550 nucleotides in length.

[0162] In some embodiments, the prestin promoter is 1000-3000, 1100-2900, 1200-2800, 1300-2700, 1400-2600, 1500-2500, 1600-2400, 1700-2300, 1800-2200, 1850-1950, 1900-1950 nucleotides in length. In some embodiments, the prestin promoter is 1900-1950 nucleotides in length. Exemplary prestin promoter (SEQ ID NO:3) Exemplary prestin promoter (SEQ ID NO: 15)

[0163] In some embodiments, the inner ear outer hair cell selective promoter is a CHRNA10 promoter. In some embodiments, the CHRNA10 promoter has 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: 4. In some embodiments, the CHRNA10 promoter has the nucleic acid sequence of SEQ ID NO: 4.

[0164] In some embodiments, the CHRNA10 promoter is 100-1200, 200-1100, 300-1000, 400-950, 500-900, 600-850, 700-800, or 700-750 nucleotides in length. In some embodiments, the CHRNA10 promoter is 740 nucleotides in length. Exemplary CHRNA10 promoter (SEQ ID NO:4) TTCAGATGCCATCATTAATGAGAACTATGACTACCTGAAGGGGTTCTTGGAAGACCTGGCAAGGAACTCCCCTTGGATTAATTGGCTTCTCTGCTTCTTTGTAGGTGGATTGCTCAGGTAATGACCTGGAGCAGTTACACATCAAAGTGACTTCACTGTGCAGTCGGATAGAGCAGATTCAGTGT CTGGTATTGGCTTTCCCTTTGTATTTTTTGAATAGAATATACCATTCAAAGCCTCCTCGCTCTTCTACTATAGTGGTTTTGTTTTTAAACCCTGAGTGACGCTTCACCTTTCTAAATCAGATTCCCTTTGTAAAGGGGATAATGATTGCTGATGTTACTTCACACAGGGCTATTTTCAAGAGGAA TCAATTGAGTAGCATGAGTACTATTCCAGATCTTATTTTGATCTGTCAAGCTGAAGATGTGAGCAAATTCCAATTAAGATTAGACCAAAGACTTCTGAGACTTTCAGGAATTCAGGGATGAGAAAGCAGAGTGGGTCAGCTCTGTTGTCTGGAACTTCCATTTAACTTAGATGCCTCAGGATAGG GGTTACTCAGCTGGAATCCCCTCCACTACTGACTCACTATGTGAACCTGAGTGAGTCACAAAACATAGTTGGACTTCCAGCAAAGAACACCTGACCTGGTTTCCTTACCAGAGGAATGTTTCAGAAAGTGAGTATGCTATAGAAATGGTTAGCTCTTAGCAGTGTTCGGAATTGTGGGCCAGGAG

[0165] In some embodiments, the inner ear outer hair cell selective promoter is a DNM3 promoter. In some embodiments, the DNM3 promoter has 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:5. In some embodiments, the DNM3 promoter has the nucleic acid sequence of SEQ ID NO:5.

[0166] In some embodiments, the DNM3 promoter is 100-2000, 200-1800, 300-1700, 400-1600, 500-1500, 600-1400, 700-1300, 800-1200, 850-1000, or 900-950 nucleotides in length. In some embodiments, the DNM3 promoter is 900-950 nucleotides in length. Exemplary DNM3 Promoter (SEQ ID NO:5) CCTTGATTCAGAGTTAAAGCTATGGGAAAGTCCTCAGGCAGAGGACAAACATTAGACAAGAAAATGCCCATATATGAAACCCTGCGAAGCATCAGTATTTGAGGAGCAGACTAAAAAGGAACCGTCTGTGGAGGCTAAGAGAAGCATGGCCATTTATCTTTGTGTCCCGATCATCAGGCACAGGACCCCACACACAGTCACTTCTCAATGTGCTAAATTTCACAGAATGCGTCCAGGGTACCTGGTTCTGGATAGATCCGGTAGAAGGAGATAGACCGGGAGGGCAAATGGCATGAGGAGTCTCACAGGCCAGAGTGATTAAAGGGGTGTATCGGGGCGGTAAACCCTACAGACTCTACCTGTGCTTATGCGGGGCTGGGGAGGACGAGTCATTACAGATGAAGAATTAAGTAAGGTCAGACCACTCAGGGCCTTAGATGGATGTCACATTGAAGAATTTAGACTCCAACAGGCCTGCCACCCTGGGAGGAGTCATCGCGGATTCTGGAGAAGGGCGTGACAGAGGAGATTTCCTTTCGGGAAGTGTAGTCTGGCAGCGGTGCCCCGGTGGTGGCGGCGGCGGTGCTGCTGTTGCTGGTGATCGTGTGGTGGTGTTAGCGGCGATAGTGCTTTCCACTGGGCTTTGGCTTGGTAGCCGCTGAAAGAGAACAACGCTGCCGCTGCTGCTGATTTCATGCCATTTCCTGACCCGGCGCTGTAACTTGGCCTCTGAGCCTTGGCCACAGAACGCAGAGGCCGTGGCATCTGGCCGCAGCTGGGCTGCAGTGCGTGCGCGCCTGGCCTGGTGGTCCGATGGGAAGCCCGGGGCGGGGCAGCCGCGGGGCGGGGGCGGGGCGTCGCGGAGATAGGCCACGCCCCTGCCCGCCCGCGCAGGCGCGCTGCGGGTCGTTAGCTGTC

[0167] In some embodiments, the inner ear outer hair cell selective promoter is a MUC15 promoter. In some embodiments, the MUC15 promoter has 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:6. In some embodiments, the MUC15 promoter has the nucleic acid sequence of SEQ ID NO:6.

[0168] In some embodiments, the MUC15 promoter is 500-2500, 600-2400, 700-2300, 800-2200, 900-2100, 1000-2000, 1100-1900, 1200-1800, 1400-1700, 1500-1650, or 1600 nucleotides in length. In some embodiments, the MUC15 promoter is 1600-1650 nucleotides in length. Exemplary MUC15 Promoter (SEQ ID NO:6)

[0169] In some embodiments, the inner ear outer hair cell selective promoter is a PLBD1 promoter. In some embodiments, the PLBD1 promoter has 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:7. In some embodiments, the PLBD1 promoter has the nucleic acid sequence of SEQ ID NO:7.

[0170] In some embodiments, the PLBD1 promoter is 100-2000, 200-1800, 300-1700, 400-1600, 500-1500, 600-1400, 700-1300, 800-1200, 850-1100, 900-1050, or 950-1000 nucleotides in length. In some embodiments, the PLBD1 promoter is 950-1000 nucleotides in length. Exemplary PLBD1 Promoter (SEQ ID NO:7) GACCCATTATTCAATGGGAGTTGTCAGGATGTCAGCAATGTACAAAATCATTGCTTAATTTGTTTGACAATGGAAATGGCCATTATGGTTTTTATGTAACTTTGCTTCTGTTACATAATTCTTGCTGACACGGTGTTTCAACCAAGGTACTTGGTAGCAAGTGTTGTACAGAAAAGGATCTGTAAGTGGTTTATGTGGTCATCAACCACAGCAAAGATTTCATCTGAGCTGTGCTATGAAGAATGTAGCTTGAGAAACACAAAATGTATCACTGGGCAAAAAGGAAGCAGAAGAAAAATACAGTTCTGCTAATGAGAGCTCTGACTGGTATCTGGAGTATAAGATGGGCCCAGCCAATGCTGAGTGAATGAATGAAATGCCTTTTGCCTACTTCACAATGTCACCTAGGGCACCCGGTGCCAACTTCACAATATCACCCAAGGCATAACTTTTGACTACTTCACAATGTCACTTTTAACTGACCCCACACAGAAATGGGGACTCCACAGAAACGTAGGAGTGTGTCTAGTGTCAGCCCCGTCTGAATCACTCTCCTGTGGTGGCTCCAGCCAACGAAGAGGAAGCAAAAAGGATAAAAAATCTGAGCTACAGCGCATGGATTTAGGTTAAACAGCCTGGGAATGAGGGGTACGCTAATCGCTGAGGAAAACGCACCTGTGGAGGCCTCTCCAGAAACAGCAGAGGATCCGAGCTGCGTGTAGGCAGGGCGCGCATGTCACCCTGGCCCGGGCGCCTGGTCCGCTGCTGGAGATAAATGGTCGACCCCGGAGGGAGAGGCTAGTAGGGGTGTTGATGTGAACTGATTCGCCCAAGCCTTGGGCCGCAAAACTGCGAAAGAAAGCGGCAGGCAGCCTCTGCATTTCCCAGAAGTGCAGCTGGGGAACTTTCCCAGACCGGCCCAGGGGTTGCTAGAGGGTCAGACGTAAAGGATCCGCCTTTCCTAGGCGGGGTGGGCCCCAGGCC

[0171] In some embodiments, the inner ear outer hair cell selective promoter is a RORB promoter. In some embodiments, the RORB promoter has 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:8. In some embodiments, the RORB promoter has the nucleic acid sequence of SEQ ID NO:8.

[0172] In some embodiments, the RORB promoter is 500-2500, 600-2400, 700-2300, 800-2200, 900-2100, 1000-2000, 1100-1900, 1200-1800, 1250-1700, 1300-1600, 1350-1550, 1400-1500, or 1400-1450 nucleotides in length. In some embodiments, the RORB promoter is 1400-1450 nucleotides in length. An exemplary RORB promoter (SEQ ID NO:8)

[0173] In some embodiments, the inner ear outer hair cell selective promoter is a STRIP2 promoter. In some embodiments, the STRIP2 promoter has 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:9. In some embodiments, the STRIP2 promoter has the nucleic acid sequence of SEQ ID NO:9.

[0174] In some embodiments, the STRIP2 promoter is 500-2000, 600-1900, 700-1800, 800-1700, 900-1600, 1000-1500, 1100-1400, 1200-1300, or 1250-1300 nucleotides in length. In some embodiments, the STRIP2 promoter is 1250-1300 nucleotides in length. Exemplary STRIP2 Promoter (SEQ ID NO:9)

[0175] In some embodiments, the inner ear outer hair cell selective promoter is the AQP11 promoter. In some embodiments, the AQP11 promoter has 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: 10. In some embodiments, the AQP11 promoter has the nucleic acid sequence of SEQ ID NO: 10.

[0176] In some embodiments, the AQP11 promoter is 500-2000, 600-1900, 700-1800, 800-1700, 900-1600, 1000-1500, 1100-1400, 1200-1300, or 1250-1300 nucleotides in length. In some embodiments, the AQP11 promoter is 1250-1300 nucleotides in length. Exemplary AQP11 promoter (SEQ ID NO:10)

[0177] In some embodiments, the inner ear outer hair cell selective promoter is a KCNQ4 promoter. In some embodiments, the KCNQ4 promoter has 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: 11. In some embodiments, the KCNQ4 promoter has the nucleic acid sequence of SEQ ID NO: 11.

[0178] In some embodiments, the KCNQ4 promoter is 500-2500, 600-2400, 700-2300, 800-2200, 900-2100, 1000-2000, 1100-1900, 1200-1800, 1300-1750, 1400-1700, 1450-1650, or 1550-1600 nucleotides in length. In some embodiments, the KCNQ4 promoter is 1550-1600 nucleotides in length. Exemplary KCNQ4 Promoter (SEQ ID NO:11)

[0179] In some embodiments, the inner ear outer hair cell selective promoter is an LBH promoter. In some embodiments, the LBH promoter has 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: 12. In some embodiments, the LBH promoter has the nucleic acid sequence of SEQ ID NO: 12.

[0180] In some embodiments, the LBH promoter is 500-2000, 600-1900, 700-1800, 800-1700, 900-1600, 1000-1500, 1100-1400, 1150-1300, or 1200-1250 nucleotides in length. In some embodiments, the LBH promoter is 1200-1250 nucleotides in length. Exemplary LBH promoter (SEQ ID NO:12)

[0181] In some embodiments, the inner ear outer hair cell selective promoter is the STRC promoter. In some embodiments, the STRC promoter has 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: 13. In some embodiments, the STRC promoter has the nucleic acid sequence of SEQ ID NO: 13.

[0182] In some embodiments, the STRC promoter is 100-2100, 200-2000, 300-1900, 400-1800, 500-1700, 600-1600, 700-1500, 800-1400, 900-1300, 1100-1200, or 1100-1150 nucleotides in length. In some embodiments, the STRC promoter is 1100-1150 nucleotides in length. An exemplary STRC promoter (SEQ ID NO: 13)

[0183] In some embodiments, the inner ear outer hair cell selective promoter is a TUBA8 promoter. In some embodiments, the TUBA8 promoter has 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: 14. In some embodiments, the TUBA8 promoter has the nucleic acid sequence of SEQ ID NO: 14.

[0184] In some embodiments, the TUBA8 promoter is 1000-2600, 1100-2500, 1200-2400, 1300-2300, 1400-2200, 1500-2100, 1600-2000, 1700-1900, 1800-1900, or 1800-1850 nucleotides in length. In some embodiments, the TUBA8 promoter is 1800-1850 nucleotides in length. Exemplary TUBA8 promoter (SEQ ID NO:14) [Table 2]

[0185] Enhancer In some embodiments, the construct may include an enhancer sequence. In some embodiments, the construct does not include an enhancer sequence. The term "enhancer" refers to a nucleotide sequence that can increase the transcription level of a nucleic acid encoding a protein (e.g., a polypeptide) of interest. Enhancer sequences (generally 50-1500 bp in length) generally increase the transcription level by providing additional binding sites for transcription-associated proteins (e.g., transcription factors). In some embodiments, enhancer sequences are found within intron sequences. Unlike promoter sequences, enhancer sequences can act much further away from the transcription start site (e.g., compared to promoters). Non-limiting examples of enhancers include RSV enhancers, CMV enhancers, and / or SV40 enhancers. In some embodiments, the construct includes a CMV enhancer 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 SEQ ID NO:18. In some embodiments, the CMV enhancer comprises the nucleic acid sequence of SEQ ID NO:18. Exemplary CMV enhancer (SEQ ID NO: 18) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG

[0186] Flanking untranslated regions, 5'UTR and 3'UTR In some aspects, any of the constructs described herein may include an untranslated region (UTR), such as a 5'UTR or a 3'UTR. The UTR of a gene is transcribed but not translated. The 5'UTR begins at the transcription start site and continues up to, but not including, the start codon. The 3'UTR begins immediately after the stop codon and continues to the transcription termination signal. The regulatory and / or control features of the UTR can be incorporated into any of the constructs, compositions, kits, or methods described herein to enhance or otherwise regulate the expression of a polypeptide.

[0187] Natural 5'UTR contains sequences that play a role in translation initiation. In some embodiments, 5'UTR can contain sequences such as the Kozak sequence, which is generally known to be involved in the process by which ribosomes initiate the translation of many genes. The Kozak sequence has the consensus sequence CCR(A / G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), which is followed by another "G". 5'UTR is also known to form secondary structures that are involved in the binding of elongation factors.

[0188] In some embodiments, a 5'UTR is included in any of the constructs described herein. Non-limiting examples of 5'UTRs can be used to enhance expression of nucleic acid molecules such as mRNAs, including 5'UTRs from the following genes: albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, and factor VIII.

[0189] In some aspects, a 5'UTR from an mRNA transcribed by cells in the cochlea may be included in any of the constructs, compositions, kits, and methods described herein. In some aspects, the 5'UTR is derived from the 5'UTR of a polynucleotide encoding a polypeptide. In some aspects, the 5'UTR is derived from an endogenous KCNQ4 5'UTR. In some aspects, the 5'UTR comprises 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:19. In some aspects, the 5'UTR comprises the nucleic acid sequence of SEQ ID NO:19.

[0190] The 3'UTR is found immediately 3' to the stop codon of the gene of interest. In some aspects, a 3'UTR from an mRNA transcribed by a cell in the cochlea may be included in any of the constructs, compositions, kits, and methods described herein. In some aspects,

[0191] In some embodiments, the 3'UTR is derived from the 3'UTR of the polynucleotide encoding the polypeptide.In some embodiments, the 3'UTR is derived from endogenous KCNQ4 3'UTR.In some embodiments, the KCNQ4 3'UTR comprises 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:45.In some embodiments, the KCNQ4 3'UTR comprises the nucleic acid sequence of SEQ ID NO:45.

[0192] In some embodiments, the 3'UTR comprises 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: 45. In some embodiments, the 3'UTR comprises the nucleic acid sequence of SEQ ID NO:45.

[0193] 3'UTRs are known to have stretches of adenosines and uridines (in RNA form) or thymidines (in DNA form) embedded within them. These AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on their sequence features and functional properties, AU-rich elements (AREs) can be separated into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995, each of which is incorporated herein by reference in its entirety): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs. Class III AREs are less well defined. These U-rich regions do not contain AUUUA motifs, and two well-studied examples of this class are c-Jun and myogenin mRNAs.

[0194] While most proteins that bind to AREs are known to destabilize messengers, members of the ELAV family, particularly HuR, have been demonstrated to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding and thus stabilization of the message in vivo.

[0195] In some embodiments, the introduction, removal, or modification of a 3'UTR ARE can be used to modulate the stability of an mRNA encoding a polypeptide, while in other embodiments, the ARE can be removed or mutated to increase the intracellular stability of the polypeptide, thus increasing its translation and production.

[0196] In other embodiments, non-ARE sequences may be incorporated into the 5' or 3' UTR. In some embodiments, introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the constructs, compositions, kits, and methods provided herein. The incorporation of intron sequences may increase protein production as well as mRNA levels. Exemplary 5'UTR Sequence (SEQ ID NO:19) CGCCGGTGGCAGGTGGAAAGGCGAGCGGCATGGAGCGCGTAATAAGAGAGTTGGAGTCGGAAAGAGCAGCCCCAGTCGCCGGGGAAGCGGGAGGTCAGTGCGGGCTCCGGCGGCCCCCAGGCTCCGAGCGCCCGCCCGCGGCCCC GGCCCGGCCCCTAGCCCCCGCCGCCCGCGCCCGCCCCGGGTCGCCCCTCTGGCCCCGGGTCCGAGCCATGCGTCTCTGAGCGCCCAGAGCGCGCCCCCGCCCCGGACCGTGCCCGGGCCCCGGCGCCCCCAGCCCGGCGCCGCCC Exemplary 3'UTR Sequence (SEQ ID NO:45)

[0197] Polyadenylation sequence In some aspects, the constructs provided herein may include a polyadenylation (poly(A)) signal sequence. Most nascent mRNAs in eukaryotes have poly(A) tails at their 3' ends that are added in a complex process involving cleavage of the primary transcript and coupled polyadenylation driven by the poly(A) signal sequence (see, e.g., Proudfoot et al., Cell 108:501-512, 2002, which is incorporated herein by reference in its entirety). The poly(A) tail confers mRNA stability and transportability (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994, which is incorporated herein by reference in its entirety). In some aspects, the poly(A) signal sequence is located 3' to the coding sequence.

[0198] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety, or modified variants thereof, to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long stretch of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) that is added to a pre-mRNA by the action of the enzyme polyadenylate polymerase. In some aspects, the poly(A) tail is added to transcripts that contain specific sequences, e.g., polyadenylation (or poly(A)) signals. The poly(A) tail and associated proteins help protect the mRNA from degradation by exonucleases. Polyadenylation also plays a role in transcription termination, nuclear export of mRNA, and translation. Polyadenylation signals typically occur in the nucleus immediately following transcription of DNA into RNA, but can occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0199] As used herein, a "poly(A) signal sequence" or "polyadenylation signal sequence" is a sequence that triggers endonucleolytic cleavage of mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0200] There are several poly(A) signal sequences that can be used, including those from bovine growth hormone (bGH) (Woychik et al., Proc. Natl. Acad Sci. US.A. 81(13):3944-3948, 1984; U.S. Pat. No. 5,122,458, each of which is incorporated herein by reference in its entirety), mouse-β-globin, mouse-α-globin (Orkin et al., EMBO J 4(2):453-456, 1985; Thein et al., Blood 71(2):313-319, 1988, each of which is incorporated herein by reference in its entirety), human collagen, polyoma virus (Batt et al., Mol. Cell 2002, 144:1111-1112, 1982), human collagen, ... Biol. 15(9):4783-4790, 1995, which is incorporated herein by reference in its entirety), herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy chain gene polyadenylation signal (US2006 / 0040354, which is incorporated herein by reference in its entirety), human growth hormone (hGH) (Szymanski et al., Mol. Therapy 15(7):1340-1347, 2007, which is incorporated herein by reference in its entirety), and the group including the SV40 poly(A) site, such as the SV40 late and early poly(A) sites (Schek et al., Mol. Cell Biol. 12(12):5386-5393, 1992, which is incorporated herein by reference in its entirety).

[0201] The poly(A) signal sequence can be AATAAA. The AATAAA sequence may be replaced with other hexanucleotide sequences that have homology to AATAAA and can signal polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e.g., WO06 / 12414, which is incorporated herein by reference in its entirety).

[0202] In some embodiments, the poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., Promega's pCl-neo expression construct, based on Levitt el al, Genes Dev. 3(7):1019-1025, 1989, which is incorporated by reference in its entirety). In some embodiments, the poly(A) signal sequence comprises or consists of an SV40 poly(A) site. In some embodiments, the poly(A) signal sequence comprises or consists of bGHpA. In some embodiments, the poly(A) signal sequence comprises 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:22. In some embodiments, the poly(A) signal sequence comprises the nucleic acid sequence of SEQ ID NO:22. In some embodiments, the poly(A) signal sequence comprises 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: 48. In some embodiments, the poly(A) signal sequence comprises the nucleic acid sequence of SEQ ID NO:48. Exemplary bGH poly(A) signal sequence (SEQ ID NO:22) CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGTGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATG Exemplary SV40 poly(A) signal sequence (SEQ ID NO:48) AACTTGTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

[0203] Additional Arrays In some embodiments, the constructs of the present disclosure may include one or more filler sequences. In some embodiments, the filler sequences may function as regulatory elements that alter the expression of the construct. In some such embodiments, the filler sequences may not be fully removed before preparation for administration to a subject. In some embodiments, the filler sequences may have functional roles, including as linker sequences, regulatory regions, or stabilizing regions. As will be understood by those skilled in the art, filler sequences may differ significantly in primary sequence while retaining their desired functions.

[0204] In some embodiments, the construct of the present disclosure may include one or more cloning sites. In some such embodiments, the cloning site may not be fully removed before manufacturing for administration to a subject. In some embodiments, the cloning site may have a functional role, including as a linker sequence, as part of a Kozak site, or as a site that codes for a stop codon. As will be understood by those skilled in the art, the cloning sites may differ significantly in primary sequence while retaining their desired function. In some embodiments, the construct may contain additional cloning sites that are less than 5 nucleotides in length.

[0205] Reporter Polypeptides, Sequences, or Elements In some aspects, the constructs provided herein may optionally include sequences encoding reporter polypeptides and / or proteins ("reporter sequences"). Non-limiting examples of reporter sequences include DNA sequences encoding beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein, mCherry fluorescent protein, yellow fluorescent protein, chloramphenicol acetyltransferase (CAT), and luciferase. Additional examples of reporter sequences are known in the art. Non-limiting examples of reporter polypeptides include beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein, mCherry fluorescent protein, yellow fluorescent protein, chloramphenicol acetyltransferase (CAT), and luciferase. Additional examples of reporter sequences are known in the art.

[0206] When associated with control elements that drive expression of the reporter sequence, the reporter sequence can provide a signal detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescent, or other spectrophotometric assays; fluorescence activated cell sorting (FACS) assays; immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0207] In some embodiments, the reporter sequence is the LacZ gene, and the presence of the construct carrying the LacZ gene in mammalian cells (e.g., cochlear hair cells) is detected by assaying for beta-galactosidase activity. If the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of the construct carrying the fluorescent protein or luciferase in mammalian cells (e.g., cochlear hair cells) may be measured by fluorescent techniques (e.g., fluorescent microscopy or FACS) or by light generation in a luminometer (e.g., a spectrophotometer or IVIS imager). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter modulating and / or controlling activity of any of the constructs described herein.

[0208] In some aspects, the reporter polypeptide is a FLAG tag (e.g., a 3xFLAG tag) and the presence of a construct carrying the FLAG tag in a mammalian cell (e.g., an inner ear cell, e.g., an outer hair cell) is detected by a protein binding or detection assay (e.g., Western blot, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry). Exemplary 3xFLAG tag sequences are provided as SEQ ID NOs: 21 and 39. Exemplary 3xFLAG tag sequence (SEQ ID NO:21) GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG Exemplary 3xFLAG tag sequence including stop codon (SEQ ID NO:39) GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTAA

[0209] AAV Capsid The present disclosure provides one or more polynucleotide constructs packaged in an AAV capsid. In some aspects, the AAV capsid is from or derived from AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rh10, rh39, rh43, or Anc80 serotypes, or one or more hybrids thereof. In some aspects, the AAV capsid is from an AAV ancestral serotype. In some aspects, the AAV capsid is an ancestral (Anc) AAV capsid. Anc capsids are generated from construct sequences that are constructed using evolutionary probability and evolutionary modeling to determine likely ancestral sequences. Thus, the Anc capsid / construct sequence is not known to have existed in nature. For example, in some aspects, the AAV capsid is an Anc80 capsid (e.g., Anc80L65 capsid). In some embodiments, the AAV capsid is generated using a template nucleotide coding sequence comprising SEQ ID NO: 40. In some embodiments, the capsid comprises a polypeptide represented by SEQ ID NO: 41. In some embodiments, the capsid comprises a polypeptide that is 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 the polypeptide represented by SEQ ID NO: 41.

[0210] As provided herein, any combination of AAV capsids and AAV constructs (e.g., including AAV ITRs) may be used in the recombinant AAV (rAAV) particles of the present disclosure. For example, wild-type or variant AAV2 ITRs and Anc80 capsid (e.g., Anc80L65 capsid), wild-type or variant AAV2 ITRs and AAV6 capsid, etc. In some embodiments of the present disclosure, the AAV particles are entirely composed of AAV2 components (e.g., the capsid and ITRs are of AAV2 serotype). In some embodiments, the AAV particles are AAV2 / 6, AAV2 / 8, or AAV2 / 9 particles (e.g., AAV constructs having AAV6, AAV8, or AAV9 capsids and AAV2 ITRs). In some aspects of the present disclosure, the AAV particle is an AAV2 / Anc80 particle that includes an Anc80 capsid (e.g., a polypeptide of SEQ ID NO: 41) that encapsidates an AAV construct having a coding sequence, e.g., AAV2 ITRs (e.g., SEQ ID NOs: 16 and 17) flanking a portion of a nucleic acid encoding a polypeptide. Other AAV particles are known in the art and are described, for example, in Sharma et al., Brain Res Bull. 2010 Feb 15; 81(2-3):273, which is incorporated herein by reference in its entirety. In some aspects, the capsid sequence is 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 the capsid nucleotide or amino acid sequence represented by SEQ ID NO: 40 or 41, respectively.

[0211] In some aspects, the composition comprises a construct comprising: (i) a 5'ITR (e.g., an AAV 5'ITR); (ii) a 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:1-15; (iii) a polynucleotide encoding a polypeptide (e.g., a therapeutic polypeptide); (v) optionally, a 3xFLAG tag (e.g., comprising the nucleic acid sequence of SEQ ID NO:39); (vi) a polyA sequence; and (vii) a 3'ITR (e.g., an AAV 3'ITR).

[0212] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a polynucleotide encoding a polypeptide; (iv) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0213] In some aspects, the composition comprises: (i) a 5' ITR; (ii) a promoter comprising any one of the nucleic acid sequences of SEQ ID NOs: 1-15; (iii) an IL-1 or IL-2 nucleotide sequence encoding actin gamma 1 (ACTG1), adenylate cyclase type 1 (ADCY1), calcium binding protein 2 (CABP2), coiled-coil domain containing 50 (CCDC50), cadherin-associated 23 (CDH23), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), chromodomain helicase DNA-binding protein 7 (CHD7), calcium and integrin binding family. member 2 (CIB2), claudin 14 (CLDN14), intracellular chloride channel 5 (CLIC5), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), clarin 1 (CLRN1), pedivakin (DFNB59), endothelin 3 (EDN3), ELMO domain-containing protein 3 (ELMOD3), epidermal growth factor receptor kinase substrate 8 (EPS8), espin (ESPN), estrogen-related receptor beta (ESRRB), eyes absent homolog 1 (EYA1), GIPC PDZ domain-containing family, member 3 (GIPC3), G protein-coupled receptor 98 (GPR98), G protein signaling modulator 2 (GPSM2), glutaredoxin, cysteine-rich 1 (GRXCR1), glutaredoxin, cysteine-rich 2 (GRXCR2), immunoglobulin-like domain-containing receptor 1 (ILDR1), lysyl-tRNA synthetase (KARS), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), lipoma HMGIC fusion partner-like 5 (LHFPL5), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT1 COMT2), tricellulin (MARVELD2), microRNA96 (MIR96), methionine sulfoxide reductase B3 (MSRB3), nonmuscle myosin heavy chain 9 (MYH9), nonmuscle myosin heavy chain 14 (MYH14), atypical myosin IIIA (MYO3A), atypical myosin VI (MYO6), atypical myosin VIIA (MYO7A), atypical myosin XVA (MYO15A), otoferlin (OTOF), otogelin-like protein (OTOGL), purinergic receptor P2X, ligand-gated ion channel, 2 (P2RX2), protocadherin 15 (PCDH15), PDZ domain-containing 7 (PDZD7), polyribonucleotide nucleotidyltransferase 1, mitochondrial (PNPT1), POU domain, class 4, transcription factor 3 (POU4 F3), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), protein tyrosine phosphatase, receptor type Q (PTPRQ), radixin (RDX), scaffold-containing ankyrin repeats and SAM domain (SANS), serpin peptidase inhibitor, clade B, member 6 (SERPINB6), SIX homeobox 1 (SIX1), SIX homeobox 5 (SIX5), prestin (SLC26A5), second mitochondrial-derived caspase activator (SMAC / DIABLO), small muscle protein, x-linked (SMPX), stereocillin (STRC), nesprin-4 (SYNE4), TBC1 domain family, member 24 (TBC / D24), tight junction protein XO (v) a polynucleotide encoding an outer hair cell polypeptide comprising a gene selected from: (i) transmembrane channel-like protein 2 (TJP2), (ii) transmembrane inner ear expressed protein (TMIE), transmembrane protease, serine 3 (TMPRSS3), (iii) tapelin (TPRN), (iv) TRIO and F-actin binding protein (TRIOBP), thrombospondin-type laminin G domain and EAR repeats (TSPEAR), (v) harmonin (USH1C), (v) usherin (USH2A), (v) wolframin (WFS1), and whirlin (WHRN), or any combination thereof; (vi) optionally, a 3×FLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence; and (vii) a 3′ITR.

[0214] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a polynucleotide encoding a polypeptide; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0215] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a polynucleotide encoding a polypeptide; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0216] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a polynucleotide encoding a polypeptide; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0217] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a polynucleotide encoding a polypeptide; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0218] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a polynucleotide encoding a polypeptide; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45; (viii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (ix) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0219] In some embodiments, the rAAVAnc80 particle comprises: (i) a 5'ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising any one of the nucleic acid sequences of SEQ ID NOs: 1-15; (iii) an ITR encoding actin gamma 1 (ACTG1), adenylate cyclase type 1 (ADCY1), calcium binding protein 2 (CABP2), coiled-coil domain containing 50 (CCDC50), cadherin associated 23 (CDH23), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), chromodomain helicase DNA binding protein 7 (CHD7), calcium and integrin. Clin-binding family member 2 (CIB2), claudin 14 (CLDN14), intracellular chloride channel 5 (CLIC5), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), clarrin 1 (CLRN1), pedivakin (DFNB59), endothelin 3 (EDN3), ELMO domain-containing protein 3 (ELMOD3), epidermal growth factor receptor kinase substrate 8 (EPS8), espin (ESPN), estrogen-related receptor beta (ESRRB), eyes absent homolog 1 (EYA1), GIPC PDZ domain-containing family, member 3 (GIPC3), G protein-coupled receptor 98 (GPR98), G protein signaling modulator 2 (GPSM2), glutaredoxin, cysteine-rich 1 (GRXCR1), glutaredoxin, cysteine-rich 2 (GRXCR2), immunoglobulin-like domain-containing receptor 1 (ILDR1), lysyl-tRNA synthetase (KARS), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), lipoma HMGIC fusion partner-like 5 (LHFPL5), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT1 COMT2), tricellulin (MARVELD2), microRNA96 (MIR96), methionine sulfoxide reductase B3 (MSRB3), nonmuscle myosin heavy chain 9 (MYH9), nonmuscle myosin heavy chain 14 (MYH14), atypical myosin IIIA (MYO3A), atypical myosin VI (MYO6), atypical myosin VIIA (MYO7A), atypical myosin XVA (MYO15A), otoferlin (OTOF), otogelin-like protein (OTOGL), purinergic receptor P2X, ligand-gated ion channel, 2 (P2RX2), protocadherin 15 (PCDH15), PDZ domain-containing 7 (PDZD7), polyribonucleotide nucleotidyltransferase 1, mitochondrial (PNPT1), POU domain, class 4, transcription factor 3 (POU4 F3), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), protein tyrosine phosphatase, receptor type Q (PTPRQ), radixin (RDX), scaffold-containing ankyrin repeats and SAM domain (SANS), serpin peptidase inhibitor, clade B, member 6 (SERPINB6), SIX homeobox 1 (SIX1), SIX homeobox 5 (SIX5), prestin (SLC26A5), second mitochondrial-derived caspase activator (SMAC / DIABLO), small muscle protein, x-linked (SMPX), stereocillin (STRC), nesprin-4 (SYNE4), TBC1 domain family, member 24 (TBC / D24), tight junction protein XO(iv) optionally, a 3×FLAG tag comprising the nucleic acid sequence of SEQ ID NO:39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO:22; and (vi) a 3′ITR comprising the nucleic acid sequence of SEQ ID NO:17.

[0220] In some embodiments, the rAAVAnc80 particle comprises: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising any one of the nucleic acid sequences of SEQ ID NOs: 1-15; (iii) a 5' ITR comprising the nucleic acid sequence of cadherin-related 23 (CDH23), clarrin 1 (CLRN1), pedivakin (DFNB59), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), otoferlin (OTOF), protocadherin 15 (PCDH15), POU domain, class 4, transcription factor 3 (POU4F3), prestin (SLC26A5), stereocillin (STRC), transmembrane tyrosine kinase (TKT)-dependent phosphodiesterase (TKI)-dependent phosphodiesterase (P ... (iv) optionally, a 3×FLAG tag comprising the nucleic acid sequence of SEQ ID NO:39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO:22; and (vi) a 3′ ITR comprising the nucleic acid sequence of SEQ ID NO:17.

[0221] In some embodiments, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a polynucleotide encoding a polypeptide encoding a voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4); (iv) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0222] In some embodiments, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (iv) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0223] In some embodiments, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0224] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0225] In some embodiments, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a 3' UTR; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0226] In some embodiments, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45; (viii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (ix) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0227] In some embodiments, the rAAVAnc80 particles include a construct that is 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 any one of SEQ ID NOs: 23-38 and 49-50. In some embodiments, the rAAVAnc80 particles include a construct that includes a nucleic acid sequence of any one of SEQ ID NOs: 23-38 and 49-50. In some embodiments, the construct comprises nucleotides 12 to 4396 of SEQ ID NO:23, nucleotides 12 to 4464 of SEQ ID NO:24, nucleotides 12 to 4016 of SEQ ID NO:25, nucleotides 12 to 4521 of SEQ ID NO:26, nucleotides 12 to 3750 of SEQ ID NO:27, nucleotides 12 to 3928 of SEQ ID NO:28, nucleotides 12 to 4641 of SEQ ID NO:29, nucleotides 12 to 3994 of SEQ ID NO:30, nucleotides 12 to 4426 of SEQ ID NO:31, nucleotides 12 to 4307 of SEQ ID NO:32, nucleotides 12 to 4309 of SEQ ID NO:33, nucleotides 12 to 4465 of SEQ ID NO:34, nucleotides 12 to 4526 of SEQ ID NO:35, nucleotides 12 to 3750 of SEQ ID NO:27, nucleotides 12 to 3928 of SEQ ID NO:28, nucleotides 12 to 4641 of SEQ ID NO:29, nucleotides 12 to 3994 of SEQ ID NO:30, nucleotides 12 to 4426 of SEQ ID NO:31, nucleotides 12 to 4307 of SEQ ID NO:32, nucleotides 12 to 4309 of SEQ ID NO:33 nucleotides 12 to 4293 of SEQ ID NO: 34, nucleotides 12 to 4565 of SEQ ID NO: 34, nucleotides 12 to 4224 of SEQ ID NO: 35, nucleotides 12 to 4140 of SEQ ID NO: 36, nucleotides 12 to 4816 of SEQ ID NO: 37, or nucleotides 12 to 4915 of SEQ ID NO: 38.In some embodiments, the rAAVAnc80 particle comprises a construct comprising a nucleic acid sequence comprising any one of nucleotides 12 to 4396 of SEQ ID NO:23, nucleotides 12 to 4464 of SEQ ID NO:24, nucleotides 12 to 4016 of SEQ ID NO:25, nucleotides 12 to 4521 of SEQ ID NO:26, nucleotides 12 to 3750 of SEQ ID NO:27, nucleotides 12 to 3928 of SEQ ID NO:28, nucleotides 12 to 4641 of SEQ ID NO:29, nucleotides 12 to 3994 of SEQ ID NO:30, nucleotides 12 to 4426 of SEQ ID NO:31, nucleotides 12 to 4307 of SEQ ID NO:32, nucleotides 12 to 4293 of SEQ ID NO:33, nucleotides 12 to 4565 of SEQ ID NO:34, nucleotides 12 to 4224 of SEQ ID NO:35, nucleotides 12 to 4140 of SEQ ID NO:36, nucleotides 12 to 4816 of SEQ ID NO:37, or nucleotides 12 to 4915 of SEQ ID NO:38.

[0228] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 23. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO: 23.

[0229] In some embodiments, the rAAVAnc80 particle comprises a construct comprising 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 nucleotides 12-4396 of SEQ ID NO:23. In some embodiments, the composition comprises a construct comprising nucleotides 12-4396 of SEQ ID NO:23.

[0230] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 1; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0231] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 24. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO: 24.

[0232] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 nucleotides 12 to 4464 of SEQ ID NO: 24. In some embodiments, the rAAVAnc80 particles comprise a construct that includes nucleotides 12 to 4464 of SEQ ID NO: 24.

[0233] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 2; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0234] In some embodiments, the rAAVAnc80 particle comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 2, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0235] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 25. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:25.

[0236] In some embodiments, the rAAVAnc80 particle 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 nucleotides 12-4016 of SEQ ID NO:25. In some embodiments, the rAAVAnc80 particle comprises a construct comprising nucleotides 12-4016 of SEQ ID NO:25.

[0237] In some embodiments, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 1; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0238] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 26. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:26.

[0239] In some embodiments, the rAAVAnc80 particles comprise a construct comprising 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 nucleotides 12 to 4521 of SEQ ID NO: 26. In some embodiments, the rAAVAnc80 particles comprise a construct comprising nucleotides 12 to 4521 of SEQ ID NO: 26.

[0240] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 3; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0241] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 3; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0242] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 27. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO: 27.

[0243] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 nucleotides 12-3750 of SEQ ID NO: 27. In some embodiments, the rAAVAnc80 particles comprise a construct that includes nucleotides 12-3750 of SEQ ID NO: 27.

[0244] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a CHRNA10 promoter comprising the nucleic acid sequence of SEQ ID NO: 4; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0245] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CHRNA10 promoter comprising the nucleic acid sequence of SEQ ID NO: 4; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0246] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 28. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO: 28.

[0247] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 nucleotides 12-3928 of SEQ ID NO: 28. In some embodiments, the rAAVAnc80 particles comprise a construct that includes nucleotides 12-3928 of SEQ ID NO: 28.

[0248] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a DNM3 promoter comprising the nucleic acid sequence of SEQ ID NO: 5; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0249] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a DNM3 promoter comprising the nucleic acid sequence of SEQ ID NO: 5; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0250] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid 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 SEQ ID NO: 29. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:29.

[0251] In some embodiments, the rAAVAnc80 particles comprise a construct comprising 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 nucleotides 12-4641 of SEQ ID NO: 29. In some embodiments, the rAAVAnc80 particles comprise a construct comprising nucleotides 12-4641 of SEQ ID NO: 29.

[0252] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a MUC15 promoter comprising the nucleic acid sequence of SEQ ID NO: 6; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0253] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a MUC15 promoter comprising the nucleic acid sequence of SEQ ID NO: 6; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0254] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 30. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:30.

[0255] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 nucleotides 12-3994 of SEQ ID NO: 30. In some embodiments, the rAAVAnc80 particles comprise a construct that includes nucleotides 12-3994 of SEQ ID NO: 30.

[0256] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a PLBD1 promoter comprising the nucleic acid sequence of SEQ ID NO: 7; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0257] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a PLBD1 promoter comprising the nucleic acid sequence of SEQ ID NO: 7; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0258] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 31. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:31.

[0259] In some embodiments, the rAAVAnc80 particles comprise a construct comprising 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 nucleotides 12-4426 of SEQ ID NO: 31. In some embodiments, the rAAVAnc80 particles comprise a construct comprising nucleotides 12-4426 of SEQ ID NO: 31.

[0260] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a RORB promoter comprising the nucleic acid sequence of SEQ ID NO: 8; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0261] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a RORB promoter comprising the nucleic acid sequence of SEQ ID NO: 8; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0262] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 32. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO: 32.

[0263] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 nucleotides 12-4307 of SEQ ID NO: 32. In some embodiments, the rAAVAnc80 particles comprise a construct that includes nucleotides 12-4307 of SEQ ID NO: 32.

[0264] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a STRIP2 promoter comprising the nucleic acid sequence of SEQ ID NO: 9; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0265] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a STRIP2 promoter comprising the nucleic acid sequence of SEQ ID NO: 9; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0266] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 33. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:33.

[0267] In some embodiments, the rAAVAnc80 particles comprise a construct comprising 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 nucleotides 12-4293 of SEQ ID NO: 33. In some embodiments, the rAAVAnc80 particles comprise a construct comprising nucleotides 12-4293 of SEQ ID NO: 33.

[0268] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) an AQP11 promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0269] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) an AQP11 promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0270] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 34. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:34.

[0271] In some embodiments, the rAAVAnc80 particles comprise a construct comprising 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 nucleotides 12-4565 of SEQ ID NO: 34. In some embodiments, the rAAVAnc80 particles comprise a construct comprising nucleotides 12-4565 of SEQ ID NO: 34.

[0272] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a KCNQ4 promoter comprising the nucleic acid sequence of SEQ ID NO: 11; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0273] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a KCNQ4 promoter comprising the nucleic acid sequence of SEQ ID NO: 11; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0274] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 35. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:35.

[0275] In some embodiments, the rAAVAnc80 particles comprise a construct comprising 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 nucleotides 12-4224 of SEQ ID NO: 35. In some embodiments, the rAAVAnc80 particles comprise a construct comprising nucleotides 12-4224 of SEQ ID NO: 35.

[0276] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) an LBH promoter comprising the nucleic acid sequence of SEQ ID NO: 12; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0277] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) an LBH promoter comprising the nucleic acid sequence of SEQ ID NO: 12; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0278] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 36. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:36.

[0279] In some embodiments, the rAAVAnc80 particles comprise a construct comprising 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 nucleotides 12 to 4140 of SEQ ID NO: 36. In some embodiments, the rAAVAnc80 particles comprise a construct comprising nucleotides 12 to 4140 of SEQ ID NO: 36.

[0280] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a STRC promoter comprising the nucleic acid sequence of SEQ ID NO: 13; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0281] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a STRC promoter comprising the nucleic acid sequence of SEQ ID NO: 13; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0282] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 37. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:37.

[0283] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 nucleotides 12-4816 of SEQ ID NO: 37. In some embodiments, the rAAVAnc80 particles comprise a construct that includes nucleotides 12-4816 of SEQ ID NO: 37.

[0284] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a TUBA8 promoter comprising the nucleic acid sequence of SEQ ID NO: 14; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0285] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a TUBA8 promoter comprising the nucleic acid sequence of SEQ ID NO: 14; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0286] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 38. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:38.

[0287] In some embodiments, the rAAVAnc80 particles comprise a construct comprising 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 nucleotides 12-4915 of SEQ ID NO: 38. In some embodiments, the rAAVAnc80 particles comprise a construct comprising nucleotides 12-4915 of SEQ ID NO: 38.

[0288] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0289] In some aspects, the rAAVAnc80 particle comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0290] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 49. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO: 49.

[0291] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 nucleotides 12 to 4070 of SEQ ID NO: 49. In some embodiments, the rAAVAnc80 particles comprise a construct that includes nucleotides 12 to 4070 of SEQ ID NO: 49.

[0292] In some embodiments, the rAAVAnc80 particles comprise a construct that includes 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 SEQ ID NO: 50. In some embodiments, the rAAVAnc80 particles comprise a construct that includes the nucleic acid sequence of SEQ ID NO:50. DNA sequence of an exemplary AAV Anc80 capsid (SEQ ID NO:40) Amino acid sequence of an exemplary AAV Anc80 capsid (SEQ ID NO:41) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAKKRLNFGQTGDSESVPD PQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTANQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSN VDLDNVMITSEEEIKTTNPVATEQYGTVATNLQSSNTAPATGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL

[0293] composition In particular, the present disclosure provides a composition. In some embodiments, the composition comprises the construct described herein. In some embodiments, the composition comprises one or more constructs described herein. In some embodiments, the composition comprises a plurality of constructs described herein. In some embodiments, when more than one construct is included in the composition, each of the constructs is different.

[0294] In some embodiments, the composition comprises an AAV particle as described herein. In some embodiments, the composition comprises one or more AAV particles as described herein. In some embodiments, the composition comprises a plurality of AAV particles. In some embodiments, when more than one AAV particle is included in the composition, each of the AAV particles is different.

[0295] In some embodiments, the composition comprises a vector as described herein. In some embodiments, the composition comprises one or more vectors as described herein. In some embodiments, the composition comprises a plurality of vectors as described herein. In some embodiments, when more than one vector is included in the composition, each vector is different.

[0296] In some embodiments, the composition comprises a cell described herein. In some embodiments, the composition comprises one or more cells described herein.

[0297] In some embodiments, the composition is or comprises a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a pharmaceutic carrier. In some embodiments, the composition is or comprises a synthetic perilymph. In some embodiments, the synthetic perilymph comprises 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl2, 1-10 mM glucose, and 2-50 mM HEPES, and has a pH of about 6 to about 9.

[0298] In some aspects, the composition comprises a construct comprising: (i) a 5'ITR (e.g., an AAV 5'ITR); (ii) a 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:1-15; (iii) a polynucleotide encoding a polypeptide (e.g., a therapeutic polypeptide); (v) optionally, a 3xFLAG tag (e.g., comprising the nucleic acid sequence of SEQ ID NO:39); (vi) a polyA sequence; and (vii) a 3'ITR (e.g., an AAV 3'ITR).

[0299] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a polynucleotide encoding a polypeptide; (iv) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0300] In some aspects, the composition comprises: (i) a 5' ITR; (ii) a promoter comprising any one of the nucleic acid sequences of SEQ ID NOs: 1-15; (iii) an IL-1 or IL-2 nucleotide sequence encoding actin gamma 1 (ACTG1), adenylate cyclase type 1 (ADCY1), calcium binding protein 2 (CABP2), coiled-coil domain containing 50 (CCDC50), cadherin-associated 23 (CDH23), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), chromodomain helicase DNA-binding protein 7 (CHD7), calcium and integrin binding family. member 2 (CIB2), claudin 14 (CLDN14), intracellular chloride channel 5 (CLIC5), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), clarin 1 (CLRN1), pedivakin (DFNB59), endothelin 3 (EDN3), ELMO domain-containing protein 3 (ELMOD3), epidermal growth factor receptor kinase substrate 8 (EPS8), espin (ESPN), estrogen-related receptor beta (ESRRB), eyes absent homolog 1 (EYA1), GIPC PDZ domain-containing family, member 3 (GIPC3), G protein-coupled receptor 98 (GPR98), G protein signaling modulator 2 (GPSM2), glutaredoxin, cysteine-rich 1 (GRXCR1), glutaredoxin, cysteine-rich 2 (GRXCR2), immunoglobulin-like domain-containing receptor 1 (ILDR1), lysyl-tRNA synthetase (KARS), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), lipoma HMGIC fusion partner-like 5 (LHFPL5), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT1 COMT2), tricellulin (MARVELD2), microRNA96 (MIR96), methionine sulfoxide reductase B3 (MSRB3), nonmuscle myosin heavy chain 9 (MYH9), nonmuscle myosin heavy chain 14 (MYH14), atypical myosin IIIA (MYO3A), atypical myosin VI (MYO6), atypical myosin VIIA (MYO7A), atypical myosin XVA (MYO15A), otoferlin (OTOF), otogelin-like protein (OTOGL), purinergic receptor P2X, ligand-gated ion channel, 2 (P2RX2), protocadherin 15 (PCDH15), PDZ domain-containing 7 (PDZD7), polyribonucleotide nucleotidyltransferase 1, mitochondrial (PNPT1), POU domain, class 4, transcription factor 3 (POU4 F3), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), protein tyrosine phosphatase, receptor type Q (PTPRQ), radixin (RDX), scaffold-containing ankyrin repeats and SAM domain (SANS), serpin peptidase inhibitor, clade B, member 6 (SERPINB6), SIX homeobox 1 (SIX1), SIX homeobox 5 (SIX5), prestin (SLC26A5), second mitochondrial-derived caspase activator (SMAC / DIABLO), small muscle protein, x-linked (SMPX), stereocillin (STRC), nesprin-4 (SYNE4), TBC1 domain family, member 24 (TBC / D24), tight junction protein XO (v) a polynucleotide encoding an outer hair cell polypeptide comprising a gene selected from: (i) transmembrane channel-like protein 2 (TJP2), (ii) transmembrane inner ear expressed protein (TMIE), transmembrane protease, serine 3 (TMPRSS3), (iii) tapelin (TPRN), (iv) TRIO and F-actin binding protein (TRIOBP), thrombospondin-type laminin G domain and EAR repeats (TSPEAR), (v) harmonin (USH1C), (v) usherin (USH2A), (v) wolframin (WFS1), and whirlin (WHRN), or any combination thereof; (vi) optionally, a 3×FLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence; and (vii) a 3′ITR.

[0301] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a polynucleotide encoding a polypeptide; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0302] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a polynucleotide encoding a polypeptide; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0303] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a polynucleotide encoding a polypeptide; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0304] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a polynucleotide encoding a polypeptide; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0305] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a polynucleotide encoding a polypeptide; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45; (viii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (ix) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0306] In some aspects, the composition comprises: (i) a 5'ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising any one of the nucleic acid sequences of SEQ ID NOs: 1-15; (iii) an ITR comprising any one of actin gamma 1 (ACTG1), adenylate cyclase type 1 (ADCY1), calcium binding protein 2 (CABP2), coiled-coil domain containing 50 (CCDC50), cadherin associated 23 (CDH23), carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM16), chromodomain helicase DNA binding protein 7 (CHD7), calcium and integrin binding. Conjugated family member 2 (CIB2), claudin 14 (CLDN14), intracellular chloride channel 5 (CLIC5), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), clarin 1 (CLRN1), pedivakin (DFNB59), endothelin 3 (EDN3), ELMO domain-containing protein 3 (ELMOD3), epidermal growth factor receptor kinase substrate 8 (EPS8), espin (ESPN), estrogen-related receptor beta (ESRRB), eyes absent homolog 1 (EYA1), GIPC PDZ domain-containing family, member 3 (GIPC3), G protein-coupled receptor 98 (GPR98), G protein signaling modulator 2 (GPSM2), glutaredoxin, cysteine-rich 1 (GRXCR1), glutaredoxin, cysteine-rich 2 (GRXCR2), immunoglobulin-like domain-containing receptor 1 (ILDR1), lysyl-tRNA synthetase (KARS), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), lipoma HMGIC fusion partner-like 5 (LHFPL5), leucine-rich transmembrane and O-methyltransferase domain-containing (LRTOMT1 COMT2), tricellulin (MARVELD2), microRNA96 (MIR96), methionine sulfoxide reductase B3 (MSRB3), nonmuscle myosin heavy chain 9 (MYH9), nonmuscle myosin heavy chain 14 (MYH14), atypical myosin IIIA (MYO3A), atypical myosin VI (MYO6), atypical myosin VIIA (MYO7A), atypical myosin XVA (MYO15A), otoferlin (OTOF), otogelin-like protein (OTOGL), purinergic receptor P2X, ligand-gated ion channel, 2 (P2RX2), protocadherin 15 (PCDH15), PDZ domain-containing 7 (PDZD7), polyribonucleotide nucleotidyltransferase 1, mitochondrial (PNPT1), POU domain, class 4, transcription factor 3 (POU4 F3), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), protein tyrosine phosphatase, receptor type Q (PTPRQ), radixin (RDX), scaffold-containing ankyrin repeats and SAM domain (SANS), serpin peptidase inhibitor, clade B, member 6 (SERPINB6), SIX homeobox 1 (SIX1), SIX homeobox 5 (SIX5), prestin (SLC26A5), second mitochondrial-derived caspase activator (SMAC / DIABLO), small muscle protein, x-linked (SMPX), stereocillin (STRC), nesprin-4 (SYNE4), TBC1 domain family, member 24 (TBC / D24), tight junction protein XO(iv) optionally, a 3×FLAG tag comprising the nucleic acid sequence of SEQ ID NO:39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO:22; and (vi) a 3′ITR comprising the nucleic acid sequence of SEQ ID NO:17.

[0307] In some embodiments, the composition comprises: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; and (iii) a nucleotide sequence encoding cadherin-related 23 (CDH23), clarin 1 (CLRN1), pedivakin (DFNB59), voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4), otoferlin (OTOF), protocadherin 15 (PCDH15), POU domain, class 4, transcription factor 3 (POU4F3), prestin (SLC26A5), stereocillin (STRC), transmembrane channel. (iv) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO:39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO:22; and (vi) a 3'ITR comprising the nucleic acid sequence of SEQ ID NO:17.

[0308] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a polynucleotide encoding a polypeptide encoding a voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4); (iv) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0309] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (iv) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0310] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0311] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0312] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0313] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a 3' UTR; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0314] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45; (viii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (ix) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0315] In some embodiments, the composition comprises a construct 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: 23-38 and 49-50. In some embodiments, the composition comprises a construct comprising a nucleic acid sequence of any one of SEQ ID NOs: 23-38 and 49-50. In some embodiments, the construct comprises nucleotides 12-4396 of SEQ ID NO: 23, 12-4464 of SEQ ID NO: 24, 12-4016 of SEQ ID NO: 25, nucleotides 12-4521 of SEQ ID NO: 26, nucleotides 12-3750 of SEQ ID NO: 27, nucleotides 12-3928 of SEQ ID NO: 28, nucleotides 12-4641 of SEQ ID NO: 29, nucleotides 12-3994 of SEQ ID NO: 30, nucleotides 12-4426 of SEQ ID NO: 31, nucleotides 12-4307 of SEQ ID NO: 32, nucleotides 12-4308 of SEQ ID NO: 33, nucleotides 12-4491 of SEQ ID NO: 34, nucleotides 12-4522 of SEQ ID NO: 35, nucleotides 12-4523 of SEQ ID NO: 36, nucleotides 12-3750 of SEQ ID NO: 37, nucleotides 12-3928 of SEQ ID NO: 38, nucleotides 12-4641 of SEQ ID NO: 39, nucleotides 12-3994 of SEQ ID NO: 30, nucleotides 1 nucleotides 12 to 4293 of SEQ ID NO: 34, nucleotides 12 to 4565 of SEQ ID NO: 34, nucleotides 12 to 4224 of SEQ ID NO: 35, nucleotides 12 to 4140 of SEQ ID NO: 36, nucleotides 12 to 4816 of SEQ ID NO: 37, or nucleotides 12 to 4915 of SEQ ID NO: 38.In some embodiments, the composition comprises a construct comprising a nucleic acid sequence comprising any one of nucleotides 12 to 4396 of SEQ ID NO:23, 12 to 4464 of SEQ ID NO:24, nucleotides 12 to 4016 of SEQ ID NO:25, nucleotides 12 to 4521 of SEQ ID NO:26, nucleotides 12 to 3750 of SEQ ID NO:27, nucleotides 12 to 3928 of SEQ ID NO:28, nucleotides 12 to 4641 of SEQ ID NO:29, nucleotides 12 to 3994 of SEQ ID NO:30, nucleotides 12 to 4426 of SEQ ID NO:31, nucleotides 12 to 4307 of SEQ ID NO:32, nucleotides 12 to 4293 of SEQ ID NO:33, nucleotides 12 to 4565 of SEQ ID NO:34, nucleotides 12 to 4224 of SEQ ID NO:35, nucleotides 12 to 4140 of SEQ ID NO:36, nucleotides 12 to 4816 of SEQ ID NO:37, or nucleotides 12 to 4915 of SEQ ID NO:38.

[0316] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 23. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 23.

[0317] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12-4396 of SEQ ID NO: 23. In some embodiments, the composition comprises a construct comprising nucleotides 12-4396 of SEQ ID NO: 23.

[0318] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 1; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0319] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 24. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:24.

[0320] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4464 of SEQ ID NO:24. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4464 of SEQ ID NO:24.

[0321] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 24. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:24.

[0322] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 2; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0323] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 2, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0324] In some embodiments, the construct 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 SEQ ID NO: 25. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:25.

[0325] In some embodiments, the construct 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 nucleotides 12 to 4016 of SEQ ID NO: 25. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4016 of SEQ ID NO: 25.

[0326] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 25. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:25.

[0327] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 1; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0328] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 26. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 26.

[0329] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4521 of SEQ ID NO: 26. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4521 of SEQ ID NO: 26.

[0330] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 26. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:26.

[0331] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 3; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0332] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 3; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0333] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 27. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 27.

[0334] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12-3750 of SEQ ID NO: 27. In some embodiments, the composition comprises a construct comprising nucleotides 12-3750 of SEQ ID NO: 27.

[0335] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 27. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:27.

[0336] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a CHRNA10 promoter comprising the nucleic acid sequence of SEQ ID NO: 4; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0337] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CHRNA10 promoter comprising the nucleic acid sequence of SEQ ID NO: 4; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0338] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 28. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 28.

[0339] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 3928 of SEQ ID NO: 28. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 3928 of SEQ ID NO: 28.

[0340] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 28. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:28.

[0341] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a DNM3 promoter comprising the nucleic acid sequence of SEQ ID NO: 5; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0342] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a DNM3 promoter comprising the nucleic acid sequence of SEQ ID NO: 5; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0343] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 29. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 29.

[0344] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4641 of SEQ ID NO: 29. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4641 of SEQ ID NO: 29.

[0345] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid 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 SEQ ID NO: 29. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:29.

[0346] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a MUC15 promoter comprising the nucleic acid sequence of SEQ ID NO: 6; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0347] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a MUC15 promoter comprising the nucleic acid sequence of SEQ ID NO: 6; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0348] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 30. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:30.

[0349] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 3994 of SEQ ID NO: 30. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 3994 of SEQ ID NO: 30.

[0350] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid 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 SEQ ID NO: 30. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:30.

[0351] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a PLBD1 promoter comprising the nucleic acid sequence of SEQ ID NO: 7; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0352] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a PLBD1 promoter comprising the nucleic acid sequence of SEQ ID NO: 7; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0353] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 31. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 31.

[0354] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12-4426 of SEQ ID NO: 31. In some embodiments, the composition comprises a construct comprising nucleotides 12-4426 of SEQ ID NO: 31.

[0355] In some embodiments, the rAAVAnc80 particle comprises a nucleic acid 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 SEQ ID NO: 31. In some embodiments, the construct rAAVAnc80 particle, the nucleic acid sequence of SEQ ID NO: 31.

[0356] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a RORB promoter comprising the nucleic acid sequence of SEQ ID NO: 8; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0357] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a RORB promoter comprising the nucleic acid sequence of SEQ ID NO: 8; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0358] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 32. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 32.

[0359] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12-4307 of SEQ ID NO: 32. In some embodiments, the composition comprises a construct comprising nucleotides 12-4307 of SEQ ID NO: 32.

[0360] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 32. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:32.

[0361] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a STRIP2 promoter comprising the nucleic acid sequence of SEQ ID NO: 9; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0362] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a STRIP2 promoter comprising the nucleic acid sequence of SEQ ID NO: 9; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0363] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 33. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:33.

[0364] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4293 of SEQ ID NO: 33. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4293 of SEQ ID NO: 33.

[0365] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid 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 SEQ ID NO: 33. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:33.

[0366] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) an AQP11 promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3x FLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0367] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) an AQP11 promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0368] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 34. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:34.

[0369] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4565 of SEQ ID NO: 34. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4565 of SEQ ID NO: 34.

[0370] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 34. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:34.

[0371] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a KCNQ4 promoter comprising the nucleic acid sequence of SEQ ID NO: 11; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0372] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a KCNQ4 promoter comprising the nucleic acid sequence of SEQ ID NO: 11; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0373] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 35. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:35.

[0374] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4224 of SEQ ID NO: 35. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4224 of SEQ ID NO: 35.

[0375] In some embodiments, the rAAVAnc80 particle 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 SEQ ID NO: 35. In some embodiments, the construct rAAVAnc80 particle, the nucleic acid sequence of SEQ ID NO: 35.

[0376] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) an LBH promoter comprising the nucleic acid sequence of SEQ ID NO: 12; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0377] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) an LBH promoter comprising the nucleic acid sequence of SEQ ID NO: 12; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0378] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 36. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:36.

[0379] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4140 of SEQ ID NO: 36. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4140 of SEQ ID NO: 36.

[0380] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid 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 SEQ ID NO: 36. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:36.

[0381] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a STRC promoter comprising the nucleic acid sequence of SEQ ID NO: 13; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0382] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a STRC promoter comprising the nucleic acid sequence of SEQ ID NO: 13; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0383] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 37. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 37.

[0384] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4816 of SEQ ID NO: 37. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4816 of SEQ ID NO: 37.

[0385] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid 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 SEQ ID NO: 37. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:37.

[0386] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a TUBA8 promoter comprising the nucleic acid sequence of SEQ ID NO: 14; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0387] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a TUBA8 promoter comprising the nucleic acid sequence of SEQ ID NO: 14; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0388] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 38. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:38.

[0389] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4915 of SEQ ID NO: 38. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4915 of SEQ ID NO: 38.

[0390] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid 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 SEQ ID NO: 38. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:38.

[0391] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0392] In some aspects, the composition comprises a construct comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 15; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0393] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 49. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO: 49.

[0394] In some embodiments, the composition comprises a construct comprising 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 nucleotides 12 to 4070 of SEQ ID NO: 49. In some embodiments, the composition comprises a construct comprising nucleotides 12 to 4070 of SEQ ID NO: 49.

[0395] In some embodiments, the rAAVAnc80 particles comprise 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 SEQ ID NO: 49. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:49.

[0396] In some embodiments, the composition comprises a construct comprising 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 SEQ ID NO: 50. In some embodiments, the composition comprises a construct comprising the nucleic acid sequence of SEQ ID NO:50.

[0397] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid 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 SEQ ID NO: 50. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:50.

[0398] Dosage and Administration In some aspects, the compositions disclosed herein, e.g., one or more AAV vectors disclosed herein, are administered as a single dose or as multiple doses.

[0399] In some embodiments, the compositions disclosed herein are administered as a single dose. In some embodiments, the compositions disclosed herein are administered as multiple doses, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0400] In some embodiments, a composition disclosed herein (e.g., a composition comprising one or more rAAV constructs disclosed herein) is administered in a volume of about 0.01 mL to about 2.00 mL, about 0.05 mL to about 1.5 mL, about 0.08 mL to about 1.10 mL, or about 0.09 mL to about 1.0 mL. In some embodiments, a composition disclosed herein (e.g., a composition comprising one or more rAAV constructs disclosed herein) is administered in a volume of about 0.01 mL, about 0.02 mL, about 0.03 mL, about 0.04 mL, about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 1.00 mL, about 1.10 mL, about 1.20 mL, about 1.30 mL, about 1.40 mL, about 1.50 mL, about 1.60 mL, about 1.70 mL, about 1.80 mL, about 1.90 mL, or about 2.00 mL. In some embodiments, a composition disclosed herein is administered in a volume of about 0.01 mL. In some embodiments, a composition disclosed herein is administered in a volume of about 0.02 mL. In some embodiments, a composition disclosed herein is administered in a volume of about 0.03 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.04 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.05 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.06 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.07 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.08 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.09 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.10 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.20 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.3 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.4 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.5 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.6 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.7 mL.In some embodiments, the compositions disclosed herein are administered in a volume of about 0.8 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.9 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.00 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.10 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.20 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.30 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.40 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.50 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.60 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.70 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.80 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.90 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 2.00 mL.

[0401] In some embodiments, a composition disclosed herein (e.g., a composition comprising one or more rAAV constructs disclosed herein) is administered in a volume of about 0.01-2.00 mL, about 0.02-1.90 mL, about 0.03-1.8 mL, about 0.04-1.70 mL, about 0.05-1.60 mL, about 0.06-1.50 mL, about 0.06-1.40 mL, about 0.07-1.30 mL, about 0.08-1.20 mL, or about 0.09-1.10 mL. In some embodiments, a composition disclosed herein (e.g., a composition comprising one or more rAAV constructs disclosed herein) may comprise a volume of about 0.01-2.00 mL, about 0.02-2.00 mL, about 0.03-2.00 mL, about 0.04-2.00 mL, about 0.05-2.00 mL, about 0.06-2.00 mL, about 0.07-2.00 mL, about 0.08-2.00 mL, It is administered in volumes of approximately 0.09 to 2.00 mL, approximately 0.01 to 1.90 mL, approximately 0.01 to 1.80 mL, approximately 0.01 to 1.70 mL, approximately 0.01 to 1.60 mL, approximately 0.01 to 1.50 mL, approximately 0.01 to 1.40 mL, approximately 0.01 to 1.30 mL, approximately 0.01 to 1.20 mL, approximately 0.01 to 1.10 mL, approximately 0.01 to 1.00 mL, and approximately 0.01 to 0.09 mL.

[0402] In some embodiments, the dosing regimen comprises delivery of a volume of at least 0.01 mL, at least 0.02 mL, at least 0.03 mL, at least 0.04 mL, at least 0.05 mL, at least 0.06 mL, at least 0.07 mL, at least 0.08 mL, at least 0.09 mL, at least 0.10 mL, at least 0.11 mL, at least 0.12 mL, at least 0.13 mL, at least 0.14 mL, at least 0.15 mL, at least 0.16 mL, at least 0.17 mL, at least 0.18 mL, at least 0.19 mL, or at least 0.20 mL per cochlea. In some embodiments, the dosing regimen includes delivery of a volume of at most 0.30 mL, at most 0.25 mL, at most 0.20 mL, at most 0.15 mL, at most 0.14 mL, at most 0.13 mL, at most 0.12 mL, at most 0.11 mL, at most 0.10 mL, at most 0.09 mL, at most 0.08 mL, at most 0.07 mL, at most 0.06 mL, or at most 0.05 mL per cochlea. In some embodiments, the dosing regimen includes delivery of a volume of about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 0.10 mL, about 0.11 mL, about 0.12 mL, about 0.13 mL, about 0.14 mL, or about 0.15 mL per cochlea depending on the population.

[0403] Composition of single AAV constructs In some aspects, the present disclosure provides compositions or systems that include AAV particles that are composed of a single construct. In some such aspects, the single construct can deliver a polynucleotide that encodes a functional (e.g., wild-type or otherwise functional, e.g., codon-optimized) polypeptide. In some aspects, the construct is or includes a rAAV construct. In some aspects described herein, the single rAAV construct can express its polypeptide in target cells (e.g., inner ear outer hair cells).

[0404] In some aspects, a single construct composition or system may include any or all of the components of the exemplary constructs described herein.

[0405] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iii) a polynucleotide encoding a polypeptide, (iv) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0406] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18, (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iv) a polynucleotide encoding a polypeptide, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0407] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a promoter comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a polynucleotide encoding a polypeptide, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0408] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18, (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (v) a polynucleotide encoding a polypeptide, (vi) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0409] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a promoter comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a polynucleotide encoding a polypeptide, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45, (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0410] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18, (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (v) a polynucleotide encoding a polypeptide, (vi) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vii) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45, (viii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (ix) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0411] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a promoter comprising any one of the nucleic acid sequences of SEQ ID NOs: 1-15, (iii) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (iv) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (v) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vi) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0412] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18, (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0413] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a promoter comprising any one of the nucleic acid sequences of SEQ ID NOs: 1-15, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0414] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18, (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (vi) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0415] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a 3' UTR, (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0416] In some aspects, the construct comprises: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18; (iii) a promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-15; (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (vi) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vii) a 3' UTR comprising the nucleic acid sequence of SEQ ID NO: 45; (viii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (ix) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0417] In some embodiments, the construct comprises 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: 23 to 38 and 49 to 50. In some embodiments, the construct comprises any nucleic acid sequence of SEQ ID NOs: 23 to 38 and 49 to 50. In some embodiments, the construct comprises nucleotides 12 to 4396 of SEQ ID NO: 23, 12 to 4464 of SEQ ID NO: 24, nucleotides 12 to 4016 of SEQ ID NO: 25, nucleotides 12 to 4521 of SEQ ID NO: 26, nucleotides 12 to 3750 of SEQ ID NO: 27, nucleotides 12 to 3928 of SEQ ID NO: 28, nucleotides 12 to 4641 of SEQ ID NO: 29, nucleotides 12 to 3994 of SEQ ID NO: 30, nucleotides 12 to 4426 of SEQ ID NO: 31, nucleotides 12 to 4307 of SEQ ID NO: 32, The sequence of any one of nucleotides 12 to 4293 of SEQ ID NO:33, nucleotides 12 to 4565 of SEQ ID NO:34, nucleotides 12 to 4224 of SEQ ID NO:35, nucleotides 12 to 4140 of SEQ ID NO:36, nucleotides 12 to 4816 of SEQ ID NO:37, or nucleotides 12 to 4915 of SEQ ID NO:38, comprises 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 nucleotides 12 to 4293 of SEQ ID NO:33, nucleotides 12 to 4565 of SEQ ID NO:34, nucleotides 12 to 4224 of SEQ ID NO:35, nucleotides 12 to 4140 of SEQ ID NO:36, nucleotides 12 to 4816 of SEQ ID NO:37, or nucleotides 12 to 4915 of SEQ ID NO:38. In some embodiments, the construct comprises a nucleic acid sequence comprising any one of nucleotides 12 to 4396 of SEQ ID NO:23, nucleotides 12 to 4464 of SEQ ID NO:24, nucleotides 12 to 4016 of SEQ ID NO:25, nucleotides 12 to 4521 of SEQ ID NO:26, nucleotides 12 to 3750 of SEQ ID NO:27, nucleotides 12 to 3928 of SEQ ID NO:28, nucleotides 12 to 4641 of SEQ ID NO:29, nucleotides 12 to 3994 of SEQ ID NO:30, nucleotides 12 to 4426 of SEQ ID NO:31, nucleotides 12 to 4307 of SEQ ID NO:32, nucleotides 12 to 4293 of SEQ ID NO:33, nucleotides 12 to 4565 of SEQ ID NO:34, nucleotides 12 to 4224 of SEQ ID NO:35, nucleotides 12 to 4140 of SEQ ID NO:36, nucleotides 12 to 4816 of SEQ ID NO:37, or nucleotides 12 to 4915 of SEQ ID NO:38.

[0418] In some embodiments, the construct comprises a nucleic acid sequence that comprises 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: 23. In some embodiments, the construct comprises the nucleic acid sequence of SEQ ID NO: 23.

[0419] In some embodiments, the construct comprises a nucleic acid sequence that comprises 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 nucleotides 12 to 4396 of SEQ ID NO: 23. In some embodiments, the construct comprises nucleotides 12 to 4396 of SEQ ID NO: 23.

[0420] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid sequence that comprises 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: 23. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:23.

[0421] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18, (iii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 1, (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (vi) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17. Exemplary construct (SEQ ID NO:23) [Table 3]

[0422] In some embodiments, the oncomodulin promoter comprises a nucleic acid sequence comprising 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 nucleotides 525 to 1530 of SEQ ID NO:23.

[0423] In some embodiments, the construct comprises a nucleic acid sequence that comprises 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: 24. In some embodiments, the construct comprises the nucleic acid sequence of SEQ ID NO: 24.

[0424] In some embodiments, the construct comprises a nucleic acid sequence that comprises 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 nucleotides 12 to 4464 of SEQ ID NO: 24. In some embodiments, the construct comprises nucleotides 12 to 4464 of SEQ ID NO: 24.

[0425] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid sequence that comprises 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: 24. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:24.

[0426] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18, (iii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 2, (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (vi) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0427] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 2, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17. Exemplary construct (SEQ ID NO:24) [Table 4]

[0428] In some embodiments, the oncomodulin promoter comprises a nucleic acid sequence comprising 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 nucleotides 145 to 1598 of SEQ ID NO:24.

[0429] In some embodiments, the construct comprises a nucleic acid sequence that comprises 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: 25. In some embodiments, the construct comprises the nucleic acid sequence of SEQ ID NO: 25.

[0430] In some embodiments, the construct comprises a nucleic acid sequence that comprises 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 nucleotides 12 to 4016 of SEQ ID NO: 25. In some embodiments, the construct comprises nucleotides 12 to 4016 of SEQ ID NO: 25.

[0431] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid sequence that comprises 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: 25. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:25.

[0432] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 1, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17. Exemplary Construct (SEQ ID NO:25) [Table 5]

[0433] In some embodiments, the oncomodulin promoter comprises a nucleic acid sequence comprising 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 nucleotides 145 to 1150 of SEQ ID NO:25.

[0434] In some embodiments, the construct comprises a nucleic acid sequence that comprises 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: 26. In some embodiments, the construct comprises the nucleic acid sequence of SEQ ID NO:26.

[0435] In some embodiments, the construct comprises a nucleic acid sequence that comprises 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 nucleotides 12 to 4521 of SEQ ID NO: 26. In some embodiments, the construct comprises nucleotides 12 to 4521 of SEQ ID NO: 26.

[0436] In some embodiments, the rAAVAnc80 particles comprise a nucleic acid sequence that comprises 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: 26. In some embodiments, the rAAVAnc80 particles comprise the nucleic acid sequence of SEQ ID NO:26.

[0437] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a CMV enhancer comprising the nucleic acid sequence of SEQ ID NO: 18, (iii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 3, (iv) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (v) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (vi) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vii) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22, and (viii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 17.

[0438] In some aspects, the construct comprises (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16, (ii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 3, (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19, (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20, (v) optionally a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39, (vi) a polyA sequence comprising the nucleic a...

Claims

1. 1. A construct comprising a polynucleotide encoding a polypeptide operably linked to a promoter that causes expression of the polynucleotide in an outer hair cell, wherein the promoter is selected from the group consisting of an oncomodulin (OCM) promoter, a prestin promoter, a cholinergic receptor nicotinic alpha 10 (CHRNA10) promoter, a dynamin 3 (DNM3) promoter, a mucin 14 (MUC15) promoter, a phospholipase D (PLDB1) promoter, a RAR-related orphan receptor B (RORB) promoter, a striatin-interacting protein 2 (STRIP2) promoter, an aquaporin 11 (AQP11) promoter, a voltage-gated potassium channel subfamily Q member 4 (KCNQ4) promoter, a LBH promoter, a stereocillin (STRC) promoter, a tubulin alpha 8 (TUBA8) promoter, and any combination thereof, and wherein the polynucleotide encodes KCNQ4.

2. A construct comprising a polynucleotide encoding a polypeptide operably linked to a promoter that causes expression of the polynucleotide in an outer hair cell, 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: 1-15, and wherein the polynucleotide encodes KCNQ4.

3. The construct of claim 1 , wherein the promoter is a human prestin promoter or an oncomodulin (OCM) promoter.

4. 3. The construct of claim 2, 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 SEQ ID NO: 1-3 or 15.

5. The construct of claim 1 or 2, wherein the construct further comprises an enhancer.

6. The construct of claim 5 , wherein the enhancer is a CMV enhancer.

7. The construct of claim 1 or 2, wherein the construct further comprises a 5'UTR.

8. The construct of claim 1 or 2, wherein the construct further comprises a 3'UTR.

9. The construct of claim 1 or 2, wherein the construct further comprises a polyA tail.

10. 10. The construct of claim 9, wherein the poly(A) tail is a bovine growth hormone poly(A) tail, a mouse β-globin poly(A) tail, a mouse α-globin poly(A) tail, a human collagen poly(A) tail, a polyomavirus poly(A) tail, a herpes simplex virus thymidine kinase gene (HSV TK) poly(A) tail, an IgG heavy chain gene poly(A) tail, a human growth hormone poly(A) tail, or an SV40 late and early poly(A) tail.

11. 3. The construct of claim 1 or 2, wherein the construct comprises a nucleic acid sequence comprising any one of nucleotides 12 to 4396 of SEQ ID NO:23, 12 to 4464 of SEQ ID NO:24, nucleotides 12 to 4016 of SEQ ID NO:25, nucleotides 12 to 4521 of SEQ ID NO:26, nucleotides 12 to 3750 of SEQ ID NO:27, nucleotides 12 to 3928 of SEQ ID NO:28, nucleotides 12 to 4641 of SEQ ID NO:29, nucleotides 12 to 3994 of SEQ ID NO:30, nucleotides 12 to 4426 of SEQ ID NO:31, nucleotides 12 to 4307 of SEQ ID NO:32, nucleotides 12 to 4293 of SEQ ID NO:33, nucleotides 12 to 4565 of SEQ ID NO:34, nucleotides 12 to 4224 of SEQ ID NO:35, nucleotides 12 to 4140 of SEQ ID NO:36, nucleotides 12 to 4816 of SEQ ID NO:37, or nucleotides 12 to 4915 of SEQ ID NO:

38.

12. 3. The construct of claim 1 or 2, wherein the 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% sequence identity to any one of SEQ ID NOs: 23-38.

13. The construct of claim 1 or 2, wherein the construct is an expression cassette.

14. A vector comprising the construct of claim 1.

15. The vector of claim 14 , wherein the vector is a viral vector.

16. 16. The vector of claim 15, wherein the viral vector is selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, or a lentivirus vector.

17. The vector of claim 16, wherein the viral vector is an AAV vector.

18. 15. The construct of claim 1 or 2 or the vector of claim 14, further comprising a 5' inverted terminal repeat (ITR) and a 3' ITR.

19. 19. The construct or vector of claim 18, wherein the 5' ITR and the 3' ITR are AAV ITRs derived from a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV Anc80 ITRs.

20. 20. The construct or vector of claim 19, wherein the AAV ITRs are of or are derived from serotype AAV2.

21. 1. A construct or vector comprising: (i) a 5′ ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) a prestin promoter comprising the nucleic acid sequence of SEQ ID NO: 3; (iii) a 5′ UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3×FLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3′ ITR comprising the nucleic acid sequence of SEQ ID NO:

17.

22. A construct or vector comprising: (i) a 5' ITR comprising the nucleic acid sequence of SEQ ID NO: 16; (ii) an oncomodulin promoter comprising the nucleic acid sequence of SEQ ID NO: 1 or 2; (iii) a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 19; (iv) a KCNQ4 coding region comprising the nucleic acid sequence of SEQ ID NO: 20; (v) optionally, a 3xFLAG tag comprising the nucleic acid sequence of SEQ ID NO: 39; (vi) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 22; and (vii) a 3' ITR comprising the nucleic acid sequence of SEQ ID NO:

17.

23. An AAV particle comprising the construct of claim 1.

24. 24. The AAV particle of claim 23, 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 capsid.

25. The AAV particle of claim 24, wherein the AAV capsid is an AAV Anc80 capsid.

26. 24. A composition comprising the construct of claim 1, the vector of claim 14, or the AAV particle of claim 23.

27. 27. The composition of claim 26, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

28. 28. The composition of claim 27, wherein the pharmaceutical composition is a synthetic perilymph fluid.

29. A cell comprising the construct of claim 1, the vector of claim 14, or the AAV particle of claim 23.

30. 30. The cell of claim 29, wherein the cell is an outer hair cell.

31. 30. The cell of claim 29, wherein the cell is an ex vivo cell.

32. To the cells, a. the construct of claim 1 or the vector of claim 14, and b) The method for producing AAV particles of claim 23, comprising transducing with one or more helper plasmids collectively comprising the AAV Rep gene, the AAV Cap gene, the AAV VA gene, the AAV E2a gene, and the AAV E4 gene.

33. 33. The method of claim 32, wherein the cell is an outer hair cell.

34. 33. The method of claim 32, wherein the cells are ex vivo cells.

35. A composition comprising the construct of claim 1, the vector of claim 14, or the AAV particle of claim 23, for use in expressing a polypeptide in outer hair cells of a subject in need of expression of the polypeptide in the outer hair cells.

36. A composition comprising the construct of claim 1, the vector of claim 14, or the AAV particle of claim 23, for use in increasing expression of a polypeptide in outer hair cells of a subject in need of increased expression of the polypeptide in outer hair cells.

37. 37. The composition of claim 36, wherein the increased expression of the polypeptide in the outer hair cells of the subject is compared to endogenous expression of the polypeptide in the outer hair cells of the subject.

38. A composition comprising the construct of claim 1, the vector of claim 14, or the AAV particle of claim 23, for use in treating hearing loss in a subject suffering from or at risk of hearing loss.

39. 39. The composition of claim 38, wherein the subject has previously been identified as having a defective inner ear cell target gene, and the defective inner ear cell target gene is voltage-gated potassium channel, KQT-like subfamily, member 4 (KCNQ4).

40. 39. The composition of claim 38, wherein the subject is a human.

41. 39. The composition of claim 38, wherein the use comprises administration to the inner ear of the subject.

42. 39. The composition of claim 38, wherein the use comprises administration to the cochlea of ​​the subject.

43. 43. The composition of claim 42, wherein the use comprises administration via round window membrane injection.

44. 39. The composition of claim 38, wherein the construct of claim 1, the vector of claim 14, or the AAV particle of claim 23 is pre-loaded into the device.

45. 45. The composition of claim 44, wherein the device is a microcatheter.

46. 24. Use of a construct according to claim 1, a vector according to claim 14, or an AAV particle according to claim 23 in the manufacture of a medicament for the treatment of hearing loss.

47. 24. A construct according to claim 1, a vector according to claim 14, or an AAV particle according to claim 23 for use as a medicament.

48. 24. A construct according to claim 1, a vector according to claim 14, or an AAV particle according to claim 23 for use in the treatment of hearing loss.

49. 24. A kit comprising the construct of claim 1, the vector of claim 14, or the AAV particle of claim 23.

50. 50. The kit of claim 49, wherein the construct, the vector, or the AAV particle is pre-loaded into a device.

51. 51. The kit of claim 50, wherein the device is a microcatheter.

52. 52. The kit of claim 51, wherein the microcatheter is shaped so that it can enter the middle ear cavity via the ear canal and contact the end of the microcatheter with the RWM.

53. 53. The kit of claim 52, wherein the distal end of the microcatheter is configured with at least one microneedle having a diameter of 10 to 1,000 micrometers.

54. 52. The kit of claim 51, wherein the device comprises a needle comprising a bent portion and a beveled tip.

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