Compositions and methods for treating SLC26A4-associated hearing loss

Abstract

Provided are compositions that result in the expression of gene products involved in the development, function, and / or maintenance of inner ear cells, including supporting cells and hair cells, the administration of said compositions, and / or the use of such compositions in the treatment of hearing loss or diseases or conditions associated with hearing loss. [Solution] Constructs are provided that include a coding sequence operably linked to a promoter, the coding sequence encoding a pendrin protein. 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

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 024,466, filed May 13, 2020, the contents of which are incorporated herein in their entirety. [Background technology]

[0002] 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 nonsyndromic hearing loss are associated with permanent hearing loss caused by damage to the structures of the inner ear (sensorineural hearing loss), although some forms may involve changes in the middle ear (conductive hearing loss). The majority of human sensorineural hearing loss is due to abnormalities in the hair cells of the organ of Corti in the cochlea (decreased hair cell function). Hair cells may be abnormal at birth or may be damaged during an individual's lifetime (e.g., as a result of noise trauma or infection). Summary of the Invention [Means for solving the problem]

[0003] The present disclosure recognizes that diseases or conditions associated with hearing loss can be treated, for example, by replacing or adding specific gene products. The present disclosure further provides that gene products involved in the development, function, and / or maintenance of inner ear cells can be useful in treating diseases or conditions associated with hair cells and / or that support cell loss. Accordingly, the present disclosure provides for the administration of compositions that result in the expression of gene products involved in the development, function, and / or maintenance of inner ear cells, including supporting cells and hair cells, and / or the use of such compositions in the treatment of hearing loss or diseases or conditions associated with hearing loss. In some embodiments, the gene product can be encoded by the SLC26A4 gene or a characteristic portion thereof. In some embodiments, the gene product can be a pendrin protein or a characteristic portion thereof.

[0004] The present disclosure further provides that AAV particles can be useful for administering compositions that result in expression of gene products involved in the development, function, and / or maintenance of inner ear cells, and / or for treating hearing loss or diseases or conditions associated with hearing loss. As described herein, AAV particles comprise (i) an AAV polynucleotide construct (e.g., a recombinant AAV polynucleotide construct) and (ii) a capsid comprising capsid proteins. In some embodiments, the AAV polynucleotide construct comprises the SLC26A4 gene or a characteristic portion thereof.

[0005] The present disclosure further provides compositions comprising polynucleotide constructs comprising the SLC26A4 gene or characteristic portions thereof. In some embodiments, the constructs may further comprise regulatory elements operably linked to the coding sequence. In certain embodiments, the included regulatory elements promote tissue-specific expression at physiologically appropriate levels.

[0006] The present disclosure also provides a genetically modified mouse whose genome comprises a modified Slc26a4 gene. In some embodiments, the genetically modified mouse comprises a modified Slc26a4 gene encoding the polypeptide set forth in SEQ ID NO: 57. In some embodiments, the genetically modified mouse is of a mouse strain suitable for use in auditory analysis experiments. In some embodiments, the genetically modified mouse is of a mouse strain suitable for use in orientation analysis experiments. In some embodiments, the genetically modified mouse is not a CBA / CaJ or CBA / J strain. In some embodiments, the genetically modified mouse suitable for use in auditory analysis experiments is of the FVB strain. In some embodiments, genetically modified mice suitable for use in auditory analysis experiments include FVB, 129 / Sv-+p+Tyr-c+Mgf-SIJ / J, A / HeJ, AKR / J, BALB / cByJ, BALB / cJ, BDP / J, BXSB / MpJ, C3H / HeJ, C3H / HeOuJ, C3HeB / FeJ, C57BL / 10J, C57BL / 10SnJ, C57BL / 6ByJ, CASA / RK, CAST / Ei, CBA / J, CZECH II / Ei, DBA / 2HaSmn, FVB / NJ, HRS / J hrl+, MOLD / Rk, MOLF / Ei, MOLG / Dn, NON / LtJ, NZB / B1NJ, NZO / NIJ, NZW / LacJ, PERA / camEi, PERC / Ei, PL / J, RBA / Dn, RBF / DnJ, RF / J, RHJ / Le hrrh-J / +, RIIIS / J, SEC / 1ReJ, SENCARC / PtJ, SF / CamEi, SHR / GnEi, SJL / J, SM / J, SPRET / Ei, ST / bJ, or SWR / J strains. In some embodiments, the genetically modified mice are treated with AAV particles, constructs, or compositions described herein.

[0007] Methods for administering the constructs and compositions described herein are also provided herein. In certain embodiments, administration involves surgical intervention and delivery of rAAV particles containing the therapeutic construct. In certain embodiments, the AAV particles can be delivered to the inner ear of a subject in need thereof by surgical introduction through the round window membrane. In some embodiments, the purpose of the intervention is to treat hearing loss in the subject. In some embodiments, the effectiveness of the intervention is determined through established tests, and measurements are compared to known control measurements.

[0008] definition The scope of the present disclosure is defined by the claims appended hereto, and is not limited by the specific embodiments described herein. Those skilled in the art who read this specification will recognize various modifications that may be equivalent to such described embodiments or that may otherwise be within the scope of the claims. Generally, terms used in this specification follow their understood meanings in the art unless expressly indicated otherwise. Explicit definitions of certain terms are provided below. Throughout this specification, the meaning of these and other terms in specific instances will be apparent to those skilled in the art from the context.

[0009] The use of ordinal terms such as "first," "second," and "third" in the claims to modify claim elements does not, in itself, imply any priority, precedence, or order of one claim element relative to other elements, or the chronological order in which method actions are performed, but is used solely as a marker to distinguish one claim element having a particular name from another element having the same name (but for the use of ordinal terms) to distinguish between claim elements.

[0010] 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 deemed to be satisfied if one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless clearly indicated to the contrary or otherwise apparent 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, two or more, or all, group members are present in, used in, or otherwise relevant to a given product or process. This disclosure does not permit one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims to be incorporated into another claim (or any other related claims) dependent on the same base claim, unless otherwise indicated or unless a contradiction or inconsistency would arise, apparent to one of ordinary skill in the art. It is to be understood that the present disclosure encompasses all variations, combinations, and permutations of any element or elements contained therein. When elements are presented as lists (e.g., in a Markush group or similar format), it is to be understood that each subgroup of elements is also disclosed, and that any element(s) can be removed from the group. In general, when an embodiment or aspect is described as "comprising" certain elements, features, etc., it is to be understood that a particular embodiment or aspect "consists of" or "consists essentially of" such elements, features, etc. For simplicity, these embodiments have not been specifically described in so many words in all instances herein. It is also to be understood that any embodiment or aspect can be explicitly excluded from the scope of the claims, regardless of whether a specific exclusion is recited herein.

[0011] Throughout this specification, whenever a polynucleotide or polypeptide is represented by a series of letters (e.g., in the case of polynucleotides, A, C, G, and T, representing adenosine, cytidine, guanosine, and thymidine, respectively), such polynucleotide or polypeptide will be presented in 5' to 3' or N-terminal to C-terminal order from left to right.

[0012] Administration: As used herein, the term "administration" typically refers to administering a composition to a subject or system to achieve delivery of an agent to the subject or system. In some embodiments, the agent is or is included in the composition. In some embodiments, the agent is produced through metabolism of the composition or one or more components thereof. Those of skill in the art will recognize 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 cochlear perilymph via injection through the round window membrane or into the scala tympani, injection through the endolymph, perilymph, and / or scala media via endolymph after canalostomy. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the application of a fixed number of doses. In some embodiments, administration may involve intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0013] Allele: As used herein, the term "allele" refers to one of two or more existing genetic variants at a particular polymorphic locus.

[0014] Amelioration: As used herein, the term "amelioration" refers to the prevention, reduction or alleviation of a subject's condition, or improvement of the condition. Amelioration can include, but does not require, complete recovery or complete prevention of a disease, disorder, or condition.

[0015] Amino acid: In its broadest sense, as used herein, the term "amino acid" refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure, e.g., HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-naturally occurring amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal amino acid and / or amino-terminal amino acid in a polypeptide, can include structural modifications compared to the general structures as shown above. For example, in some embodiments, In the present specification, amino acids may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of amino groups, carboxylic acid groups, one or more protons, and / or hydroxyl groups). In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to a polypeptide containing an otherwise identical, unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to a polypeptide containing an otherwise identical, unmodified amino acid.

[0016] 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 stated or otherwise clear from the context (unless such number exceeds 100% of possible values). For example, in some embodiments, the term "approximately" or "about" may encompass values ​​that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0017] Associated: As used herein, the term "associated" describes two events or entities being "associated" with one another if the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact directly or indirectly, such that they are in and / or remain in close physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently associated by, for example, hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0018] Biologically active: As used herein, the term "biologically active" refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is assessed through detection of a direct or indirect product produced by the biological pathway or event of interest.

[0019] 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 the particular characteristic, attribute, or activity, but not in those that do not share the particular characteristic, attribute, or activity. In some embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is a portion containing a stretch of consecutive amino acids, or a collection of stretches of consecutive amino acids, that together are characteristic of the protein or polypeptide. In some embodiments, such a stretch Each extension generally 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 characteristic with the related intact substance in addition to the sequence and / or structural identity identified above. In some embodiments, a characteristic portion may be biologically active.

[0020] 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 that can therefore be used by one of skill in the art to define the members of the family.

[0021] Characteristic sequence element: As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., consecutively linked monomers). In some embodiments, a characteristic sequence element comprises at least first and second stretches of contiguous monomers separated by one or more spacer regions, which may or may not vary in length between polymers sharing the sequence element.

[0022] 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, two or more agents may be administered simultaneously. In some embodiments, two or more agents may be administered sequentially. In some embodiments, two or more agents may be administered in an overlapping dosing regimen.

[0023] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, circumstances, sets of conditions, subjects, populations, etc. that are not identical to one another, but are sufficiently similar to permit a comparison between them so that one of ordinary skill in the art would understand that reasonable conclusions can be drawn based on the observed differences or similarities. In some embodiments, an equivalent set of agents, entities, circumstances, sets of conditions, subjects, populations, etc. is characterized by multiple substantially identical characteristics and one or a few diverse characteristics. One of ordinary skill in the art will understand the degree of identity required in any given situation for two or more such agents, entities, circumstances, sets of conditions, subjects, populations, etc. to be considered equivalent in context. For example, one skilled in the art will understand that sets of agents, entities, circumstances, sets of conditions, subjects, populations, etc. are equivalent to one another when they are characterized by a sufficient number and kind of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, stimuli, agents, entities, situations, sets of conditions, subjects, populations, etc. are caused by or indicate variations in those characteristics that vary.

[0024] Construct: As used herein, the term "construct" refers to a composition comprising a polynucleotide capable of carrying at least one heterologous polynucleotide. In some embodiments, a construct is 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 recombinant vector. ), or P1-derived artificial chromosome (PAC)) or viral construct, and any Gateway® plasmid. A construct can include, for example, sufficient cis-acting elements for expression; other elements for expression can be supplied by the host primate cell or in an in vitro expression system. A construct can include any genetic element (e.g., a plasmid, transposon, cosmid, artificial chromosome, or viral construct) capable of replication when associated with appropriate regulatory elements. Thus, in some embodiments, 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).

[0025] Conservative: As used herein, the term "conservative" refers to illustrative examples of conservative amino acid substitutions, including the replacement of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution will not substantially alter the desired functional property of a protein, e.g., the ability of a receptor to bind to a ligand. Examples of amino acid groups 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), glutamate / aspartate (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, conservative amino acid substitutions can be substitutions of any naturally occurring residue in a protein with alanine, such as those used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445 (incorporated herein by reference in its entirety). In some embodiments, the substitution is a moderately conservative substitution, and the substitution has a non-negative value in the PAM250 log-likelihood matrix.Those skilled in the art will understand that changes (e.g., substitutions, additions, deletions, etc.) in amino acids that are not conserved between the same proteins from different species are unlikely to affect the function of the protein, and therefore these amino acids should be selected for mutation. Amino acids that are conserved between the same proteins from different species should not be altered (e.g., deleted, added, substituted, etc.) because these mutations are likely to result in a change in the function of the protein. [Table 1]

[0026] Control: As used herein, the term "control" refers to the art-understood meaning of "control," which is a standard against which results are compared. Typically, controls are used to enhance consistency in experiments by isolating a variable in order to draw conclusions about such variable. In some embodiments, a control is a reaction or assay run 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, the "control," i.e., the variable being tested, is not applied. In some embodiments, a control is a historical control (e.g., a previously performed test or assay, or a previously known amount or result). In some embodiments, a control is or includes a printed or otherwise kept record. In some embodiments, a control is a positive control In some embodiments, the control is a negative control.

[0027] Determining, Measuring, Evaluating, Assessing, Assaying, and Analyzing: As used herein, the terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” may be used interchangeably to refer to any form of measurement, including determining whether an element is present or not. These terms include both quantitative and / or qualitative determinations. Assaying may be relative or absolute. For example, in some embodiments, “assaying for the presence of” can be determining the amount of something present and / or determining whether it is present or not.

[0028] Engineered: Generally, as used herein, the term "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a cell or organism is considered "engineered" if it has been manipulated so that its genetic information has been altered (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutations or by mating protocols). As is common practice and understood by those skilled in the art, the progeny of an engineered polynucleotide or cell are typically referred to as "engineered," even if the actual manipulation was performed on the previous entity.

[0029] Excipient: As used herein, the term "excipient" refers to an inactive (e.g., non-therapeutic) agent that may be included in a pharmaceutical composition to, for example, provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.

[0030] 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 embodiments, the gene product can be a transcription product. In some embodiments, the gene product can be a polypeptide. In some embodiments, 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.

[0031] Functional: As used herein, the term "functional" describes something that exists in a form in which it exhibits a property and / or activity by which it is characterized. For example, in some embodiments, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized. In some such embodiments, a functional biomolecule is characterized relative to another biomolecule that is non-functional in that the "non-functional" version does not exhibit the same or equivalent property and / or activity as the "functional" molecule. A biomolecule may have one function, two functions (i.e., bifunctional), or many functions (i.e., multifunctional).

[0032] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a gene product (e.g., an RNA product, e.g., a polypeptide product). In some embodiments, a gene comprises coding sequence (i.e., a sequence that encodes a specific product). In some embodiments, a gene comprises non-coding sequence. In some particular embodiments, a gene may comprise both coding (e.g., exon) and non-coding (e.g., intron) sequence. In some embodiments, a gene may comprise one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that, for example, can control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.). As used herein, the term "gene" generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof. The term may optionally encompass regulatory sequences, as will be clear to one of skill in the art from the context. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but rather to clarify that in most cases the term as used in this document refers to a polypeptide-encoding nucleic acid. In some embodiments, a gene may encode a polypeptide, but the polypeptide may not be functional; for example, a gene variant may encode a polypeptide that does not function in the same way as the wild-type gene, or may not function at all. In some embodiments, a gene may encode a transcript that may, in some embodiments, be toxic above a threshold level. In some embodiments, a gene may encode a polypeptide, but the polypeptide may not be functional and / or may be toxic above a threshold level.

[0033] Hearing loss: As used herein, the term "hearing loss" may refer to the partial or complete inability of a living ear to hear. In some embodiments, the hearing loss may be acquired. In some embodiments, the hearing loss may be hereditary. In some embodiments, the hearing loss may be genetic. In some embodiments, the hearing loss may be the result of disease or trauma (e.g., physical trauma, treatment with one or more drugs that cause hearing loss, etc.). In some embodiments, the hearing loss may be due to one or more known genetic causes and / or syndromes. In some embodiments, the hearing loss may be of unknown etiology. In some embodiments, the hearing loss may or may not be alleviated by the use of hearing aids or other treatments.

[0034] Heterologous: As used herein, the term "heterologous" may be used in reference to one or more regions of a particular molecule as compared to another region and / or another molecule. For example, in some embodiments, heterologous polypeptide domains refer to the fact that the polypeptide domains do not naturally occur together (e.g., within the same polypeptide). For example, in a fusion protein created by the hand of man, a polypeptide domain from one polypeptide may be fused to a polypeptide domain from a different polypeptide. In such a fusion protein, the two polypeptide domains would be considered "heterologous" to each other because they do not naturally occur together.

[0035] 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 to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into 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, alignment for comparison is performed using a sequence-specific sequence-specific sequence. The length of the referenced sequence 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 then the nucleotides at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the two molecules (i.e., the first and second 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 needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17, incorporated herein by reference in its entirety) as incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons performed with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0036] Improve, increase, enhance, inhibit, or reduce: As used herein, the terms "improve," "increase," "enhance," "inhibit," "reduce," or their grammatical equivalents refer to a value relative to a baseline or other reference measurement. In some embodiments, the value is 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., of a single individual) in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in an equivalent system known or expected to respond in a particular way in the presence of the relevant agent or treatment. In some embodiments, a suitable reference is a negative reference. In some embodiments, a suitable reference is a positive reference.

[0037] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As is clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA, and in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a 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 embodiments, a 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- The nucleic acid may be, comprise, or consist of: 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, intercalated bases, and combinations thereof. In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to sugars in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from natural sources, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in recombinant cells or systems, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 residues in length or longer. In some embodiments, the nucleic acid is partially or entirely single-stranded, and in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is complementary to a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0038] Operably linked: As used herein, refers to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control elements. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest. In some embodiments, the control element acts in trans with or otherwise interacts with the functional element of interest. In some embodiments, "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that effects expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In some embodiments, for example, functional linkage can include transcriptional control. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, for example, where necessary to join two protein-coding regions, in the same reading frame.

[0039] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for administration, such as, for example, an injectable formulation that is an aqueous or non-aqueous solution or suspension, or drops designed to be administered into the ear canal. In some embodiments, the pharmaceutical composition may be formulated for administration by injection, either in a specific organ or compartment (e.g., directly into the ear) or systemically (e.g., intravenously). In some embodiments, the formulation is in the form of a drench (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granules, paste, capsule, powder, etc. In some embodiments, the active agent may be or comprise an isolated, purified, or pure compound.

[0040] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" may be used in reference to, for example, a carrier, diluent, or excipient used to formulate a pharmaceutical composition disclosed herein, means the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0041] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in the carrying or transport of a subject compound from one organ or part of the body 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 function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as powdered tragacanth, malt, gelatin, talc, 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.

[0042] Polyadenylation: As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. In some embodiments, a 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to a pre-mRNA through the action of the enzyme polyadenylate polymerase. In higher eukaryotes, a poly(A) tail can be added to transcripts containing a specific sequence, a polyadenylation signal or "poly(A) sequence." The poly(A) tail and its associated proteins help protect the mRNA from degradation by exonucleases. Polyadenylation can affect transcription termination, mRNA export from the nucleus, and translation. Typically, polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand can be cleaved through 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 mRNA is cleaved, adenosine residues can be added to the free 3' end of the cleavage site. As used herein, a "poly(A) sequence" is a sequence that triggers endonuclease cleavage of mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0043] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide is designed and / or produced by the action of man. In some embodiments, polypeptides have amino acid sequences that are engineered in that they are engineered to conform to the intended structure. In some embodiments, polypeptides may comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, polypeptides may 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 embodiments, such pendant groups or modifications may be acetylation, amidation, lipidation, methylation, pegylation, etc. (including combinations thereof). In some embodiments, polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. In some embodiments, useful modifications may be or include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0044] Polynucleotide: As used herein, the term "polynucleotide" refers to any polymeric chain of nucleic acid. In some embodiments, a polynucleotide is or comprises RNA. In some embodiments, a polynucleotide is or comprises DNA. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, polynucleotide analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a polynucleotide has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, polynucleotides comprise one or more modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to sugars in natural nucleic acids.In some embodiments, the polynucleotide has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, the polynucleotide includes one or more introns. In some embodiments, the polynucleotide is prepared by one or more of the following: isolation from a natural source, enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template, reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, the polynucleotide is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 3 The polynucleotide may be 75, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 residues or more in length. In some embodiments, the polynucleotide is partially or entirely single-stranded, and in some embodiments, the polynucleotide is partially or entirely double-stranded. In some embodiments, the polynucleotide has a nucleotide sequence containing at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, the polynucleotide has enzymatic activity.

[0045] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., at least two groups of amino acids linked together by a peptide bond). A protein may include moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be processed or modified in other ways. One of skill in the art will understand that a "protein" can be an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. One of skill in the art will understand that a protein can include two or more polypeptide chains, for example, linked by one or more disulfide bonds or associated by other means.

[0046] Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide expressed using a recombinant expression construct transfected into a host cell, a polypeptide isolated from a recombinant, combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise engineered to express one or more genes or genetic components that encode and / or direct the expression of the polypeptide, or one or more component(s), portion(s), element(s), or domain(s) thereof; or a polypeptide prepared, expressed, produced, or isolated by any other means, including splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating nucleic acids that encode and / or direct the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources such as the germline of a source organism of interest (e.g., human, mouse, etc.).

[0047] Reference: As used herein, the term "reference" refers to 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 previously established reference or control, optionally embodied in a tangible medium. Typically, as understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. Those of skill in the art will understand when sufficient similarity exists to justify reliance on 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.

[0048] Regulatory element: As used herein, the term "regulatory element" or "regulatory sequence" refers to a non-coding region of DNA that regulates in some way the expression of one or more specific genes. In some embodiments, such genes are juxtaposed to or "proximal" to a given regulatory element. In some embodiments, such genes are located significantly farther from a given regulatory element. In some embodiments, a regulatory element impairs or enhances transcription of one or more genes. In some embodiments, a regulatory element can be located in cis with respect to the gene being regulated. In some embodiments, a regulatory element can be located in trans with respect to the gene being regulated. For example, in some embodiments, a regulatory sequence refers to a nucleic acid sequence that regulates the expression of a gene product operably linked to the regulatory sequence. In some such embodiments, this sequence may be an enhancer sequence or other regulatory element that regulates expression of the gene product.

[0049] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained from 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 includes a cell or organism, such as a microorganism (e.g., a virus), a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, semen, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural effusion, pus, catarrhal 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 embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological fluid may be or include plant exudates. In some embodiments, the biological tissue or sample can be obtained by, for example, 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 epithelial, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto), for example, filtering using a semipermeable membrane.Such "processed samples" may include, for example, nucleic acids or proteins extracted from a sample, or nucleic acids or proteins obtained by subjecting a primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of specific components, etc.

[0050] 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 is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is one who has one or more traits of susceptibility to 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 a diagnosis and / or therapy is administered.

[0051] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a desired characteristic or property. Those skilled in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0052] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapy that partially or completely alleviates, relieves, eliminates, reverses, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the occurrence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of subjects who do not exhibit signs of the associated disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, such treatment may be of subjects who exhibit one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who have been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who are known to have one or more susceptibility factors that statistically correlate with an increased risk of developing a given disease, disorder, and / or condition.

[0053] Variant: As used herein, the term "variant" refers to something (e.g., a version of 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 from that reference version. In some embodiments, a variant can have the same or a different (e.g., increased or decreased) level of activity or functionality as the wild-type sequence. For example, in some embodiments, a variant can have improved functionality compared to the wild-type sequence, for example, when codon-optimized to resist degradation by an inhibitory nucleic acid (e.g., miRNA). Such mutants are referred to herein as gain-of-function variants. In some embodiments, a variant has a reduction or elimination of activity or functionality that results in a negative outcome, or an altered activity (e.g., increased electrical activity that results in chronic depolarization that leads to cell death). Such variants are referred to herein as loss-of-function variants. For example, in some embodiments, the SLC26A4 gene sequence is a wild-type sequence that encodes a functional protein and is present in the majority of members of a species whose genome contains the SLC26A4 gene. In some such embodiments, a gain-of-function variant may be a gene sequence of SLC26A4 that contains one or more nucleotide differences compared to the wild-type SLC26A4 gene sequence. In some embodiments, a gain-of-function variant is a codon-optimized sequence that encodes a transcript or polypeptide that may have improved properties (e.g., less susceptible to degradation, e.g., less susceptible to miRNA-mediated degradation) than the corresponding wild-type (e.g., non-codon-optimized) version. In some embodiments, 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. For example, in some embodiments, mutations in the SLC26A4 sequence result in a non-functional or otherwise defective pendrin protein. [Brief explanation of the drawings]

[0054] [Figure 1] Panel (A) shows a simplified endogenous AAV genome, and panel (B) shows a simplified recombinant AAV (rAAV) construct capable of expressing the SLC26A4 gene. [Figure 2] 1 shows an exemplary nucleotide construct sequence map. [Figure 3] An exemplary rAAV construct containing the SLC26A4 gene is shown. [Figure 4] An exemplary rAAV construct containing the SLC26A4 gene is shown. [Figure 5] 1 shows the expression of pendrin protein in HEK293FT cells transfected with exemplary rAAV constructs. [Figure 6] 1 shows SLC26A4 mRNA expression in HEK293FT cells transduced with exemplary rAAV constructs and in wild-type neonatal CD1 explants. [Figure 7] FIG. 1 shows inner ear morphology of wild-type neonatal CD1 explants transduced with exemplary rAAV constructs. [Figure 8] Panel (A) shows the inner ear morphology of a P21-day-old C57BL / 6J mouse, and panel (B) shows the inner ear morphology of a P21-day-old C57BL / 6J Slc26a4tm1Dontuh / tm1Dontuh mouse that received a unilateral intracochlear injection of a composition containing an exemplary rAAV construct on day P3. [Figure 9]Panel (A) shows control hearing levels in C57BL / 6J heterozygous Slc26a4tm1Dontuh / + mice; Panel (B) shows auditory brainstem response (ABR) results from P21-day-old C57BL / 6J Slc26a4tm1Dontuh / tm1Dontuh mice that received a unilateral intraauricular injection of a composition comprising an exemplary rAAV construct on day P0; Panel (C) is a graphical representation of ABR data from test mice that were injected with control and exemplary rAAV construct-containing compositions on day P0 or day P3; and Panel (D) shows auditory brainstem response results from P21-day-old C57BL / 6J Slc26a4tm1Dontuh / tm1Dontuh mice that received a unilateral intracochlear injection of an exemplary rAAV construct-containing composition on day P3. [Figure 10] Figure 1 shows eGFP protein expression in HEK293T cells under the influence of various exemplary promoters; cells were sorted and quantified 72 hours after transfection. [Figure 11] ABR results from control homozygous Slc26a4 mutant mice (Slc26a4L236P / L236P), control WT mice (Slc26a4WT / WT), and homozygous Slc26a4L236P / L236P mice delivered with constructs as shown in Figure 4 by round window membrane (RWM) injection on day P2 are shown. The Y-axis is the ABR threshold in dB SPL in response to a click stimulus, and the X-axis represents the age at the time of measurement ranging from P30 to P180. The injected ear is recorded as "treated" and the uninjected ear is "contralateral." [Figure 12] ABR results from homozygous Slc26a4L236P / L236P mutant mice delivered with constructs as shown in Figure 4 by RWM injection on day P2 (N=4) are shown. The Y-axis shows the ABR threshold in dB SPL, and the X-axis represents the delivered noise stimulus (click at the indicated frequency, or pure tone). Measurements were taken on P30; the injected ear is labeled "treated" and the uninjected ear is labeled "contralateral." [Figure 13A]ABR results from homozygous Slc26a4L236P / L236P mutant mice delivered with constructs as shown in Figure 4 via RWM injection through a posterior semicircular canal (PSCC) fenestration on day P23 are shown. The Y-axis shows ABR thresholds in dB SPL, and the X-axis represents the noise stimulus delivered (click or pure tone at the indicated frequency). Pre-injection measurements were taken on P22, and post-injection measurements were taken on P50; the injected ear is labeled "treated," and the uninjected ear is labeled "contralateral." [Figure 13B] ABR results from homozygous Slc26a4L236P / L236P mutant mice delivered with the construct shown in Figure 4 via RWM injection through a PSCC fenestration on day P23 are shown. The Y-axis shows the ABR threshold in dB SPL, and the X-axis represents the delivered noise stimulus (click or pure tone at the indicated frequency). Pre-injection measurements were taken on P22, and post-injection measurements were taken on P50; the injected ear is labeled "treated," and the uninjected ear is labeled "contralateral." [Figure 14A] ABR results from four groups of untreated homozygous Slc26a4 L236P / L236P mutant mice are shown over time (P30–P150). The Y-axis shows ABR thresholds in dB SPL in response to click stimuli, and the X-axis represents age over time. Mice are grouped based on hearing level. Phenotypes such as circling were observed in groups of mice with degenerative hearing or stable levels of poor hearing. [Figure 14B] ABR results from one group of untreated homozygous Slc26a4 L236P / L236P mutant mice are shown over time (P21–P150). The Y-axis shows the ABR threshold in dB SPL, and the X-axis represents the noise stimulus (click or pure tone at the indicated frequency) provided. Animals in this group had a stable level of severe hearing loss over time and exhibited circling behavior. [Figure 14C]ABR results from one group of untreated homozygous Slc26a4 L236P / L236P mutant mice are shown over time (P21–P150). The Y-axis shows the ABR threshold in dB SPL, and the X-axis represents the noise stimulus (click or pure tone at the indicated frequency) provided. Animals in this group had stable levels of hearing loss over time and exhibited circling behavior. [Figure 14D] ABR results from one group of untreated homozygous Slc26a4 L236P / L236P mutant mice over time (P30–P150) are shown. The Y-axis shows ABR thresholds in dB SPL, and the X-axis represents the noise stimulus (click or pure tone at the indicated frequency) presented. Animals in this group had degenerated and impaired hearing by P60, at which point hearing stabilized at an impaired level and animals exhibited circling behavior. [Figure 14E] ABR results from one group of untreated homozygous Slc26a4 L236P / L236P mutant mice are shown over time (P30–P150). The Y-axis shows the ABR threshold in dB SPL, and the X-axis represents the noise stimulus (click or pure tone at the indicated frequency) provided. Animals in this group had stable hearing and did not exhibit circling behavior. [Figure 15] 1 shows a perspective view of a device for delivering fluid to the inner ear according to an aspect of the present disclosure. [Figure 16] 1 illustrates a side view of a bent needle subassembly according to an aspect of the present disclosure. [Figure 17] 1 shows a perspective view of a device for delivering fluid to the inner ear according to an aspect of the present disclosure. [Figure 18] 1 shows a perspective view of a bent needle subassembly coupled to the distal end of a device according to an aspect of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0055] hearing loss In general, the ear can be described as including the outer ear, middle ear, inner ear, auditory (sound) nerve, and auditory system (which processes sound as it travels from the ear to the brain). The ear not only detects sound but also helps maintain balance. Thus, in some embodiments, disorders of the inner ear can cause hearing loss, tinnitus, dizziness, imbalance, or a combination thereof.

[0056] Hearing loss can be the result of genetic factors, environmental factors, or a combination of genetic and environmental factors. Approximately half of people with tinnitus—a hallucination in the auditory system (ringing, buzzing, chirping, humming, or throbbing)—have a hypersensitivity or reduced tolerance to certain sound frequencies and volume ranges (also known as hyperacusis, also spelled hyperacousis). Various nonsyndromic and syndrome-associated hearing losses are known to those skilled in the art (e.g., DFNB4 and Pendred syndromes, respectively). Environmental causes of hearing impairment or loss include, for example, certain medications, certain prenatal or postnatal infections, and / or prolonged exposure to loud noise. In some embodiments, hearing loss can result from noise affecting specific parts of the ear, ototoxic agents, presbycusis, disease, infection, or cancer. In some embodiments, ischemic injury can cause hearing loss through pathophysiological mechanisms. In some embodiments, intrinsic abnormalities, such as congenital mutations in genes that play important anatomical or physiological roles in the cochlea, or genetic or anatomical changes in supporting cells and / or hair cells, can cause or contribute to hearing loss.

[0057] Hearing loss and / or hearing loss are among the most common sensory disorders in humans and can occur for many reasons. In some embodiments, subjects may be born with hearing loss or without hearing loss, while others may slowly lose their hearing over time. Approximately 36 million American adults report some degree of hearing loss, with one in three people over the age of 60 and half of people over the age of 85 experiencing hearing loss. Approximately 1.5 children in every 1,000 are born with profound hearing loss, and two to three children in every 1,000 are born with partial hearing loss (Smith et al., 2005, Lancet 365:879-890, incorporated herein by reference in its entirety). More than half of these cases are due to genetic causes (Di Domenico, et al., 2011, J. Cell. Physiol. 226:2494-2499, incorporated herein by reference in its entirety).

[0058] 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, incorporated herein by reference in their entireties). Recent research in this field has focused on cochlear hair cell regeneration, which is applicable to the most common forms of hearing loss, including presbycusis, noise injury, infection, and ototoxicity. There remains a need for effective treatments, such as gene therapy, that can repair and / or alleviate the source of hearing impairment (see, e.g., WO2018 / 039375, WO2019 / 165292, and PCT Application No. US2019 / 060328, each of which is incorporated herein by reference in its entirety).

[0059] In some embodiments, nonsyndromic hearing loss and / or hearing loss is not associated with other signs and symptoms. In some embodiments, syndromic hearing loss and / or hearing loss occurs in conjunction with abnormalities in other parts of the body. Approximately 70 to 80 percent of cases of hereditary hearing loss and / or hearing loss are nonsyndromic, with the remaining cases often caused by specific genetic syndromes. Nonsyndromic hearing loss and / or hearing loss can have different patterns of inheritance and can occur at any age. Types of nonsyndromic hearing loss and / or hearing loss are generally named according to their inheritance pattern. For example, the autosomal dominant type is called DFNA, the autosomal recessive type is called DFNB, and the X-linked type is called DFN. Each type is numbered in the order in which it was first described. For example, DFNA1 was the first autosomal dominant type of nonsyndromic hearing loss. Seventy-five to eighty percent of cases of genetically caused hearing loss and / or hearing loss are inherited in an autosomal recessive pattern, meaning that both copies of the gene in each cell carry a mutation. Typically, each parent of an individual with autosomal recessive hearing loss and / or hearing loss carries one copy of the mutated gene but is unaffected by this form of hearing loss. Another 20 to 25 percent of nonsyndromic hearing loss and / or hearing loss cases are autosomal dominant, meaning that one copy of the mutated gene in each cell is sufficient to cause hearing loss and / or hearing loss. People with autosomal dominant hearing loss and / or hearing loss almost always inherit an mutated copy of the gene from a parent with hearing loss and / or hearing loss. One to two percent of cases of hearing loss and / or hearing loss exhibit an X-linked inheritance pattern, meaning that the mutated gene causing the condition is located on the X chromosome (one of the two sex chromosomes). Males with X-linked nonsyndromic hearing impairment and / or hearing loss inherit two copies of the same gene mutation. Men tend to develop severe hearing loss earlier than women. A characteristic of X-linked inheritance is that fathers cannot pass on X-linked genes to their sons. Mitochondrial nonsyndromic hearing loss, caused by alterations to mitochondrial DNA, occurs in less than 1 percent of cases in the United States. The altered mitochondrial DNA is passed from mother to all sons and daughters. This form of hearing loss is not inherited from the father. The causes of syndromic and nonsyndromic hearing loss and / or hearing loss are complex. Researchers have identified more than 30 genes that, when altered, are associated with syndromic and / or nonsyndromic hearing loss and / or hearing loss, although some of these genes have not been fully characterized. Different mutations in the same gene can be associated with different types of hearing loss and / or hearing loss, and some genes are associated with both syndromic and nonsyndromic hearing loss and / or hearing loss.

[0060] 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 the structures of the inner ear (sensorineural hearing loss). In some embodiments, sensorineural hearing loss can result from poor hair cell function. In some embodiments, sensorineural transauricular damage involves the eighth cranial nerve (vestibulocochlear nerve) or auditory portion of the brain. In some such embodiments, only the auditory center of the brain is affected. In such situations, cortical hearing loss can occur, in which sounds can be heard at normal thresholds, but the perceived quality of the sound is so poor that speech cannot be understood. Hearing loss resulting from changes in the middle ear is called conductive hearing loss. Some forms of non-syndromic hearing loss and / or hearing loss involve changes in both the inner and middle ear, called mixed hearing loss. Hearing loss and / or hearing loss that is present before a child learns to speak can be classified as prelingual or congenital hearing loss and / or hearing loss. Hearing loss and / or hearing loss that occurs after speech development can be classified as postlingual hearing loss and / or hearing loss. Most autosomal recessive loci associated with syndromic or nonsyndromic hearing loss cause severe to profound prelingual hearing loss.

[0061] As known to those skilled in the art, hair cells are sensory receptors in both the auditory and vestibular systems of vertebrate ears. Hair cells detect movement in the environment, and in mammals, hair cells are located in the cochlea, the organ of Corti, of the ear. It is known that there are two types of hair cells in mammalian ears: inner hair cells and outer hair cells. Outer hair cells can amplify low-level sound frequencies through either mechanical movement of the hair cell bundle or electrically driven movement of the hair cell corpuscles. Inner hair cells convert vibrations in the cochlear fluid into electrical signals that the auditory nerve transmits to the brain. In some embodiments, hair cells may be abnormal at birth or damaged during an individual's lifetime. In some embodiments, outer hair cells may be able to regenerate. In some embodiments, inner hair cells cannot regenerate after disease or injury. In some embodiments, sensorineural hearing loss results from abnormalities in hair cells.

[0062] As known to those skilled in the art, hair cells do not occur in isolation; their function is supported by a wide variety of cells that may be collectively referred to as supporting cells. Supporting cells may perform numerous functions and comprise numerous cell types, including, but not limited to, Hensen's cells, Deiters' cells, Pillar cells, Claudius' cells, inner phalangeal cells, and border cells. In some embodiments, sensorineural hearing loss results from abnormalities in supporting cells. In some embodiments, supporting cells may be abnormal at birth or damaged during an individual's lifetime. In some embodiments, supporting cells may be capable of regenerating. In some embodiments, certain supporting cells may be capable of regenerating.

[0063] Solute Carrier Family 26 Member 4 (SLC26A4) The SLC26A4 gene is highly conserved and encodes the pendrin protein. The SLC26A4 gene is located on chromosome 7q22. The gene contains 21 exons spanning approximately 57 kilobases (kb) (NCBI accession number NG_008489.1). The full-length wild-type pendrin protein expressed from the human SLC26A4 gene is approximately 780 amino acids long.

[0064] Pendrin is an anion exchange protein that functions as a sodium-independent chloride-iodide exchanger, as well as a formate and bicarbonate exchanger. Pendrin shares homology with sulfate transporters. In the inner ear, pendrin functions as an ion exchanger, specifically a chloride and bicarbonate exchanger, where it is thought to help control the pH of the endolymph. The lack of properly functioning pendrin protein can result in an imbalance of certain ions. The resulting ion imbalance can interfere with the development and / or function of the thyroid gland and structures within the inner ear. In mammalian inner ears, the lack of properly functioning pendrin results in endolymphatic acidification, severe degeneration of sensory cells in the organ of Corti and the vestibular macula, and malformation of the otoliths and otolithic membranes.

[0065] The pendrin protein has a complex tertiary structure and is thought to be difficult to fold. Some mutations in SLC26A4 and / or pendrin are thought to lead to misfolding / defective transport and subsequent degradation. Some pendrin variants reach the plasma membrane and exhibit impaired transport function.

[0066] SLC26A4 protein is expressed in multiple nonsensory cell populations in the cochlea, vestibular labyrinth, and endolymphatic sac and duct. Without being limited by current theory, in the inner ear, pendrin is thought to be expressed within the epithelium of the endolymphatic sac and duct, on the apical membrane of transitional cells in the sac, urethra, and ampulla, and within a variety of cell types in the cochlea (inner and outer hair cells, Deiter's strips, Claudius cells, spiral ligament, spiral ganglion, spiral eminence, outer spiral sulcus cells), as well as in peripheral, intermediate, and basal cells.

[0067] Mutations in the SLC26A4 gene have been associated with hearing loss and hearing impairment (Albert et al., Eur. J. Hum. Genet. 14:773-779, 2006, and Qing et al., Genet. Test Mol. Biomarkers 19(1):52-58, 2015 (each of which is incorporated herein by reference in its entirety). Mutations in the SLC26A4 gene alter the structure or function of pendrin, disrupting its intrinsic function. More than 200 mutations in SLC26A4 have been reported to be associated with hearing loss. For example, point mutations E29Q, V138F, L236P, G209V, L236P, V239D, V250A, D266N, E303Q, F345S, N392Y, R409H, T410M, T416P, L445W, L597S, D697, K715N, H723R, and E737D have been reported in patients worldwide (e.g., at least in Chinese, Taiwanese, Mongolian, Turkish, Pakistani, French, Spanish, Czech, Iranian, Dutch, German, British, and / or North American patients) and have been associated with syndromic or nonsyndromic hearing loss (Dai et al., Physiol Genomics 38(3):281-290, 2015, and Tsukada et al., Ann Otol Rhinol Laryngol. 2015 May;124 Suppl 1:61S-76S. (each of which is incorporated herein by reference in its entirety). Further exemplary mutations in the SLC26A4 gene detected in subjects with nonsyndromic sensorineural hearing loss or syndromic sensorineural hearing loss, and methods for sequencing nucleic acids encoding SLC26A4, are described, for example, in Albert et al., Eur. J. Hum. Genet. 14:773-779, 2006, and Qing et al., Genet. Test Mol. Biomarkers 19(1):52-58, 2015 (each of which is incorporated herein by reference in its entirety). Methods for detecting mutations in genes are well known in the art. Non-limiting examples of such techniques include real-time polymerase chain reaction (RT-PCR), PCR, Sanger sequencing, next-generation sequencing, Southern blotting, and Northern blotting.

[0068] Mutations in the SLC26A4 gene and the encoded pendrin protein are associated with Pendred syndrome, an autosomal recessive genetic disorder characterized by congenital sensorineural hearing loss, cochlear abnormalities (enlarged vestibular aqueduct or Mondini malformation), and thyroid enlargement (goiter). Approximately 8% of congenital hearing loss cases are estimated to be attributable to Pendred syndrome, with a prevalence of approximately 8-16 per 100,000 live births in the United States or EU5, representing a population of approximately 40,000-80,000 affected individuals. In most individuals with Pendred syndrome, severe-to-profound hearing loss caused by alterations in the inner ear is evident before, at, or shortly after birth. Classically, hearing loss is bilateral, severe-to-profound, and congenital (or prelingual). However, hearing loss can be slow-onset and progressive, progression can be rapid in early childhood, and can be associated with head injury or infection.

[0069] Mutations in the SLC26A4 gene are also known to cause autosomal recessive hearing loss with enlargement of the vestibular aqueduct (EVA)-4 (DFNB4), another congenital cause of hearing loss. This disease state is sometimes called nonsyndromic enlarged vestibular aqueduct (NSEVA). While certain mutations in SLC26A4 are likely to cause DFNB4, other mutations are more associated with Pendred syndrome (Azaiez, et al. (December 2007), Hum. Genet. 122(5):451-7 (incorporated herein by reference in its entirety)). Patients with DFNB4 generally lack the additional goiter symptoms of Pendred syndrome.

[0070] While a correlation between Pendred syndrome and the presence of two mutant SLC26A4 alleles has been reported, DFNB4 may be associated with either one or even none of the mutant SLC26A4 alleles. Rarely, DFNB4 can also arise through bigenic inheritance of mutations in the FOXI1 gene or heterozygous mutations in the SLC26A4 gene and interacting genes FOXI1 or KCNJ10. For individuals with DFNB4 hearing loss, the degree and manifestation of hearing impairment can vary. For example, people with DFNB4 are typically born with normal hearing and gradually develop hearing loss during childhood. The majority of DFNB4 patients (approximately 80%) report fluctuating hearing.

[0071] Certain mouse models homozygous for Slc26A4 knockout (KO) have been reported to exhibit endolymphatic dilation, severe degeneration of sensory cells in the organ of Corti and vestibular macula by embryonic day 15 and postnatal week 2, resulting in malformations of the otoliths and otolithic membranes. In mice, loss of pendrin is associated with endolymphatic acidification.

[0072] As mentioned above, hundreds of SLC26A4 gene mutations have been identified, and in recent years, various mouse models have accelerated our understanding of the pathogenesis of DFNB4 and Pendred syndromes associated with such gene mutations (e.g., A. Nishio, et al., Slc26a4 expression prevents fluctuation of hearing in a mouse model of large vestibular aqueduct syndrome, Neuroscience 329(2016)74e82, T. Ito, et al., Progressive irreversible hearing loss is caused by stria vascularis degeneration in an Slc26a4-insufficient mouse model of large vestibular aqu educt syndrome,Neuroscience 310(2015)188e197, YCLu,et al.,Differences in the pathogenicity of the p.H723R mutation of the common deafness-associated SLC26A4 gene in humans and mice,PLoS One 8(6)(2014),e64906, T.Ito,et al.,Slc26a4-insufficiency causes fluctuating hearing loss and stria vascularis dysfunction,Neurobiol.Dis.66(2014)53e65, P.Wangemann,Mouse models for pendrin-associated loss of cochlear and vestibular function,Cell.Physiol.Biochem.32(7)(2013)157e165, and X.Li,et al.,SLC26A4 targeted to the endolymphatic sac rescues hearing and balance in SLC26A4 mutant mice, PLoS Genet. 9(7) (2013), e1003641 (each of which is incorporated herein by reference in its entirety).

[0073] In some circumstances, patients with different SLC26A4 mutations are associated with different clinical phenotypes (see, e.g., H. Azaiez, et al., Genotype-phenotype correlations for SLC26A4-related deafness, Hum. Genet. 122(5)(2007)451e457 (incorporated herein by reference in its entirety)). Similar to humans, mice with different mutations have been reported to have different phenotypes. For example, pds- / - mice were completely deaf and showed signs of vestibular dysfunction, with severe hair cell degeneration in both the organ of Corti and the vestibule, and malformations of the otoliths and otolithic membranes in the vestibule. Slc26a4 with the p.S408F mutation loop / loop Mice exhibit profound hearing loss, abnormal vestibular behavior, and malformations of the otoliths, but normal vestibular hair cell morphology. However, Slc26a4 carrying the p.H723R mutation tm2Dontuh / tm2Dontuh Mice can exhibit normal speech and vestibular phenotypes and inner ear morphology. Cell line studies have shown that certain SLC26A4 mutations only partially impair pendrin function, and see BY Choi, et al., "Hypo-functional SLC26A4 variants associated with nonsyndromic hearing loss and enlargement of the vestibular aqueduct: genotype-phenotype correlation or coincidental polymorphisms? Hum. Mutat. 30(4)(2009)599e608 (incorporated herein by reference in its entirety)," indicating that in some circumstances, the pathology associated with each distinct mutation is distinct. In addition, patients typically present with moderate to profound sensorineural hearing impairment (e.g., Y. Yuan, et al., "Molecular Hearing Loss and Enlargement of the Vestibular Aqueduct: Genotype-Phenotype Correlation or Coincidental Polymorphisms?"). epidemiology and functional assessment of novel allelic variants of SLC26A4 in non-syndromic hearing loss patients with enlarged vestibular aqueduct in China, PLoS One 7(11)(2012), e49984 (incorporated herein by reference in its entirety), however, in almost all cases, existing Slc26a4 mutant mouse strains (e.g., Slc26a4 tm2Dontuh / tm2Dontuh ) have profound loss of hearing and vestibular function, as well as severe inner ear malformations that do not necessarily mimic the human phenotype.

[0074] In some embodiments described herein, CRISPR / Cas technology is utilized to express Slc26a4 L236P / L236P Mice were generated to mimic the most common SLC26A4 mutation in Caucasians (see, e.g., J.S. Yoon, et al., Heterogeneity in the processing defect of SLC26A4 mutants, J. Med. Genet. 45(7) (2008) 411e419, which is incorporated herein by reference in its entirety). L236P / L236P The mice have a variable phenotypic profile, mimicking the human spectrum of disease manifestations. In some embodiments, L236P mice exhibited moderate to profound hearing loss (see, e.g., Figure 14A). L236P / L236P Mutant mice can mimic the human disease state and provide a useful tool for elucidating the pathogenesis of Pendred syndrome and the efficacy of potential gene therapies to alleviate symptoms associated with Pendred syndrome. In some embodiments, results generated in such models may more accurately mimic the human disease state and the outcomes of potential therapeutic interventions compared to previously described mouse models.

[0075] SLC26A4 polynucleotides Among other things, the present disclosure provides polynucleotides, eg, polynucleotides comprising the SLC26A4 gene or characteristic portions thereof, as well as compositions comprising such polynucleotides, and methods utilizing such polynucleotides and / or compositions.

[0076] In some embodiments, the polynucleotide comprising the SLC26A4 gene or a characteristic portion thereof can be DNA or RNA. In some embodiments, the DNA can be genomic DNA or cDNA. In some embodiments, the RNA can be mRNA. In some embodiments, the polynucleotide comprises exons and / or introns of the SLC26A4 gene.

[0077] In some embodiments, the gene product is expressed from a polynucleotide comprising the SLC26A4 gene or a characteristic portion thereof. In some embodiments, the expression of such a polynucleotide can utilize one or more regulatory elements (e.g., promoters, enhancers, splice sites, polyadenylation sites, translation initiation sites, etc.). Thus, in some embodiments, the polynucleotides provided herein can include one or more regulatory elements.

[0078] In some embodiments, the SLC26A4 gene is a mammalian SLC26A4 gene. In some embodiments, the Slc26a4 gene is a mouse Slc26a4 gene. In some embodiments, the SLC26A4 gene is a primate SLC26A4 gene. In some embodiments, the SLC26A4 gene is a human SLC26A4 gene. An exemplary human SLC26A4 cDNA sequence is or includes the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. An exemplary human SLC26A4 genomic DNA sequence can be found in SEQ ID NO: 3. An exemplary human SLC26A4 cDNA sequence including an untranslated region is or includes the sequence of SEQ ID NO: 4 or 5. Exemplary Human SLC26A4 cDNA Coding Sequence (SEQ ID NO: 1) ATGGCAGCGCCAGGCGGCAGGTCGGAGCCGCCGCAGCTCCCCGAGTACAGCTGCAGCTACATGGTGTCGCGGCCGGTCTACTCGGAGCTAGCTTTCCAGCAACAGCACGAGCGGCGCCTGCAGGAGCGCAAGACGCTGCGGGAGAGCCTGGCCAAGTGCTGCAGTTGTTCAAGAAAGAGAGCCTTTGGTGTGCTAAAGACTCTTGTGCCCATCTTGGAGTGGCTCCCCAAATACCGAGTCAAGGAATGGCTGCTTAGTGACGTCATTTCGGGAGTTAGTACTGGGCTAGTGGCCACGCTGCAAGGGATGGCATATGCCCT CTATGCGTACACTTGCATCCTAA Exemplary Human SLC26A4 cDNA Coding Sequence (SEQ ID NO:2) AACTCTGAGCTTCCAGTCAAAGTGAACGTTCCCAAAGTGCCAATCCATAGCCTTGTGCTTGACTGTGGAGCTATATCTTTCCTGGACGTTGTTGGAGTGAGATCACTGCGGGTGATTGTCAAAGAATTCCAAAGAATTGATGTGAATGTGTATTTTGCATCACTTCAAGATTATGTGATAGAAAAGCTGGAGCAATGCGGGTTCTTTGACG ACAACATTAGAAAGGACACATTCTTTTTGACGGTCCATGATGCTATACTCTATCTACAGAACCAAGTGAAATCTCAAGAGGGTCAAGGTTCCATTTTAGAAACGATCACTCTCATTCAGGATTGTAAAGATACCCTTGAATTAATAGAAACAGAGCTGACGGAAGAAGAACTTGATGTCCAGGATGAGGCTATGCGTACACTTGCATCCTGA Exemplary Human SLC26A4 Genomic DNA Sequence (SEQ ID NO:3) TGCTCCGTAAATAAAACGTCCCACTGCCTTCTGAGAGCGCTATAAAGGCAGCGGAAGGGTAGTCCGCGGGGCATTCCGGGCGGGGCGCGAGCAGAGACAGGTGAGTTCGCCCTGAAGATGCCCACACCGCCCGGCCCGGGCTCCACTCCCGGGGAGGCCTCGAGGGTTGCGGATGGGACTCTTAAGTGGTCACGGATCAGGTGGGCAGGGGGCAGTACAGCTTTCTTTCTGAGACGCCGAGAGCGAACAGGCTGCTCGGAAAACAGGACGAGGGGAGAGACTTGCTCAATAAGCTGAAAGTTCTGCCCCCGAGAGGGCTG AAGCGGTCTTAGGGGATCTTGAATAGCTTCCTGGAAGTTGAATGTCAAAGCTGAGTTTCCCTGATGGCTAAGTCTGAGTTTGCCAAGTGATGGGAAGTAGGGTGGGGTGGGGATGGCTTGAAAATAGACTATTTCAGAAGCTAGTAATGAATGTTGAGCAATATTGTGAGCTAAAATTTGGCTATGGAGCTTTCAAGATTGCTCAGATCAGGCCAGGTAGATGCCCGAAGAGAAGGAGGATGGCCTATGAACCCAGGGTGGAGCCTCAGCCTGATCAAGCATGTTCTTAATCACAAAGGTGGACGGGAGAGAGACTAGGATGGGCTGTGTGGTTCCTAAGTTAAACAATGCCAAGTATTCTACACTGTGCTCTAAGGGCTGTGACATGGTGACACCCTGT GACTCCTGGGGGACAGGAACCATGCCTCATTCATGTTTGTTCAGCAGGTGCCTAGCACAGAGACAGGCACATAATAGATGGTATATTTAGTGAAAAAGATTGAATTGCATTCCTGATAATGAATCTTCTTTATATATAAAAAATGGTGGTTCATGCTCCATGTCTCCCCAGTTTTCATAGACAAGTATCTTATAAGAATTCATTTTGTTTATAAGCATGAGACACTATGCTAAGTAAGAATGTTGCCAGCTATCTCATTTAATCCTCATAACAATCCTGCTAGGTAGGTCTATTATTATTTCATTTTCAGTAGGTGAAAAAATCTATTAAATCTTGCCCAGGGTCACGTGGCTGGTAAGTGGTACAGCTGGGATTTTAATCCGGTGTCTATTGTGTAGTTC A TTCATATTCTGGCTCTACTGTCTACTAGCCATGTGAGCCAATCACCATGCCTCAGTTTCCTTCTCTGCAAAATTGGGTTAGTAATAGTATATGCCTCATTGGATTGTTAAATAAGAATAGATAAGCACTTGGCCCAGGGGCTGGTACATAACAAGTACTCTAAATAAAGGAGTTATTTTGAAATGATTATTTCAAGCCATCTTTTCTTTTTTATTGGTGAAATGAAATGATTGGAGATGTATCTCTAAAAGCTTTTTCTAACCATAAGAGTCCGTAAAATGCATAATGTAAATGTCTCAACAATTATAAATGAAAAGGAACATTAGATTCAGAGATGATTCACCATGCAAAAGAAATGCA AAAGCAGGATGTAGATCACACTAATTAGATTTGAAAAAGGTTTTGGATCTAAAAATGTGTTCTACATAAAGCATGTTATTGATGCTTGAAAAATGGTGATAATAGTAACATGATGTCATCCTAGCTTGGGGGGAATGATACCATACAAAAATAACGTGTCAACATTAAGAATGGAGTTTGAGTCTTTAAAGACCCACCTATTAGCTTCTAAAACAATGTAAGGCCATGTACACATATCTAAGAATTGGTGAGTATGTCAATAATTGCTCAATTTTATAACGGTGCCATAATCCCATACTATTACACTCCATCCATTCAACAAATATTACCAAGTGCTTACTACAGGCCAGGTACTATGCT TTTTTCAAGTTTTAATTTTTGCATCTTGAGTTGAAAAGTACACCTTTAATGAATAGACAATTCATCAAAATGTTATACCTAGCTCCTGAATTAATTAGGGTCATTCATCAGAAAACTGATTATGTAAAGGACCAAACAGCAATGATAACCTTCAAGCCTCTGGGATAGGTAGGGCTGTTTGCTCCTGTGCTAAATGCCCTAAGTTCTTGTTATCGTGTCTGCTGGTGGTAGTTATCCCATTTTCTTTTTCACTCTGCTGTCCCTGGCATCATCTTGTTTCTGTCAGTGTAAATGCCTTTAGCAATTTTCAGTGACTCTGAAAACCAGACAAAGTGAATTTGTTAGTACTGGATTTCAAGAAAGAGCAAAAGTACCCTCTGAGTGAAAGTTCAAGAGTCTTC TCCCTCCCTTTCCTCCCCTTCCTTCCTTCCTTCCTCTCTCTCTTTTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTTCTTTCTTTCCTTCTTTCTTTCTTTTTCTTTTCCTTTCTTTCCTTTTCTGAAACAGAACCTCACTCTGTCACCCAGCATGGAATGCAGTGGCATGATCTCGGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACAACAGGCACACACTGCCATGCCTGGCTAATTTTTAGTAGAGACGGGGTTTCACCA TTGGTCCCACAGAACAACAGTTTGCTAGCCAAGAATATTTTTGTTGTAACATTTTTGGCATTTCTTCCTGAGTAAGATTTATGTTGTTGAAGTTCTGCATGTGGGTTTAATCTTATCTTTTAAATGTGGTTTGCAGTTGTTGCCTTTACAAGGTGGCCAAAAAGCCAGCATCCTAGTTAATCTCTGGATAATCCCTCCTTCAGTGTTCAGAAAGCTCAGGAGCCATACTCAAAGGCCACTCTTTCCAGCAGGACACAGTCGAAGAGACCACACCAAGCAGATGGGCACAAGAGACAGACTCAGGAATTTTGCTTCTGTCTCTTATGCCTTGAGGTCCTTATCTTCCCAACACAGAAAGAACTATTATTTAGGAGTAAGAAGTGCATTGAGACTCCAAAGAACAACAAACCCAAACTACATAGTGCAATACACACACACACACACACACACACACACACACACATGCACACACACATGCACAGTCTTCACAGTCTTCAAATGGGTTTTACTAAGGCGGGGACACTTAAAAAATAAATATAAGTCGGGCACAGTGGCTCATGCTTGTAATCCCAGCACTTTGGGAGGCTGAGGTGGGCGGATCAATTGAGGTCAGGAGTTCGAGACTAACATGGCCAACATGGTGAAACCCCTTCTCTACTAAAAATGCAAAAATTAGCCAGGTGTGGTGGTAGATGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGTGGAGGTTGCAGTGAGCCAATATCGTGCCACTACATTCCAGCCTGGGCG C GCTTACCAAGGAACAGTGTGTAGGTCTTTTGGATAATTTGATATGAATGGTTGAAAGATTTCAAATCTTTGACAATTAAGTTGACAGTGTTTTCTTCGTTTAGAATGGCATCATAAGTGATGCTGTTTCAACAAATAATGCTTTTGAGCCTGATGAGGATATTGAAGATCTGGAGGAACTTGATATCCCAACCAAGGAAATAGAGATTCAAGTGGATTGGAACTCTGAGCTTCCAGTCAAAGTGAACGTTCCCAAAGTGCCAATCCATAGCCTTGTGCTTGACTGTGGAGCTATATCTTTCCTGGACGTTGTTGGAGTGAGATCACTGCGGGTGGTAAGGTTCTGGGTTTTCTGAATTATA CATTTGGAGCTTTGGCAATAGTAAAATGATGTGGGTTGTCCAGTATTGCAACAGGGCAAATACATGGGCTTTGTAATTTTTCTAGGTGAATGCTTTTGTAAAAAAGTGTAATATTTTAAAGCATAGGCTCTGGAGCCAGACTACCTGGGGGAGATACTGGCTTCACCACTTACTAGCAGTACGACCCTGGGCAAGTTGCTTAATCTGTCTATATCTCAGTTTCTTCATCTGTAATATGGAGGTAATGATGGTATCTACCTTCACAGGTTGTTACAAGGATTAAATAAGCTAATAGATATAAGGTGTTTAGAAGAGTGTCTGGTTCAGGCTGGGCATGGTGGCTCACGCCTGTAATCCCAG GTGAGACCATGTCTCCAAAAAAATAATTGCTTTGATAGAAATTTTGACAAGGAATCTCAGATTGGACTTTTTAAAACGTCTTGATGCTATGAAGTCAAACCAAGGCAGACATTAGGCTTTGCCTGATGTATCTAATATCTTGAAGTTACTGGGCCTCCCAGGAAGGAACGACTTTTTATTCACTCATTGTAAGGCTAGCAGCCCTTGAAGCCAGGAATTCTGTGCACATTTTCAAATATGATATTCTATTCAAAGCCTTGATAATATAACCAATGTTTTCCAATTGTATTCTATTTAAAAGAACAGATTCTATTGAACTTTCATGTAAATAATCATATTGCCATAAAAATAAGAATACTCACAAAGAGTTTCCAAATTCTGGAAGGATCAGGTAGAGAGGAA TGTCCTTTTCCATGCAAAACACAAAGCCAAAATTGTCGTTGGTTTCCTCTGTGTAGAAGATTAAATTACATGCCACCTCTAAACAGTAGAGCTTTTCTGAATAACCAACTTGGTCCATAGACATTGGTTTCCATCTCCAATAGAATTAATTTCCACCCAATTCCATTTGTGGCTGTTTTTTGTCATCAGTGACAAGCTCTTCACTGTGCATTCATTGCACACTCAACGCTGTGCTAAGTGCTCTTAGCTTAGCCATTGAGAGATGCACTATTGACTGCTGAATCATTTAGGCAGAGGGGGTGACTTGTTAAGAGGCATAC CTGACATAAAAACAGTGCTATTCTGAGTGAAAATTTTTTTGATGTGCTTACATAACCATGGTGATTAAAATGAGTTTATATTTTTTCTCAAAAATTTTAGCAGTGTGTAAAGTAAGTAATCTTTAACTGAACTCTGACCACTTAAAAAAAAATCTAAAAATTGAACTACCTATAGTAGTCTGTGTTTAAAGTGAATTTTTAAAGACAAAGCATTCTAAATGAACTCAATATAAAAACATTCATTTGGAATGTACATACTGAAAAATACAGGTTTTTTTGACCAAAAGTTTTTATATCTTTTCTTTTTATTTATTTTTTTCCTAAGTGCCAACAATTTTCTAGATATTATATACAACACAGGCTTTGATCTTGGGGACTTTTCCCATATATTTCACACTGG AGTGAATGAAGTTGTACTTCATTTCTAGAGAAAGTTATACCCAGGTCCCCAATTGAGAATGTCTTGCTTGATTGAAAACGACATCATCCCTTGGTATACTCCAGGGATTGGTTTCAGGACCCCTGCATTTACCAAAATTTGTGCACACTCAAGTCCTGCAGTCACCCCTGCCTAAAGATAGAATGGCTTCTCTGTTTTTCTTCTGAAATACAACCAGAAACAATGTGTCTATTTCTGAAAGAATAGGATTAATGATCATACAAATGGGTTAATCCTGAATTCTGGTTGTAAATCTGGTTACAGCATAACTAGGATTATAATGCTGCCTCATTTTCACAGCACTACTTGCTTATATTGACAACAAATCATCTCGCTAAAGAGTGAATGTAGGCCAGGCGCG

Table 2

[0079] The present disclosure recognizes that certain changes to a polynucleotide sequence do not affect its expression or the protein encoded by the polynucleotide. In some embodiments, the polynucleotide comprises an SLC26A4 gene having one or more silent mutations. In some embodiments, the present disclosure provides polynucleotides comprising an SLC26A4 gene having one or more silent mutations, for example, an SLC26A4 gene having a sequence different from SEQ ID NO: 1, 2, 3, 4, or 5 but encoding the same amino acid sequence as a functional SLC26A4 gene. In some embodiments, the present disclosure provides polynucleotides comprising an SLC26A4 gene having a sequence different from SEQ ID NO: 1, 2, 3, 4, or 5 and encoding an amino acid sequence containing one or more mutations (e.g., a different amino acid sequence compared to that produced from a functional SLC26A4 gene), wherein the one or more mutations are conservative amino acid substitutions. In some embodiments, the present disclosure provides a polynucleotide comprising an SLC26A4 gene that has a sequence different from SEQ ID NO: 1, 2, 3, 4, or 5 and encodes an amino acid sequence that includes one or more mutations (e.g., a different amino acid sequence compared to that produced from a functional SLC26A4 gene), wherein the one or more mutations are not within a characteristic portion of the SLC26A4 gene or the encoded pendrin protein. In some embodiments, a polynucleotide according to the present disclosure comprises an SLC26A4 gene that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 1, 2, 3, 4, or 5 ... As can be understood in the art, SEQ ID NO: 1, 2, 3, 4, or 5 can be optimized (e.g., codon-optimized) to achieve increased or optimal expression in an animal, e.g., a mammal, e.g., a human.

[0080] Polypeptide encoded by the SLC26A4 gene In particular, the present disclosure provides a polypeptide encoded by the SLC26A4 gene or a characteristic portion thereof. In some embodiments, the SLC26A4 gene is a mammalian SLC26A4 gene. In some embodiments, the Slc2a4 gene is a mouse Slc2a4 gene. In some embodiments, the SLC26A4 gene is a primate SLC26A4 gene. In some embodiments, the SLC26A4 gene is a human SLC26A4 gene.

[0081] In some embodiments, the polypeptide comprises a pendrin protein or characteristic portion thereof. In some embodiments, the pendrin protein or characteristic portion thereof is a mammalian pendrin protein or characteristic portion thereof, e.g., a primate pendrin protein or characteristic portion thereof. In some embodiments, the pendrin protein or characteristic portion thereof is a human pendrin protein or characteristic portion thereof.

[0082] In some embodiments, the polypeptides provided herein comprise post-translational modifications. In some embodiments, the pendrin proteins or characteristic portions thereof provided herein comprise post-translational modifications. In some embodiments, the post-translational modifications can include, but are not limited to, glycosylation (e.g., N-linked glycosylation, O-linked glycosylation), phosphorylation, acetylation, amidation, hydroxylation, methylation, ubiquitination, sulfation, and / or combinations thereof.

[0083] An exemplary human pendrin protein sequence is or comprises the sequence of SEQ ID NO: 6. An exemplary human pendrin protein sequence with a C-terminal flag tag is or comprises the sequence of SEQ ID NO: 7. Exemplary Human Pendrin Protein Sequence (SEQ ID NO:6) MAAPGGRSEPQLPEYSCSYMVSRPVYSELAFQQQHERRLQERKTLRESLAKCCSCSRKRAFGVLKTLVPILEWLPKYRVKEWLLSDVISGVSTGLVATLQGMAYALLAAVPVGYGLYSAFFPILTYFIFGTSRHISVGPFPVVSLMVGSVVLSMAPDEHFLVSSSNGTVLNTTMIDTAARDTARVLIASALTLL VGIIQLIFGGLQIGFIVRYLADPLVGGFTTAAAFQVLVSQLKIVLNVSTKNYNGVLSIIYTLVEIFQNIGDTNLADFTAGLLTIVVCMAVKELNDRFRHKIPVPIPIEVIVTIIATAISYGANLEKNYNAGIVKSIPRGFLPPELPPVSLFSEMLAASFSIAVVAYAIAVSVGKVYATKYDYTIDGNQEFIAFGI SNIFSGFFSCFVATTALSRTAVQESTGGKTQVAGIISAAIVMIAILALGKLLEPLQKSVLAAVVIANLKGMFMQLCDIPRLWRQNKIDAVIWVFTCIVSIILGLDLGLLAGLIFGLLTVVLRVQFPSWNGLGSIPSTDIYKSTKNYKNIEEPQGVKILRFSSPIFYGNVDGFKKCIKSTVGFDAIRVYNKRLKAL RKIQKLIKSGQLRATKNGIISDAVSTNNAFEPDEDIEDLEELDIPTKEIEIQVDWNSELPVKVNVPKVPIHSLVLDCGAIISFLDVVGVRSLRVIVKE FQRIDVNVYFASLQDYVIEKLEQCGFFDDNIRKDTFFLTVHDAILYLQNQVKSQEGQGSILETITLIQDCKDTLELIETELTEEELDVQDEAMRTLAS Exemplary human pendrin protein sequence with C-terminal flag tag (SEQ ID NO: 7) MAAPGGRSEPQLPEYSCSYMVSRPVYSELAFQQQHERRLQERKTLRESLAKCCSCSRKRAFGVLKTLVPILEWLPKYRVKEWLLSDVISGVSTGLVATLQGMAYALLAAVPVGYGLYSAFFPILTYFIFGTSRHISVGPFPVVSLMVGSVVLSMAPDEHFLVSSSNGTVLNTTMIDTAARDTARVLIASALTLLVGIIQL IFGGLQIGFIVRYLADPLVGGFTTAAAFQVLVSQLKIVLNVSTKNYNGVLSIIYTLVEIFQNIGDTNLADFTAGLLTIVVCMAVKELNDRFRHKIPVPIPIEVIVTIIATAISYGANLEKNYNAGIVKSIPRGFLPPELPPVSLFSEMLAASFSIAVVAYAIAVSVGKVYATKYDYTIDGNQEFIAFGISNIFSGFFSCFVA TTALSRTAVQESTGGKTQVAGIISAAIVMIAILALGKLLEPLQKSVLAAVVIANLKGMFMQLCDIPRLWRQNKIDAVIWVFTCIVSIILGLDLGLLAGLI FGLLTVVLRVQFPSWNGLGSIPSTDIYKSTKNYKNIEEPQGVKILRFSSPIFYGNVDGFKKCIKSTVGFDAIRVYNKRLKALRKIQKLIKSGQLRATKNGI ISDAVSTNNAFEPDEDIEDLEELDIPTKEIEIQVDWNSELPVKVNVPKVPIHSLVLDCGAIISFLDVVGVRSLRVIVKEFQRIDVNVYFASLQDYVIEKLEQ CGFFDDNIRKDTFFLTVHDAILYLQNQVKSQEGQGSILETITLIQDCKDTLELIETELTEEELDVQDEAMRTLASGSRADYKDHDGDYKDHDIDYKDDDDK Exemplary Mouse Pendrin Protein Sequence (SEQ ID NO: 56) MAARGGRSEPQLAEYSCSYTVSRPVYSELAFQQQRERRLPERRTLRDSLARSCSCSRKRAFGVVKTLLPILDWLPKYRVKEWLLSDIISGVSTGLVGTLQGMAYALLAAVPVQFGLYSAFFPILTYFVFGTSRHISVGPFPVVSLMVGSVVLSMAPDDHFLVPSGNGSALNSTTLDTGTRDAARVLLASTLTLL VGIIQLVFGGLQIGFIVRYLADPLVGGFTTAAAFQVLVSQLKIVLNVSTKNYNGILSIIYTLIEIFQNIGDTNIADFIAGLLTIIVCMAVKELNDRFKHRIPVPIPIEVIVTIIATAISYGANLEKNYNAGIVKSIPSGFLPPVLPSVGLFSDMLAASFSIAVVAYAIAVSVGKVYATKHDYVIDGNQEFIAFGI SNVFSGFFSCFVATTALSRTAVQESTGGKTQVAGLISAVIVMVAIVALGRLLEPLQKSVLAAVVIANLKGMFMQVCDVPRLWKQNKTDAVIWVFTCIMSIILGLDLGLLAGLLFALLTVVLRVQFPSWNGLGSVPSTDIYKSITHYKNLEEPEGVKILRFSSPIFYGNVDGFKCCINSTVGFDAIRVYNKRLKAL RRIQKLIKKGQLRATKNGIISDIGSSNNAFEPDEDVEEPEELNIPTKEIEIQVDWNSELPVKVNVPKVPIHSLVLDCGAVSFLDVVGVRSLRMIVKEFQRIDVNVYFALLQDDVLEKMEQCGFFDDNIRKDRFFLTVHDAILHLQNQVKSREGQDSLLETVARIRDCKDPLDLMEAEMNAEELDVQDEAMRRLAS Exemplary Mouse Mutant Pendrin Protein Sequence (SEQ ID NO: 57) MAARGGRSEPPQLAEYSCSYTVSRPVYSELAFQQQRERRLPERRTLRDSLARSCSCSRKRAFGVVKTLLPILDWLPKYRVKEWLLSDIISGVSTGLVGTLQGMAYALLAAVPVQFGLYSAFFPILTYFVFGTSRHISVGPFPVVSLMVGSVVLSMAPDDHFLVPSGNGSALNSTTLDTGTRDAARVLLASTLTLLVGIIQLVFGGLQIGFIVRYLADPLVGGFTTAAAFQVLVSQPKIVL NVSTKNYNGILSIIYTLIEIFQNIGDTNIADFIAGLLTIIVCMAVKELNDRFKHRIPVPIPIEVIVTIIATAISYGANLEKNYNAGIVKSIPSGFLPPVLPSVGLFSDMLAASFSIAVVAYAIAVSVGKVYATKHDYVIDGNQEFIAFGISNVFSGFFSCFVATTALSRTAVQESTGGKTQVAGLISAVIVMVAIVALGRLLEPLQKSVLAAVVIANLKGMFMQVCDVPRLWKQNKTDAVIWVFTCIMSIILGLDLGLLAGLLFALLTVVLRVQFPSWNGLGSVPSTDIYKSITHYKNLEEPEGVKILRFSSPIFYGNVDGFKKCINSTVGFDAIRVYNKRLKALRRIQKLIKKGQLRATKNGIISDIGSSNNAFEPDEDVEEPEELNIPTKEIEIQVDWNSELPVKVNVPKVPIHSLVLDCGAVSFLDVVGVRSLRMIVKEFQRIDVNVYFALLQDDVLEKMEQCGFFDDNIRKDRFFLTVHDAILHLQNQVKSREGQDSLLETVARIRDCKDPLDLMEAEMNAEELDVQDEAMRRLAS

[0084] The present disclosure recognizes that certain mutations in the amino acid sequence of a polypeptide described herein (e.g., comprising pendrin or a characteristic portion thereof) do not affect the expression, folding, or activity of the polypeptide. In some embodiments, a polypeptide (e.g., comprising pendrin or a characteristic portion thereof) comprises one or more mutations, wherein the one or more mutations are conservative amino acid substitutions. In some embodiments, a polypeptide in accordance with the present disclosure comprises a pendrin or a characteristic portion thereof that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:6. In some embodiments, a polypeptide in accordance with the present disclosure comprises a pendrin or a characteristic portion thereof that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:7. In some embodiments, a polypeptide in accordance with the present disclosure comprises a pendrin protein or a characteristic portion thereof that is identical to the sequence of SEQ ID NO:7.

[0085] construct In particular, the present disclosure provides that some polynucleotides described herein are polynucleotide constructs. Polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viral constructs (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus constructs) that incorporate a polynucleotide comprising the SLC26A4 gene or a characteristic portion 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 embodiments, the construct is a plasmid (i.e., a circular DNA molecule that can replicate autonomously within a cell). In some embodiments, the construct can be a cosmid (e.g., the pWE or sCos series).

[0086] In some embodiments, the construct is a viral construct. In some embodiments, the viral construct is a lentiviral, retroviral, adenoviral, or adeno-associated viral construct. In some embodiments, the construct is an adeno-associated viral (AAV) construct (see, e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, incorporated herein by reference in its entirety). In some embodiments, the viral construct is an adenoviral construct. In some embodiments, the viral construct may be based on or derived from an alphavirus. Alphaviruses include Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Beval virus, Cadbury virus, and the like. These include Assou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kizilagach virus, Mayaro virus, Me Tri virus, Middleburg virus, Mosso das Pedras virus, Mucambo virus, Nudum 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, Benebela equine encephalitis virus, Western equine encephalitis virus, and Wataroa virus. Generally, the genomes of such viruses encode nonstructural (e.g., replicon) 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. Patent Publication Nos. 20150050243, 20090305344, and 20060177819; the constructs and methods for their production are incorporated herein by reference in their entirety.

[0087] 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.

[0088] In some embodiments, the construct is a viral construct and can have a total nucleotide number of up to 10 kb. In some embodiments, the viral construct may be from about 1 kb to about 2 kb, 1 kb to about 3 kb, from about 1 kb to about 4 kb, from about 1 kb to about 5 kb, from about 1 kb to about 6 kb, from about 1 kb to about 7 kb, from about 1 kb to about 8 kb, from about 1 kb to about 9 kb, from about 1 kb to about 10 kb, from about 2 kb to about 3 kb, from about 2 kb to about 4 kb, from about 2 kb to about 5 kb, from about 2 kb to about 6 kb, from about 2 kb to about 7 kb, from about 2 kb to about 8 kb, from about 2 kb to about 9 kb, from about 2 kb to about 10 kb, from about 3 kb to about 4 kb, from about 3 kb to about 5 kb, from about 3 kb to about 6 kb, from about 3 kb to about 7 kb, from about 3 kb to about 8 kb, from about 3 kb to about 9 kb, The total number of nucleotides may be 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.

[0089] In some embodiments, the construct is a lentiviral construct and can have a total nucleotide number of up to 8 kb. In some examples, the lentiviral construct can be about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 8 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 3 kb to about 11 kb, about 3 kb to about 12 kb, about 3 kb to about 13 kb, about 3 kb to about 14 kb, about 3 kb to about 15 kb, about 3 kb to about 16 kb, about 3 kb to about 17 kb, about 3 kb to about 18 kb, about 3 kb to about 19 kb, about 3 k The total number of nucleotides can be from about 3 kb to about 7 kb, from about 3 kb to about 8 kb, from about 4 kb to about 5 kb, from about 4 kb to about 6 kb, from about 4 kb to about 7 kb, from about 4 kb to about 8 kb, from about 5 kb to about 6 kb, from about 5 kb to about 7 kb, from about 5 kb to about 8 kb, from about 6 kb to about 8 kb, from about 6 kb to about 7 kb, or from about 7 kb to about 8 kb.

[0090] In some embodiments, the construct is an adenoviral construct and can have a total nucleotide number of up to 8 kb. In some embodiments, the adenoviral construct can be about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, It can have a total number of nucleotides 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.

[0091] Any of the constructs described herein can further comprise regulatory sequences selected from, 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 embodiments, the promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of regulatory sequences are described herein.

[0092] AAV particles Among other things, the present disclosure provides AAV particles comprising a construct encoding the SLC26A4 gene or a characteristic portion thereof described herein and a capsid described herein. In some embodiments, the AAV particles can be described as having a serotype, which is a description of the construct strain and capsid strain. For example, in some embodiments, the AAV particles can be described as AAV2, where the particle has an AAV2 capsid and a construct comprising characteristic AAV2 inverted terminal repeats (ITRs). In some embodiments, the AAV particles can be described as pseudotyped, where the capsid and construct are derived from different AAV strains, for example, AAV2 / 9 refers to an AAV particle comprising a construct utilizing AAV2 ITRs and an AAV9 capsid.

[0093] AAV constructs The present disclosure provides a polynucleotide construct comprising the SLC26A4 gene or a characteristic portion thereof. In some embodiments described herein, the polynucleotide comprising the SLC26A4 gene or a characteristic portion thereof can be included in an AAV particle.

[0094] In some embodiments, the polynucleotide construct comprises one or more components derived from or modified from a naturally occurring AAV genome construct. In some embodiments, the sequence derived from the AAV construct is an AAV1 construct, an AAV2 construct, an AAV3 construct, an AAV4 construct, an AAV5 construct, an AAV6 construct, an AAV7 construct, an AAV8 construct, an AAV9 construct, an AAV2.7m8 construct, an AAV8BP2 construct, an AAV293 construct, or an AAV Anc80 construct. Additional exemplary AAV constructs that can be used herein are known in the art. For example, see 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).

[0095] In some embodiments, the provided constructs include a coding sequence, e.g., the SLC26A4 gene or a characteristic portion thereof, one or more regulatory and / or control sequences, and optionally 5' and 3' AAV-derived inverted terminal repeats (ITRs). In some embodiments in which 5' and 3' AAV-derived ITRs are utilized, the polynucleotide construct may be referred to as a recombinant AAV (rAAV) construct. In some embodiments, the provided rAAV constructs are packaged into AAV capsids to form AAV particles.

[0096] In some embodiments, the AAV-derived sequences (included in the polynucleotide construct) typically include cis-acting 5' and 3' ITR sequences (see, e.g., B.J. Carter, in "Handbook of Parvoviruses," ed., P. Tijsser, CRC Press, pp. 155-168, 1990, incorporated herein by reference in its entirety). A typical AAV2-derived ITR sequence is approximately 145 nucleotides in length. In some embodiments, at least 80% (e.g., 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., texts 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 herein by reference in its entirety.) In some embodiments, any of the coding sequences and / or constructs described herein are flanked by 5' and 3' AAV ITR sequences. ITR sequences can be obtained from any known AAV, including currently identified AAV types.

[0097] In some embodiments, polynucleotide constructs, as described in accordance with the present disclosure and in a pattern known in the art (e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, incorporated herein by reference in its entirety), typically consist of a coding sequence or portion thereof, at least one and / or regulatory sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). In some embodiments, the provided constructs can be packaged into capsids to produce AAV particles. The AAV particles may be delivered to selected target cells. In some embodiments, the provided constructs include an additional optional coding sequence that is a nucleic acid sequence heterologous to the construct sequence (e.g., an inhibitory nucleic acid sequence) that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product of interest. In some embodiments, the nucleic acid coding sequence is operably linked to and / or controls components in a manner that allows for coding sequence transcription, translation, and / or expression in cells of the target tissue.

[0098] As shown in panel (A) of Figure 1, an unmodified AAV endogenous genome includes two open reading frames, "cap" and "rep," flanked by ITRs. As shown in panel (B) of Figure 1, an exemplary rAAV construct similarly includes ITRs flanking a coding region, e.g., a coding sequence (e.g., the SLC26A4 gene). In some embodiments, the rAAV construct also contains ITRs flanking a coding region, e.g., a coding sequence (e.g., the SLC26A4 gene). In some embodiments, the rAAV construct also contains ITRs flanking a coding region, e.g., a coding sequence (e.g., the SLC26A4 gene). In some embodiments, the rAAV construct also contains ITRs flanking a coding region, e.g., a coding sequence (e.g., the SLC26A4 gene) ... The rAAV constructs include conventional regulatory elements operably linked to the coding sequence in a manner that allows for transcription, translation, and / or expression. In some embodiments, the rAAV constructs optionally include a promoter (shown in panel (B) of Figure 1), an enhancer, an untranslated region (e.g., 5'UTR, 3'UTR), a Kozak sequence, an internal ribosome entry site (IRES), a splicing site (e.g., acceptor site, donor site), a polyadenylation site (shown in panel (B) of Figure 1), or any combination thereof. Such additional elements are further described herein.

[0099] In some embodiments, the construct is a rAAV construct. In some embodiments, the rAAV construct can comprise at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, or at least 4.5 kb. In some embodiments, the AAV construct can comprise up to 7.5 kb, up to 7 kb, up to 6.5 kb, up to 6 kb, up to 5.5 kb, up to 5 kb, up to 4.5 kb, up to 4 kb, up to 3.5 kb, up to 3 kb, or up to 2.5 kb. In some embodiments, the AAV construct can 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.

[0100] Any of the constructs described herein can further comprise regulatory and / or control sequences, e.g., 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 embodiments, the promoter can 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.

[0101] 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, incorporated herein by reference in its entirety). Generally, ITRs are capable of forming hairpins. The ability to form hairpins can contribute to the ITRs' ability to self-prime, allowing for primase-independent synthesis of the second DNA strand. ITRs aid in the efficient encapsidation of AAV constructs within AAV particles.

[0102] rAAV particles (e.g., AAV2 / Anc80 particles) of the present disclosure can include rAAV constructs containing coding sequences (e.g., the SLC26A4 gene) and associated elements flanking the 5' and 3' AAV ITR sequences. In some embodiments, the ITRs are or include approximately 145 nucleic acids. In some embodiments, all or substantially all of the ITR-encoding sequences are used. AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the ITRs are AAV2 ITRs.

[0103] An example of a construct molecule used in this disclosure is a "cis-acting" construct containing a transgene, in which the 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 sequences relative to the entire construct read from left to right in the sense orientation. For example, in some embodiments, In embodiments, the 5' or left ITR is the ITR closest to the promoter of a given construct (different from the polyadenylation sequence) when the construct is displayed linearly 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 closest to the polyadenylation sequence of a given construct (different from the promoter sequence) when the construct is displayed linearly in the sense orientation. The ITRs provided herein are listed in 5' to 3' order according to the sense strand. Therefore, those skilled in the art will understand that when converting from sense to antisense, an ITR in the 5' or "left" orientation can also be depicted as a 3' or "right" ITR. Furthermore, converting a given sense ITR sequence (e.g., a 5' / left AAV ITR) to an antisense sequence (e.g., a 3' / right ITR sequence) is well within the capabilities of one skilled in the art. One of skill in the art would know how to modify a given ITR sequence for use as either a 5' / left or 3' / right ITR, or an antisense version thereof.

[0104] For example, an ITR (e.g., a 5' ITR) can have a sequence according to SEQ ID NO: 10. In some embodiments, an ITR (e.g., a 3' ITR) can have a sequence according to SEQ ID NO: 11. 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., 127, 130, 134, or 141 nucleotides. For example, in some embodiments, an 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, an ITR (e.g., a 5' ITR) can have a sequence according to SEQ ID NO: 12. In some embodiments, an ITR (e.g., a 3' ITR) can have a sequence according to SEQ ID NO: 13.

[0105] A non-limiting example of a 5' AAV ITR sequence is SEQ ID NO: 10. A non-limiting example of a 3' AAV ITR sequence is SEQ ID NO: 11. In some embodiments, the rAAV constructs of the present disclosure comprise a 5' AAV ITR and / or a 3' AAV ITR. In some embodiments, the 5' AAV ITR sequence is SEQ ID NO: 12. In some embodiments, the 3' AAV ITR sequence is SEQ ID NO: 13. In some embodiments, the 5' and 3' AAV ITRs (e.g., SEQ ID NOs: 10 and 11, or 12 and 13) flank a portion of a coding sequence, e.g., all or a portion of the SLC26A4 gene (e.g., SEQ ID NO: 1 or 2). 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 See texts such as "Laboratory Manual," 2nd 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 sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the 5' ITR sequence set forth in SEQ ID NO: 10 or 12. In some embodiments, the 3' ITR sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the 3' ITR sequence set forth in SEQ ID NO: 11 or 13. Exemplary 5' AAV ITR (SEQ ID NO: 10) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT Exemplary 3' AAV ITR (SEQ ID NO: 11) AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGGGAGTGGCCAA Exemplary 5' AAV ITR (SEQ ID NO: 12) CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT Exemplary 3' AAV ITR (SEQ ID NO: 13) AGGAACCCCTAGTGATGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0106] promoter In some embodiments, a construct (e.g., an 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., the SLC26A4 gene). For example, a promoter typically refers to a nucleotide sequence to which, for example, RNA polymerase and / or any associated factors can bind and initiate transcription therefrom. Thus, in some embodiments, a construct (e.g., an rAAV construct) comprises a promoter operably linked to one of the non-limiting exemplary promoters described herein.

[0107] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an artificial promoter, a tissue-specific 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, the HI promoter, the human U6 promoter, the mouse U6 promoter, or the porcine U6 promoter. The promoter is generally a promoter capable of promoting transcription in inner ear cells. In some embodiments, the promoter is a cochlea-specific or cochlea-directed promoter. In some embodiments, the promoter is a hair cell-specific or supporting cell-specific promoter.

[0108] Various promoters are known in the art and can be used herein. Non-limiting examples of promoters that can be used herein include human EF1α, human cytomegalovirus (CMV) (U.S. Pat. No. 5,168,062, the entire contents of which are incorporated herein by reference), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40), β-globin, β-actin, α-fetoprotein, γ-globin, β-interferon, γ-glutamyltransferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g., human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, mouse MX gene, glucose-regulated proteins (GRP78 and GRP94), α-2-macroglobulin, vimentin, MHC class I gene H-2 K b, HSP70 , proliferin, tumor necrosis factor, thyroid-stimulating hormone alpha gene, immunoglobulin light chain, T cell receptor, HLA DQα and DQ, interleukin 2 receptor, MHC class II, MHC class II HLA-DRα, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponin I (TN I), Duchenne muscular dystrophy, human immunodeficiency virus, and Gibbon Ape Leukemia Virus (GALV) promoters. Further examples of promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology, Freeman and Company, New York 2007 (each of which is incorporated herein by reference in its entirety). In some embodiments, the promoter is a CMV immediate early promoter. In some embodiments, the promoter is a CAG promoter or a CAG / CBA promoter. In some embodiments, the promoter comprises or consists of SEQ ID NO: 14. In some embodiments, the promoter comprises or consists of SEQ ID NO: 15. In certain embodiments, the promoter comprises a CMV / CBA enhancer / promoter construct exemplified by SEQ ID NO: 16. In certain embodiments, the promoter comprises a CMV / CBA enhancer / promoter construct exemplified by SEQ ID NO: 17. In certain embodiments, the promoter comprises a CAG promoter or a CMV / CBA / SV-40 enhancer / promoter construct exemplified by SEQ ID NO: 43. In certain embodiments, the promoter comprises a CAG promoter or a CMV / CBA / SV-40 enhancer / promoter construct exemplified by SEQ ID NO: 44. In some embodiments, the promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the promoter sequence represented by SEQ ID NO: 14 or 15.In some embodiments, the enhancer-promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer-promoter sequence set forth in SEQ ID NO: 16, 17, 43, or 44.

[0109] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a nucleic acid encoding a protein (e.g., pendrin protein), causes transcription of RNA from the nucleic acid in a cell under most or all physiological conditions.

[0110] Examples of constitutive promoters include the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (e.g., Boshart et al, Cell 41:521-530, 1985 (incorporated herein in its entirety), SV40 promoter, dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1-alpha promoter (Invitrogen).

[0111] Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions (e.g., acute phase, specific differentiation states of cells, or only in replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Further examples of inducible promoters are known in the art.

[0112] Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088, incorporated herein by reference in its entirety); The tetracycline-inducible system (Gossen et al., Science 268:1766-1769, 1995; see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518, 1998; each of which is incorporated by reference in its entirety)), the RU486-inducible system (Wang et al., Nat. Biotech. 15:239-243, 1997; and Wang et al., Gene Ther. 4:432-441, 1997 (each of which is incorporated herein by reference in its entirety), and the rapamycin-inducible system (Magari et al. J Clin. Invest. 100:2865-2872, 1997 (each of which is incorporated herein by reference in its entirety).

[0113] The term "tissue-specific" promoter refers to a promoter that is active only in certain cell types and / or tissues (e.g., transcription of a particular gene occurs only in cells that express a transcriptional regulatory and / or control protein that is linked to the tissue-specific promoter).

[0114] In some embodiments, the regulatory and / or control sequences confer tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory and / or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.

[0115] In some embodiments, the tissue-specific promoter is a cochlear-specific promoter. In some embodiments, the tissue-specific promoter is a cochlear hair cell-specific promoter. Non-limiting examples of cochlear hair cell-specific promoters include, but are not limited to, the ATOH1 promoter, the POU4F3 promoter, the LHX3 promoter, the MYO7A promoter, the MYO6 promoter, the α9ACHR promoter, and the α10ACHR promoter. In some embodiments, the promoter is a cochlear hair cell-specific promoter, such as the PRESTIN promoter or the ONCOMOD promoter. See, e.g., Zheng et al., Nature 405:149-155, 2000; Tian et al., Dev. Dyn. 23 1:199-203, 2004; and Ryan et al., Adv. Otorhinolaryngol. 66:99-115, 2009.

[0116] In some embodiments, the tissue-specific promoter is an ear cell-specific promoter. In some embodiments, the tissue-specific promoter is an inner ear cell-specific promoter. Non-limiting examples of inner ear non-sensory cell-specific promoters include, but are not limited to, GJB2, GJB6, SLC26A4, TECTA, DFNA5, COCH, NDP, SYN1, GFAP, PLP, TAK1, or SOX21. In some embodiments, the cochlear non-sensory cell-specific promoter can be an inner ear supporting cell-specific promoter. Non-limiting examples of inner ear supporting cell-specific promoters include, but are not limited to, SOX2, FGFR3, PROX1, GLAST1, LGR5, HES1, HES5, NOTCH1, JAG1, CDKN1A, CDKN1B, SOX10, P75, CD44, HEY2, LFNG, or S100b.

[0117] In some embodiments, the provided AAV construct comprises a promoter sequence selected from a CAG, CBA, CMV, or CB7 promoter. In some embodiments of any of the therapeutic compositions described herein, the first or sole AAV construct is , cochlea- and / or inner ear-specific promoters. Exemplary CBA Promoter (SEQ ID NO: 14) GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGG CGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG Exemplary CBA Promoter (SEQ ID NO: 15) GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGCGCGCCAGGCGGGGC GGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG Exemplary CMV / CBA Enhancer / Promoter (SEQ ID NO: 16) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG Exemplary CMV / CBA enhancer / promoter (SEQ ID NO: 17) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG GGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG Exemplary CAG enhancer / promoter (SEQ ID NO: 43) Exemplary CAG enhancer / promoter (SEQ ID NO: 44) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT

[0118] In certain embodiments, the promoter is the endogenous human ATOH1 enhancer-promoter set forth in SEQ ID NO: 18. In some embodiments, the enhancer-promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer-promoter sequence set forth in SEQ ID NO: 18. Exemplary Human ATOH1 Enhancer-Promoter (SEQ ID NO: 18) CTATGGAGTTTGCATAAAAAAACGTTTGGCAGCTCGCTCTCTTACACTCCATTAACAAGCTGTAACATATAGCTGCAGGTTGCTATAATCTCATTAATATTTTGGAAACTTGAATATTGAGTATTTCTGAGTGCTCATTCCCCATATGCCAGCCACTTCTG

[0119] In certain embodiments, the promoter is the endogenous human SLC26A4 immediate promoter set forth in SEQ ID NO: 45 or 46. In certain embodiments, the promoter is the endogenous human SLC26A4 enhancer-promoter set forth in SEQ ID NO: 47, 48, or 50. In some embodiments, the enhancer-promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the promoter or enhancer-promoter sequence represented by SEQ ID NO: 45, 46, 47, 48, or 50. In certain embodiments, the promoter is the human SLC26A4 endogenous enhancer-promoter sequence contained within SEQ ID NO: 47, 48, or 50. Exemplary human SLC26A4 immediate early promoter (SEQ ID NO: 45) CTGCCTTCTGAGAGCGCTATAAAAGGCAGCGGAAGGGTAGTCCGCGGGGCATTCCGGGCGG Exemplary human SLC26A4 immediate early promoter (SEQ ID NO: 46) CTCTAGGCGGGCTCTGCTCTTCTTTAAGGAGTCCCACAGGGCCTGGCCCGCCCCTGACCT Exemplary Human SLC26A4 Enhancer-Promoter (SEQ ID NO: 47) TGGCCCGCCCCTGACCTCGCAACCCTTGAGATTAGTAACGGGATGAGTGAGGATCCGGGTGGCCCCTGCGTGGCAGCCAGTAAGAGTCTCAGCCTTCCCGGTTCGGGAAAGGGGAAGAATGCAGGAGGGGTAGGATTTCTTTCCTGATAGGATCGGTTGGGAAAGACCGCAGCCTGTGTGTGTCTTTCCCTTCGACCAAGGTGTCTGTTGCTCCGTAAATAAAACGTCCCACTGCCTTCTGAGAGCGCTATAAAGGCAGCGGAAGGGTAGTCCGCGGGGC Exemplary Human SLC26A4 Enhancer-Promoter (SEQ ID NO: 48) CGGAAGGTTGATGTACAGAGGTCTGTATTTTGGAGCCTCTTCTGTATTTACTTCAGAACACTAACAATCAGGCGAGAATGTTCTGGTTTATCAAACCCTTCCTTCTGCCTTTCATCTTAACCATGCATTAGTTTTAACAAAGTTCATCCCAACAGAAGACAAAACACTGATGAGGTAGGATAGCTCCAGCTCCTCCTCCCTCTCTTCTAGTCTTGATTTCCATGTAGTCCAGTTTATTCC TTCCCTGATTGTCCAGGAGAATGAGAAAAAGAAAAAACAGAGTCTAGTGGGTAAGAAAGGGCCACCTGGACGGCTTGATTTGGATTGTGAAATAAAACACACACACATGCACACGTAGAATAAGTGGCTAAAATCTGAGTAAATCGTGAACTCTCTGTATCCTCCACCCATTGAATACTCCTAAAAGACTTTCTAGAAATTCAAGGACTTATTAATATAGAAACCTGGCCATTGTTCCTCTTCTCCTCCCCATGTGGTATGAGAGCACCTGTGGCAGGCTCCCAGAGACCACGGACCTCTTCCTCTAGGCGGGCTCTGCTCTTCTTTAAGGAGTCCCACAGGGCCTGGCCCGCCCCTGACCTCGCAACCCTTGAGATTAGTAACGGGATGAGTGAGGATCCGGGTGGCCCCTGCGTGGCAGCCAGTAAGAGTCTCAGCCTTCCCGGTTCGGGAAAGGGGAAGAATGCAGGAGGGGTAGGATTTCTTTCCTGATAGGATCGGTTGGGAAAGACCGCAGCCTGTGTGTGTCTTTCCCTTCGACCAAGGTGTCTGTTGCTCCGTAAATAAAACGTCCCACTGCCTTCTGAGAGCGCTATAAAGGCAGCGGAAGGGTAGTCCGCGGGGCATTCCGGGCGGGGCGCGAGCAGAGACAGGTGAGTT

[0120] In certain embodiments, the promoter is the human LGR5 enhancer-promoter set forth in SEQ ID NO: 51. In some embodiments, the enhancer-promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer-promoter sequence set forth in SEQ ID NO: 51. In some embodiments, the promoter is the human LGR5 endogenous enhancer-promoter sequence contained within SEQ ID NO: 51. Exemplary Human LGR5 Enhancer-Promoter (SEQ ID NO: 51) AGGGCTATTTGTACCTCAACGAGGGCTTCTCTCCAAGAAAGCCCTGAATCCTTTTCCTCCTTTTTCCTGCAGATTCACTATAGGACACTTTTTGAAGCAAGAGCATGCATTTTCCCCCTGGCGCTCTGCAGCGGTTCTCAGAGCCCAGTGTCACTCACATAGGTGGGACTGCTCTCAGTTCAGAGAGCGCTGGGACACTTAAGATGAAAAGTCCCTGGAAGTTAGCAAACAGCCATCTGTCACTCTGGCATCGATTTACTAAAAGTGACTTCTAGGGTATTCTAAACCACTTTTAAAAAACAAATGAGTCACTTCGACTTCCTCACCCCGCAAGAGATAGGAAGGCAGCAGTGGAGTGCTCGCTCAGGAGCTGTATTTGTTTAGCGATTAGCCTAGAGCTTTGATTTTAGGGCAAAAGCGAGCCAGACAGTGCGGCAGACGTAAGGATCAAAAAGGCCACCTATCATTCGCCGGGGACGCCTGCCTCCTTACCCTGATAACGTAACTATTTCTCTGCATAGGATTTTAGTTTTTGTGTTTTTGTTTTGTTTTATTCTGTTTAATCACTTCAAGTATCTCATCCATTATTTGAAGCGGGCTCGGAGGAAACGTGCCGCATCCTCCAGTCCTTGTGCGTCTGTTTAGGTCTCTCCGAAGCAGGTCCCTCTCGACTCTTAGATCTGGGTCTCCAGCACGCATGAAGGGGTAAGGGTGGGGGGGTCCCCTATTCCGGCGCGCGGCGTTGAGCACTGAATCTTCCAGGCGGAGGCTCAGTGGGAGCGCCGAGAACTCGCCAGTACCGCGCGCTGCCTGCTGCCTGCTGCCTCCCAGCCCAGGACTTGGGAAAGGAGGGAGGGGACAAGTGGAGGGAAAGTGGGGCCGGGCGGGGGGTGCCTGGGAAGCCAGGCTGCGCTGACGTCACTGGGCGCGCAATTCGGGCTGGAGCGCTTTAAAAAACGAGCGTGCAAGCAGAGATGCTGCTCCACACCGCTCAGGCCGC。 GAGCAGCAGCAAGGCGCACCGCCACTGTCGCCGCTGCAGCCAGGGCTGCTCCGAAGGCCGGCGTGGCGGCAACCGGCACCTCTGTCCCCGCCGCGCTTCTCCTCGCCGCCACCGCCGTGGGGTCAGGAACGCGG CGTCTGGCGCTGCAGACGCCCGCTGAGTTGCAGAAGCCCACGGAGCGGCGCCCGGCGCGCCACGGCCCGTAGCAGTCCGGTGCTGCTCCGCCCGCGTCCGGCTCGTGGCCCCCTACTTCGGGCACCGACCGGT

[0121] In certain embodiments, the promoter is the human SYN1 enhancer-promoter set forth in SEQ ID NO: 52. In some embodiments, the enhancer-promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer-promoter sequence represented by SEQ ID NO: 52. In some embodiments, the promoter is the human SYN1 endogenous enhancer-promoter sequence contained within SEQ ID NO: 52. Exemplary Human SYN1 Enhancer-Promoter (SEQ ID NO: 52) TGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCCCACCGCCGC CTCAGCACTGAAGGCGCCGCTGACGTCACTCGCCGGTCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGCAGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGTCGAGAA

[0122] In certain embodiments, the promoter is the human GFAP enhancer-promoter set forth in SEQ ID NO: 53. In some embodiments, the enhancer-promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer-promoter sequence set forth in SEQ ID NO: 53. In some embodiments, the promoter is the human GFAP endogenous enhancer-promoter sequence contained within SEQ ID NO: 53. Exemplary Human GFAP Enhancer-Promoter (SEQ ID NO: 53) CCCACCTCCCTCTCTGTGCTGGGACTCACAGAGGGAGACCTCAGGAGGCAGTCTGTCCATCACATGTCCAAATGCAGAGCATACCCTGGGCTGGGCGCAGTGGCGCACAACTGTAATTCCAGCACTTTGGGAGGCTGATGTGGAAGGATCACTTGAGCCCAGAAGTTCTAGACCAGCCTGGGCAACATGGCAAGACCCTATCTCTACAAAAAAAGTTAAAAAATCAGCCACGTGTGGTGACACACACCTGTAGTCCCAGCTATTCAGGAGGCTGAGGTGAGGGGATCACTTAAGGCTGGGAGGTTGAGGCTGCAGTGAGTCGTGGTTGCGCCACTGCACTCCAGCCTGGGCAACAGTGAGACCCTGTCTCAAAAGACAAAAAAAAAAAAAAAAAAAAAAAGAACATATCCTGGTGTGGAGTAGGGGACGCTGCTCTGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGGAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTCGGGGT

[0123] Enhancer In some cases, the construct may include an enhancer sequence. The term "enhancer" refers to a nucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., a pendrin protein). Enhancer sequences (generally 50-1500 bp in length) generally increase the level of transcription by providing additional binding sites for transcription-related proteins (e.g., transcription factors). In some embodiments, enhancer sequences are found within intron sequences. Unlike promoter sequences, enhancer sequences are located further from the transcription start site (e.g., compared to a promoter). It can act at a greater distance. Non-limiting examples of enhancers include an RSV enhancer, a CMV enhancer, and / or an SV40 enhancer. In some embodiments, the construct comprises a CMV enhancer exemplified by SEQ ID NO: 19. In some embodiments, the enhancer sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer sequence represented by SEQ ID NO: 19. In some embodiments, the SV-40-derived enhancer is an SV-40 T intron sequence exemplified by SEQ ID NO: 20. In some embodiments, the enhancer sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer sequence represented by SEQ ID NO: 20. Exemplary CMV enhancer (SEQ ID NO: 19) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG Exemplary SV-40 Synthetic Intron (SEQ ID NO: 20)

[0124] Adjacent untranslated regions 5'UTR and 3'UTR In some embodiments, any of the constructs described herein may include a 5'UTR or a 3'UTR. A gene can include untranslated regions (UTRs), such as 5'UTRs. UTRs of a gene are transcribed but not translated. The 5'UTR starts at the transcription start site and continues up to, but not including, the start codon. The 3'UTR starts immediately after the stop codon and continues to the transcription termination signal. UTR regulatory and / or control mechanisms can be incorporated into any of the constructs, compositions, kits, or methods described herein to enhance or otherwise regulate expression of the pendrin protein.

[0125] Naturally occurring 5'UTRs contain sequences that play a role in translation initiation. In some embodiments, 5'UTRs can contain sequences, such as the Kozak sequence, that are commonly known to be involved in the process by which ribosomes initiate 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), followed by another "G" after the start codon. 5'UTRs are also known to form secondary structures involved in elongation factor binding.

[0126] 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 mRNA, including those from the following genes: albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, and factor VIII.

[0127] In some embodiments, a 5'UTR from an mRNA transcribed by cells in the cochlea can be included in any of the constructs, compositions, kits, and methods described herein. In some embodiments, the 5'UTR is derived from the endogenous SLC26A4 locus and can include all or a portion of the endogenous sequence exemplified by SEQ ID NO: 21. In some embodiments, the 5'UTR sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the 5'UTR sequence represented by SEQ ID NO: 21.

[0128] The 3'UTR is found immediately 3' to the stop codon of the gene of interest. In some embodiments, a 3'UTR from an mRNA transcribed by cells in the cochlea can be included in any of the constructs, compositions, kits, and methods described herein. In some embodiments, the 3'UTR is derived from the endogenous SLC26A4 locus and can include all or a portion of the endogenous sequence exemplified by SEQ ID NO:22. In some embodiments, the 3'UTR sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the 3'UTR sequence represented by SEQ ID NO:22.

[0129] 3'UTRs are known for having stretches of adenosine and uridine (in RNA form) or thymidine (in DNA form) embedded within them. These AU-rich features are found in genes with particularly high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al., Mal. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mal. 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 a 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 containing class II AREs. Class III AREs are less well defined. Their U-rich regions do not contain the AUUUA motif, and two well-studied examples of this class are c-Jun and myogenin mRNAs.

[0130] Most proteins that bind to AREs are known to destabilize messengers. While ELAV family members, most notably 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 message stabilization in vivo.

[0131] In some embodiments, the introduction, removal, or modification of a 3'UTR ARE can be used to modulate the stability of the mRNA encoding the pendrin protein, hi other embodiments, the ARE can be removed or mutated to increase intracellular stability and therefore increase translation and production of the pendrin protein.

[0132] In other embodiments, a non-ARE sequence may be incorporated into the 5' or 3' UTR. In some embodiments, an intron or a portion of an intron sequence may be incorporated into a flanking region of a polynucleotide in any of the constructs, compositions, kits, and methods provided herein. Incorporation of an intron sequence may increase protein production and mRNA levels. Exemplary 5'UTR sequence (SEQ ID NO:21) CTCAGCCTTCCCGGTTCGGGAAAGGGGAAGAATGCAGGAGGGGTAGGATTTCTTTCCTGATAGGATCGGTTGGGAAAGACCGCAGCCTGTGTGTGTCTTTCCCTTCGACCAAGGTGTCTGTTGCTCCGTAAATAAAACGTCCCACTGCCTTCTGAGAGCGCTATAAAGGCAGCGGAAGGGTAGTCCGCGGGGCATTCCGGGCGGGGCGCGAGCAGAGACAGGTC Exemplary 3'UTR sequence (SEQ ID NO: 22)

[0133] Internal ribosome entry site (IRES) In some embodiments, the construct encoding the pendrin protein can include an internal ribosome entry site (IRES), which forms a complex secondary structure that allows translation to begin anywhere with the mRNA immediately downstream from where the IRES is located (see, e.g., Pelletier and Sonenberg, Mal. Cell. Biol. 8(3):1103-1112, 1988).

[0134] There are several IRES sequences known to those of skill in the art, including, for example, those derived from foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), and poliovirus (PV). See, e.g., Alberts, Molecular Biology of the Cell, Garland Science, 2002, and Hellen et al., Genes Dev. 15(13):1593-612, 2001, each of which is incorporated herein by reference in its entirety.

[0135] In some embodiments, the IRES sequence incorporated into the construct encoding the pendrin protein or the C-terminal portion of the pendrin protein is the foot-and-mouth disease virus (FMDV) 2A sequence, a small peptide (approximately 18 amino acids in length) that has been shown to mediate polyprotein cleavage (Ryan, MD et al., EMBO 4:928-933, 1994; Mattion et al., J Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999, each of which is incorporated herein by reference in its entirety). The cleavage activity of 2A sequences has previously been demonstrated in artificial systems, including plasmids and gene therapy constructs (AAV and retrovirus) (Ryan et al., EMBO 4:928-933, 1994; Mattion et al., J Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999; de Felipe et al., Gene Therapy 6:198-208, 1999; de Felipe et al., Human Gene Therapy II:1921-1931, 2000, and Klump et al., Gene Therapy 8:811-817, 2001, each of which is incorporated herein by reference in its entirety.

[0136] An IRES can be utilized in an AAV construct. In some embodiments, the construct encoding the C-terminal portion of the pendrin protein can include an internal ribosome entry site (IRES). In some embodiments, the IRES can be part of a composition comprising two or more constructs. In some embodiments, the IRES is used to produce two or more polypeptides from a single gene transcript.

[0137] Splice site In some embodiments, any of the constructs provided herein can include a splice donor sequence and / or a splice acceptor sequence that is functional during RNA processing that occurs during transcription. In some embodiments, the splice site is involved in trans-splicing. Exemplary splice donor intron (SEQ ID NO: 41) GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGGCGTTTCT Exemplary splice acceptor intron (SEQ ID NO: 42) GATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG

[0138] Polyadenylation sequence In some embodiments, the constructs provided herein can include a polyadenylation (poly(A)) signal sequence. Most nascent eukaryotic mRNAs have a poly(A) tail added to their 3' ends in a complex process involving cleavage of the primary transcript and coupled polyadenylation driven by a poly(A) signal sequence (see, e.g., Proudfoot et al., Cell 108:501-512, 2002, incorporated herein by reference in its entirety). The poly(A) tail confers mRNA stability and translocation (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994 (incorporated herein by reference in its entirety). In some embodiments, a poly(A) signal sequence is provided relative to the coding sequence. and positioned at the 3' end.

[0139] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or its modified variants to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) adenine nucleotide sequence added to pre-mRNA by the action of the enzyme polyadenylate polymerase. In some embodiments, the poly(A) tail is added to transcripts containing specific sequences, such as poly(A) signals. The poly(A) tail and associated proteins help protect mRNA from exonuclease degradation. Polyadenylation also plays a role in transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also 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.

[0140] As used herein, a "poly(A) signal sequence" or "polyadenylation signal sequence" is a sequence that triggers endonucleolytic cleavage of an mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0141] 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, polyomavirus (Batt et al., Mal. Cell Biol. 15(9):4783-4790, 1995, each of which is incorporated herein by reference in its entirety), herpes simplex virus thymidine kinase gene (HSV Several poly(A) signal sequences can be used, including those derived from human growth hormone (hGH) (Szymanski et al., Mal. Therapy 15(7):1340-1347, 2007, which is incorporated herein by reference in its entirety), SV40 poly(A) sites, such as the SV40 late and early poly(A) sites (Schek et al., Mal. Cell Biol. 12(12):5386-5393, 1992, which is incorporated herein by reference in its entirety).

[0142] The poly(A) signal sequence can be AATAAA. The AATAAA sequence can be substituted 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, incorporated herein by reference in its entirety).

[0143] In some embodiments, the poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., Levitt el al, Genes Dev. 3(7):1019- 1025, 1989 (incorporated herein by reference in its entirety). In some embodiments, the poly(A) signal sequence is the soluble neuropilin-1 (sNRP) polyadenylation signal (AAATAAAATACGAAATG (SEQ ID NO: 23)) (see, e.g., WO 05 / 073384 (incorporated herein by reference in its entirety)). In some embodiments, the poly(A) signal sequence comprises or consists of the SV40 poly(A) site. In some embodiments, the poly(A) signal comprises or consists of SEQ ID NO: 25. In some embodiments, the poly(A) signal sequence comprises or consists of bGHpA. In some embodiments, the poly(A) signal comprises or consists of SEQ ID NO: 24. Further examples of poly(A) signal sequences are known in the art. In some embodiments, the poly(A) sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the poly(A) sequence set forth in SEQ ID NO:24 or 25. Exemplary bGH poly(A) signal sequence (SEQ ID NO:24) CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGTGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG Exemplary SV40 poly(A) signal sequence (SEQ ID NO:25) AACTTGTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

[0144] Additional arrays In some embodiments, constructs of the present disclosure may include a T2A element or sequence. In some embodiments, constructs of the present disclosure may include one or more cloning sites. In some such embodiments, the cloning sites may not be completely removed prior to preparation for administration to a subject. In some embodiments, the cloning sites may have a functional role, such as as a linker sequence or as part of a Kozak site. As will be understood by those skilled in the art, cloning sites may vary significantly in primary sequence while retaining desired function. In some embodiments, constructs may contain any combination of cloning sites, with exemplary cloning sites represented by SEQ ID NOS: 26-33. Exemplary Cloning Site A (SEQ ID NO: 26) TTGTCGACGCGGCCGCACGCGT Exemplary Cloning Site B (SEQ ID NO: 27) CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTCGCTAGCCACC Exemplary Cloning Site C (SEQ ID NO: 28) TAAGAGCTCGCTGATCAGCCTCGA Exemplary Cloning Site D (SEQ ID NO: 29) AAGCTTGAATTCAGCTGACGTGCCTCGGACCGTCCTAGG Exemplary Cloning Site E (SEQ ID NO: 30) GCGGCCGCACGCGT Exemplary cloning site F (SEQ ID NO: 31) CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC Exemplary Cloning Site G (SEQ ID NO: 32) TAAGAGCTCGCTGATCAGCCTCGA Exemplary cloning site H (SEQ ID NO: 33) AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT

[0145] Destabilizing domains In some embodiments, any of the constructs provided herein can optionally include a sequence encoding a destabilization domain ("destabilization sequence") for temporal control of protein expression. Non-limiting examples of destabilization sequences include sequences encoding FK506 sequences, dihydrofolate reductase (DHFR) sequences, or other exemplary destabilization sequences.

[0146] In the absence of a stabilizing ligand, the protein sequence operably linked to the destabilizing sequence is degraded by ubiquitination. In contrast, in the presence of a stabilizing ligand, proteolysis is inhibited, thereby allowing the protein sequence operably linked to the destabilizing sequence to be actively expressed. As a positive control for stabilization of protein expression, protein expression can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting (FACS) assays, immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0147] Further examples of destabilizing sequences are known in the art. In some embodiments, the destabilizing sequence is an FK506-binding protein and rapamycin-binding protein (FKBP12) sequence, and the stabilizing ligand is Shield-1 (Shld1) (Banaszynski et al. (2012) Cell 126(5):995-1004, incorporated herein by reference in its entirety). In some embodiments, the destabilizing sequence is a DHFR sequence, and the stabilizing ligand is trimethoprim (TMP) (Iwamoto et al. (2010) Chem Biol 17:981-988, incorporated herein by reference in its entirety).

[0148] In some embodiments, the destabilization sequence is an FKBP12 sequence, and the presence of an AAV construct carrying the FKBP12 gene in a target cell (e.g., a supporting cochlear outer hair cell) is detected by Western blotting. In some embodiments, the destabilization sequence can be used to verify the temporal-specific activity of any of the AAV constructs described herein. Exemplary DHFR destabilizing amino acid sequence (SEQ ID NO: 34) MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR Exemplary DHFR destabilizing nucleotide sequence (SEQ ID NO: 35) GGTACCATCAGTTCTGATTGCGGCGTTAGCGGTAGATTACGTTATCGGCATGGAAAACGCCATGCCGTGGAACCTGCCTGCCGATCTCGCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGGCCGCCATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGCAGTCAACC GAGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGATGAAGCCATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTGATTGGCGGCGGTCGCGTTATTGAACAGTTCTTGCCAAAAGCGCAAAAACTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGATTACGAGCCGGATGACT GGGAATCGGTATTCAGCGAATTCCACGATGCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGATAA Exemplary Destabilization Domain (SEQ ID NO: 36) ATCAGTCTGATTGCGGCGTTAGCGGTAGATTACGTTATCGGCATGGAAAACGCCATGCCGTGGAACCTGCCTGCCGATCTCGCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGCCGCCATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGCAGTCAACCGAGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGATGAA GCCATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTGATTGGCGGCGGTCGCGTTATTGAACAGTTCTTGCCAAAAGCGCAAAAACTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGATTACGAGCCGGATGACTGGGAATCGGTATTCAGCGAATTCCACGATGCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGA Exemplary FKBP12 destabilizing peptide amino acid sequence (SEQ ID NO: 37) MGVEKQVIRPGNGPKPAPGQTVTVHCTGFGKDGDLSQKFWSTKDEGQKPFSFQIGKGAVIKGWDEGVIGMQIGEVARLRCSSDYAYGAGGFPAWGIQPNSVLDFEIEVLSVQ

[0149] Reporter sequence or element In some embodiments, the constructs provided herein can optionally include a sequence encoding a reporter polypeptide and / or protein ("reporter sequence"). 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. Further examples of reporter sequences are known in the art. Reporter sequences, when associated with regulatory elements that drive their expression, can provide a signal detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting (FACS) assays, immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0150] In some embodiments, the reporter sequence is a LacZ gene, and the presence of a 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 a construct carrying the fluorescent protein or luciferase in mammalian cells (e.g., cochlear hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or light production in a luminometer (e.g., a spectrophotometer or IVIS imaging instrument). In some embodiments, reporter sequences can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulation and / or regulatory activity of any of the constructs described herein.

[0151] In some embodiments, the reporter sequence is a FLAG tag (e.g., a 3×FLAG tag), and the presence of a construct bearing a FLAG tag in a mammalian cell (e.g., an inner ear cell, e.g., a cochlear hair cell or supporting cell) is detected by a protein binding or detection assay (e.g., Western blot, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry). An exemplary 3xFLAG tag sequence is provided as SEQ ID NO:38. Exemplary 3xFLAG tag sequence (SEQ ID NO: 38) GGATCCCGGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG

[0152] AAV capsid The present disclosure provides one or more polynucleotide constructs packaged in an AAV capsid. In some embodiments, the AAV capsid is from or derived from an AAV capsid of an AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rh10, rh39, rh43, or Anc80 serotype, or one or more hybrids thereof. In some embodiments, the AAV capsid is derived from an AAV ancestral serotype. In some embodiments, the AAV capsid is an ancestral (Anc) AAV capsid. Anc capsids are generated from assembly sequences constructed using evolutionary probability and evolutionary modeling to determine likely ancestral sequences. In this manner, the Anc capsid / construct sequence is not known to have existed in nature. For example, in some embodiments, the AAV capsid is an Anc80 capsid (e.g., an Anc80L65 capsid). In some embodiments, the AAV capsid is made using a template nucleotide coding sequence comprising SEQ ID NO: 8. In some embodiments, the capsid comprises a polypeptide represented by SEQ ID NO: 9. In some embodiments, the capsid comprises a polypeptide having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the polypeptide represented by SEQ ID NO: 9.

[0153] As provided herein, any combination of AAV capsids and AAV constructs (e.g., containing AAV ITRs) can be used in the recombinant AAV (rAAV) particles of the present disclosure. For example, a wild-type or variant AAV2 ITR and Anc80 capsid, a wild-type or variant AAV2 ITR and AAV6 capsid, etc. In some embodiments of the present disclosure, the AAV particle is composed entirely of AAV2 components (e.g., the capsid and ITRs are of the AAV2 serotype). In some embodiments, the AAV particle is an AAV2 / 6, AAV2 / 8, or AAV2 / 9 particle (e.g., an AAV6, AAV8, or AAV9 capsid with an AAV construct having AAV2 ITRs). In some embodiments of the present disclosure, the AAV particle is an AAV2 / Anc80 particle comprising an Anc80 capsid (e.g., comprising the polypeptide of SEQ ID NO: 9) that encapsidates an AAV construct with AAV 2 ITRs (e.g., SEQ ID NOs: 10 and 11) adjacent to a portion of a coding sequence, e.g., the SLC26A4 gene or a characteristic portion thereof (e.g., SEQ ID NOs: 1, 2, 3, 4, or 5). 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 (incorporated herein by reference in its entirety). In some embodiments, the capsid sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the capsid nucleotide or amino acid sequence set forth in SEQ ID NO: 8 or 9, respectively. Exemplary AAV Anc80 Capsid DNA Sequence (SEQ ID NO:8) ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGA Exemplary AAV Anc80 Capsid Amino Acid Sequence (SEQ ID NO:9) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQD *

[0154] composition Among other things, the present disclosure provides compositions. In some embodiments, the compositions comprise a construct described herein. In some embodiments, the compositions comprise one or more constructs described herein. In some embodiments, the compositions comprise a plurality of constructs described herein. In some embodiments, when two or more constructs are included in the composition, the constructs are different from each other.

[0155] In some embodiments, the composition comprises an AAV particle described herein. In some embodiments, the composition comprises one or more AAV particles described herein. In some embodiments, the composition comprises a plurality of AAV particles. In some embodiments, when two or more AAV particles are included in the composition, the AAV particles are each different.

[0156] In some embodiments, the composition comprises a pendrin protein. In some embodiments, the composition comprises a cell.

[0157] In some embodiments, the composition is or comprises a pharmaceutical composition.

[0158] Single AAV construct composition In some embodiments, the present disclosure provides compositions or systems comprising AAV particles comprised of a single construct. In some such embodiments, the single construct may deliver a polynucleotide encoding a functional (e.g., wild-type or other functional, e.g., codon-optimized) copy of the SLC26A4 gene. In some embodiments, the construct is or comprises an rAAV construct. In some embodiments described herein, the single rAAV construct is capable of expressing full-length SLC26A4 messenger RNA or its characteristic proteins in target cells (e.g., inner ear cells). In some embodiments, the single construct (e.g., any of the constructs described herein) comprises a sequence encoding a functional pendrin protein (e.g., any construct that produces a functional pendrin protein). In some embodiments, the single construct (e.g., any of the constructs described herein) comprises a sequence encoding a functional pendrin protein (e.g., any construct that produces a functional pendrin protein) and Optionally, sequences encoding additional polypeptide sequences (eg, regulatory and / or reporter sequences) can be included.

[0159] In some embodiments, a single construct composition or system can include any or all of the exemplary construct components described herein. In some embodiments, an exemplary single construct is represented by SEQ ID NO: 39. In some embodiments, an exemplary single construct is represented by SEQ ID NO: 40. In some embodiments, an exemplary single construct is at least 85%, 90%, 95%, 98%, or 99% identical to the sequence represented by SEQ ID NO: 39 or 40. One of skill in the art will recognize that constructs can be subject to additional modifications, including codon optimization, introduction of novel but functionally equivalent sequences (e.g., silent mutations), addition of reporter sequences, and / or other routine modifications.

[0160] In some embodiments, an exemplary construct comprises a 5' ITR exemplified by SEQ ID NO: 10, optionally a cloning site exemplified by SEQ ID NO: 26, a CMV enhancer exemplified by SEQ ID NO: 19, a CBA promoter exemplified by SEQ ID NO: 14, a chimeric intron exemplified by SEQ ID NO: 20, optionally a cloning site exemplified by SEQ ID NO: 27, a SLC26A4 coding region exemplified by SEQ ID NO: 1, optionally a cloning site exemplified by SEQ ID NO: 28, a poly(A) site exemplified by SEQ ID NO: 24, optionally a cloning site exemplified by SEQ ID NO: 29, and a 3' ITR exemplified by SEQ ID NO: 12.

[0161] In some embodiments, an exemplary construct comprises a 5' ITR exemplified by SEQ ID NO:11, optionally a cloning site exemplified by SEQ ID NO:30, a CMV enhancer exemplified by SEQ ID NO:19, a CBA promoter exemplified by SEQ ID NO:15, a chimeric intron exemplified by SEQ ID NO:20, optionally a cloning site exemplified by SEQ ID NO:31, a SLC26A4 coding region exemplified by SEQ ID NO:1, optionally a reporter sequence exemplified by SEQ ID NO:38, optionally a cloning site exemplified by SEQ ID NO:32, a poly(A) site exemplified by SEQ ID NO:24, optionally a cloning site exemplified by SEQ ID NO:34, and a 3' ITR exemplified by SEQ ID NO:13. Exemplary single construct sequence (SEQ ID NO: 39) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTGTCGACGCGGCCGCACGCGTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCG Exemplary single construct sequence with FLAG reporter (SEQ ID NO: 40) CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGG TGAAATCTCCAAGAGGGTCAAGGTTCCATTTTAGAAACGATCACTCTCATTCAGGATTGTAAAGATACCCTTGAATTAATAGAAACAGAGCTGACGGAAGAAGAACTTGATGTCCAGGATGAGGCTATGCGTACACTTGCATCCGGATCCCGGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATCAGATGACAAGTAAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTCCCCTCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCA CTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGCAAGGGGGAGGATTGGGAAGACATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAAGCTTGAATTCAGCTGACGTGCCTCGGACCGCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGGCGCAG

[0162] Multiple AAV constructs The present disclosure recognizes that some coding sequences encoding proteins (e.g., pendrin proteins) may be delivered by dividing the coding sequence into multiple portions, each contained in a different construct. In some embodiments, the present invention provides compositions or systems comprising at least two different constructs (e.g., two, three, four, five, or six). In some embodiments, each of the at least two different constructs comprises a coding sequence that encodes a different portion of the coding region (e.g., encoding a target protein, e.g., an inner ear target protein, e.g., a pendrin protein), and each of the coding portions is at least 10 amino acids (e.g., at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 110 amino acids, at least about 120 amino acids, at least about 130 amino acids, at least about 140 amino acids, at least about 150 amino acids, at least about 160 amino acids, at least about 170 amino acids, at least about 180 amino acids, at least about 190 amino acids, at least about 200 amino acids, at least about 210 amino acids, at least about 220 amino acids, at least about 230 amino acids, at least about 240 amino acids, at least about 250 amino acids, at least about 260 amino acids, at least about 270 amino acids, at least about 280 amino acids, at least about 290 amino acids, at least about 300 amino acids, at least about 310 amino acids, at least about 320 amino acids, at least about 330 amino acids, at least about 340 amino acids, at least about 350 amino acids, at least about 360 amino acids, at least about 370 amino acids, at least about 380 amino acids, at least about 390 amino acids, at least about 400 amino acids, at least about 410 amino acids, at least about 420 amino acids, at least about 430 amino acids, at least about 440 amino acids, at least about 450 amino acids, at least about 460 amino 00 amino acids, at least about 210 amino acids, at least about 220 amino acids, at least about 230 amino acids, at least about 240 amino acids, at least about 250 amino acids, at least about 260 amino acids, at least about 270 amino acids, at least about 280 amino acids, at least about 290 amino acids, at least about 300 amino acids, at least about 310 amino acids, at least about 320 amino acids, at least about 330 amino acids, at least about 340 amino acids, at least about 350 amino acids, at least about 360 amino acids, at least about 370 amino acids, at least about 380 amino acids, at least about 390 amino acids, at least about 400 amino acids, at least about 410 amino acids, at least about 420 amino acids, at least about 430 amino acids, at least about 440 amino acids, at least about 450 amino acids,at least about 460 amino acids, at least about 470 amino acids, at least about 480 amino acids, at least about 490 amino acids, at least about 500 amino acids, at least about 510 amino acids, at least about 520 amino acids, at least about 530 amino acids, at least about 540 amino acids, at least about 550 amino acids, at least about 560 amino acids, at least about 570 amino acids, at least about 580 amino acids, at least about 590 amino acids, at least about 600 amino acids, at least about 610 amino acids, at least about 620 amino acids, at least about 630 amino acids, At least about 640 amino acids, at least about 650 amino acids, at least about 660 amino acids, at least about 670 amino acids, at least about 680 amino acids, at least about 690 amino acids, at least about 700 amino acids, at least about 710 amino acids, at least about 720 amino acids, at least about 730 amino acids, at least about 740 amino acids, at least about 750 amino acids, at least about 760 amino acids, at least about 770 amino acids, at least about 780 amino acids, at least about 790 amino acids, at least about 800 amino acids, at least about 810 amino acids, or at least about 820 amino acids), wherein the amino acid sequence of each of the encoded portions may optionally overlap with a different amino acid sequence of the encoded portion, wherein a single construct of the at least two different constructs does not encode an active target protein, and wherein the at least two different constructs, when introduced into a subject cell (e.g., an animal cell, e.g., a primate cell, e.g., a human cell), undergo homologous recombination with each other, wherein the recombinant nucleic acid encodes an active target protein (e.g., a gene product encoded by the SLC26A4 gene, or a characteristic portion thereof). In some embodiments, one of the nucleic acid constructs can include a coding sequence that encodes a portion of a target protein (e.g., an inner ear target protein, e.g., a pendrin protein), wherein the coding portion is at most about 820 amino acids (e.g., at most about 10 amino acids, at most about 20 amino acids, at most about 30 amino acids, at most about 60 amino acids, at most about 70 amino acids, at most about 80 amino acids, at most about 90 amino acids, at most about 100 amino acids, At most about 110 amino acids, at most about 120 amino acids, at most about 130 amino acids, at most about 140 amino acids, at most about 150 amino acids, at most about 160 amino acids, at most about 170 amino acids, at most about 180 amino acids, at most about 190 amino acids, at most about 200 amino acids, at most about 210 amino acids, at most about 220 amino acids, at most about 230 amino acids, at most about 240 amino acids, at most about 250 amino acids,At most about 260 amino acids, at most about 270 amino acids, at most about 280 amino acids, at most about 290 amino acids, at most about 300 amino acids, at most about 310 amino acids, at most about 320 amino acids, at most about 330 amino acids, at most about 340 amino acids, at most about 350 amino acids, at most about 360 amino acids, at most about 370 amino acids, at most about 380 amino acids, at most about 390 amino acids, at most about 400 amino acids, at most about 410 amino acids, at most about 420 amino acids, at most about 430 amino acids, at most about 440 amino acids, at most about 450 amino acids, at most about 460 amino acids, at most about 470 amino acids, at most about 480 amino acids, at most about 490 amino acids, at most about 500 amino acids, at most about 510 amino acids, at most about 520 amino acids, at most about 530 amino acids, at most about 540 amino acids amino acids, at most about 550 amino acids, at most about 560 amino acids, at most about 570 amino acids, at most about 580 amino acids, at most about 590 amino acids, at most about 600 amino acids, at most about 610 amino acids, at most about 620 amino acids, at most about 630 amino acids, at most about 640 amino acids, at most about 650 amino acids, at most about 660 amino acids, at most about 670 amino acids, at most about 680 amino acids, at most at most about 690 amino acids, at most about 700 amino acids, at most about 710 amino acids, at most about 720 amino acids, at most about 730 amino acids, at most about 740 amino acids, at most about 750 amino acids, at most about 760 amino acids, at most about 770 amino acids, at most about 780 amino acids, at most about 790 amino acids, at most about 800 amino acids, at most about 810 amino acids, or at most about 820 amino acids).

[0163] In some embodiments, at least one of the constructs comprises two adjacent exons of a target genomic DNA (e.g., an inner ear target genomic DNA, e.g., an SLC26A4 genomic DNA fragment). It contains a nucleotide sequence spanning the 2 exons (NA) and lacks any naturally occurring intron sequence between the two adjacent exons.

[0164] In some embodiments, the amino acid sequence of each coding portion of the constructs does not overlap, even partially, with the different amino acid sequences of the coding portions. In some embodiments, the amino acid sequence of the coding portion of the constructs partially overlaps with the amino acid sequence of the coding portion of a different construct. In some embodiments, the amino acid sequence of the coding portion of each construct partially overlaps with the amino acid sequence of the coding portion of at least one different construct.In some embodiments, the overlapping amino acid sequences are between about 10 amino acid residues and about 820 amino acids, or any subrange of this range (e.g., about 10 amino acids, about 20 amino acids, about 30 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, about 110 amino acids, about 120 amino acids, about 130 amino acids, about 140 amino acids, about 150 amino acids, about 160 amino acids, about 170 amino acids, about 180 amino acids, or any subrange of this range). about 190 amino acids, about 200 amino acids, about 210 amino acids, about 220 amino acids, about 230 amino acids, about 240 amino acids, about 250 amino acids, about 260 amino acids, about 270 amino acids, about 280 amino acids, about 290 amino acids, about 300 amino acids, about 310 amino acids, about 320 amino acids, about 330 amino acids, about 340 amino acids, about 350 amino acids, about 360 amino acids, about 370 amino acids, about 380 amino acids, about 390 amino acids, about 400 amino acids, about 410 amino acids, about 420 amino acids, about 430 amino acids, about 440 amino acids, about 450 amino acids, about 460 amino acids, about 470 amino acids, about 480 amino acids, about 490 amino acids, about 500 amino acids, about 510 amino acids, about 520 amino acids, about 530 amino acids, about 540 amino acids, about 550 amino acids, about 560 amino acids, about 570 amino acids, about 580 amino acids, about 590 amino acids, about 600 amino acids, about 610 amino acids, about 620 amino acids, about 630 amino acids, about 640 amino acids, about 650 amino acids, about 660 amino acids, about 670 amino acids, about 680 amino acids, about 690 amino acids, about 700 amino acids, about 710 amino acids, about 720 amino acids, about 730 amino acids, about 740 amino acids, about 750 amino acids, about 760 amino acids, about 770 amino acids, about 780 amino acids, about 790 amino acids, about 800 amino acids, about 810 amino acids, or about 820 amino acids in length).

[0165] In some examples, a desired gene product (e.g., a therapeutic gene product) is encoded by at least two different constructs. In some embodiments, each of the at least two different constructs comprises a different segment of an intron, where the intron comprises a nucleotide sequence of an intron present in the target genomic DNA (e.g., inner ear cell target genomic DNA (e.g., SLC26A4 genomic DNA) (e.g., any of the exemplary introns in SEQ ID NO: 3 described herein). In some embodiments, the different intron segments overlap. In some embodiments, the different intron segments are at most about 12,000 nucleotides long (e.g., at most about 100 nucleotides, at most about 200 nucleotides, at most about 300 nucleotides, at most about 600 nucleotides, at most about 700 nucleotides, at most about 800 nucleotides, at most about 900 nucleotides, at most about 1000 nucleotides, at most about 12000 nucleotides, at most about 12000 nucleotides). about 700 nucleotides, at most about 800 nucleotides, at most about 900 nucleotides, at most about 1,000 nucleotides, at most about 1,100 nucleotides, at most about 1,200 nucleotides, at most about 1,300 nucleotides, at most about 1,400 nucleotides, at most about 1,500 nucleotides, at most about 1,600 nucleotides, at most about 1,700 nucleotides, at most about 1,800 nucleotides, at most about 1,900 nucleotides, at most about 2,000 nucleotides, at most about 2,100 nucleotides, at most about 2,200 nucleotides, at most about 2,300 nucleotides, at most about 2,400 nucleotides, at most about 2,500 nucleotides nucleotides, at most about 2,600 nucleotides, at most about 2,700 nucleotides, at most about 2,800 nucleotides, at most about 2,900 nucleotides, at most about 3,000 nucleotides, at most about 3,100 nucleotides, at most about 3,200 nucleotides, at most about 3,300 nucleotides, at most about 3,400 nucleotides, at most about 3,500 nucleotides, at most about 3,600 nucleotides, at most about 3,700 nucleotides, at most about 3,800 nucleotides, at most about 3,900 nucleotides, at most about 4,000 nucleotides 00 nucleotides, at most about 4,100 nucleotides, at most about 4,200 nucleotides, at most about 4,300 nucleotides, at most about 4,400 nucleotides, at most about 4,500 nucleotides, at most about 4,600 nucleotides, at most about 4,700 nucleotides, at most about 4,800 nucleotides, at most about 4,900 nucleotides, at most about 5,000 nucleotides, at most about 5,100 nucleotides, at most about 5,200 nucleotides, at most about 5,300 nucleotides, at most about 5,400 nucleotides, at most about 5,500 nucleotides, at most about 5,600 nucleotides, at most about 5,700 nucleotides, at most about 5,800 nucleotides, at most about 5,900 nucleotides, at most about 6,000 nucleotides, at most about 6,100 nucleotides, at most about 6,200 nucleotides, at most about 6,300 nucleotides, at most about 6,400 nucleotides, at most about 6,500 nucleotides, at most about 6,600 nucleotides, at most about 6,700 nucleotides, at most about 6,800 nucleotides, at most about 6,900 nucleotides nucleotides, at most about 7,000 nucleotides, at most about 7,100 nucleotides, at most about 7,200 nucleotides, at most about 7,300 nucleotides, at most about 7,400 nucleotides, at most about 7,500 nucleotides, at most about 7,600 nucleotides, at most about 7,700 nucleotides, at most about 7,800 nucleotides, at most about 7,900 nucleotides, at most about 8,000 nucleotides, at most about 8,100 nucleotides, at most about 8,200 nucleotides, at most about 8,300 nucleotides, at most about 8,400 nucleotides, at most about 8,500 nucleotides, at most about 8,600 nucleotides, at most about 8,700 nucleotides, at most about 8,800 nucleotides, at most about 8,900 nucleotides, at most about 9,000 nucleotides, at most about 9,100 nucleotides, at most about 9,200 nucleotides, at most about 9,300 nucleotides, at most about 9,400 nucleotides, at most about 9,500 nucleotides, at most about 9,600 nucleotides, at most about 9,700 nucleotides, at most about 9,800 nucleotides, at most about 9,900 nucleotides, at most about 10,000 nucleotides, at most about 10,100 nucleotides, at most about 10,200 nucleotides, at most about 10,300 nucleotides 0 nucleotides, at most about 10,400 nucleotides, at most about 10,500 nucleotides, at most about 10,600 nucleotides, at most about 10,700 nucleotides, at most about 10,800 nucleotides, at most about 10,900 nucleotides, at most about 11,000 nucleotides, at most about 11,100 nucleotides, at most about 11,200 nucleotides, at most about 11,300 nucleotides, at most about 11,400 nucleotides, at most about 11,500 nucleotides, at most about 11,600 nucleotides, at most about 11,700 nucleotides, at most about 11,800 nucleotides, at most about 11,900 nucleotides, or at most about 12,000 nucleotides). In some embodiments, the overlapping nucleotide sequence in any two of the different constructs can include part or all of one or more exons of a target gene (e.g., an inner ear cell target gene (e.g., the SLC26A4 gene) (e.g., any one or more of the exemplary exons in SEQ ID NO: 3 described herein).

[0166] In some embodiments, the composition or system is or comprises two, three, four, or five different constructs. In a composition where the number of different constructs in the composition is two, the first of the two different constructs is a protein (e.g., a pendrin protein). The 5' portion may include a coding sequence encoding the N-terminal portion of an inner ear cell protein (e.g., the N-terminal portion of an inner ear cell protein, e.g., the N-terminal portion of a pendrin protein), which may be referred to as the lead portion, first construct, or 5' portion (e.g., the N-terminal portion of an inner ear cell protein, e.g., the N-terminal portion of a pendrin protein). In some examples, the N-terminal portion of the target gene is at least about 10 amino acids (e.g., at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 110 amino acids, at least about 120 amino acids, at least about 130 amino acids, at least about 140 amino acids, at least about 150 amino acids, at least about 160 amino acids, at least about 170 amino acids, at least about 180 amino acids, at least about 190 amino acids, at least about 200 amino acids, at least about 210 amino acids, at least about 220 amino acids, at least about 230 amino acids, at least about 240 amino acids, at least about 250 amino acids, at least about 260 amino acids, at least about 270 amino acids, at least about 280 amino acids). amino acids, at least about 290 amino acids, at least about 300 amino acids, at least about 310 amino acids, at least about 320 amino acids, at least about 330 amino acids, at least about 340 amino acids, at least about 350 amino acids, at least about 360 amino acids, at least about 370 amino acids, at least about 380 amino acids, at least about 390 amino acids, at least about 400 amino acids, at least about 410 amino acids, at least about 420 amino acids, at least about 430 amino acids, at least about 440 amino acids, at least about 450 amino acids, at least about 460 amino acids, at least about 470 amino acids, at least about 480 amino acids, at least about 490 amino acids, at least about 500 amino acids, at least about 510 amino acids, at least about 520 amino acids, at least about 530 amino acids, at least about 540 amino acids, at least about 550 amino acids, at least about 560 amino acids,The polypeptide has a length of at least about 570 amino acids, at least about 580 amino acids, at least about 590 amino acids, at least about 600 amino acids, at least about 610 amino acids, at least about 620 amino acids, at least about 630 amino acids, at least about 640 amino acids, at least about 650 amino acids, at least about 660 amino acids, at least about 670 amino acids, at least about 680 amino acids, at least about 690 amino acids, at least about 700 amino acids, at least about 710 amino acids, at least about 720 amino acids, at least about 730 amino acids, at least about 740 amino acids, at least about 750 amino acids, at least about 760 amino acids, at least about 770 amino acids, at least about 780 amino acids, at least about 790 amino acids, at least about 800 amino acids, at least about 810 amino acids, or at least about 820 amino acids. In some examples, the first construct includes one or both of a promoter (e.g., any of the promoters described herein or known in the art) and a Kozak sequence (e.g., any of the exemplary Kozak sequences described herein or known in the art). In some examples, the first construct includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter. In some instances, the second of the two different constructs includes a coding sequence that encodes a C-terminal portion of a protein, which may be referred to as a terminal portion, a second construct, or a 3' portion (e.g., a C-terminal portion of an inner ear cell-targeted protein, e.g., a C-terminal portion of a pendrin protein). In some examples, the C-terminal portion of the target protein is at least about 10 amino acids (e.g., at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 110 amino acids, at least about 120 amino acids, at least about 130 amino acids, at least about 140 amino acids,at least, About 150 amino acids, at least about 160 amino acids, at least about 170 amino acids, at least about 180 amino acids, at least about 190 amino acids, at least about 200 amino acids, at least about 210 amino acids, at least about 220 amino acids, at least about 230 amino acids, at least about 240 amino acids, at least about 250 amino acids, at least about 260 amino acids, at least about 270 amino acids, at least about 280 amino acids, at least about 290 amino acids, at least about 300 amino acids , at least about 310 amino acids, at least about 320 amino acids, at least about 330 amino acids, at least about 340 amino acids, at least about 350 amino acids, at least about 360 amino acids, at least about 370 amino acids, at least about 380 amino acids, at least about 390 amino acids, at least about 400 amino acids, at least about 410 amino acids, at least about 420 amino acids, at least about 430 amino acids, at least about 440 amino acids, at least about 450 amino acids, at least about 460 amino acids, at least about 470 amino acids, at least about 480 amino acids, at least about 490 amino acids, at least about 500 amino acids, at least about 510 amino acids, at least about 520 amino acids, at least about 530 amino acids, at least about 540 amino acids, at least about 550 amino acids, at least about 560 amino acids, at least about 570 amino acids, at least about 580 amino acids, at least about 590 amino acids, at least about 600 amino acids, at least about 610 amino acids, at least about 620 amino acids, at least about 630 amino acids, at least about 640 amino acids, at least about 650 amino acids, at least about 660 amino acids, at least about 670 amino acids, at least about 680 amino acids, at least about 690 amino acids, at least about 700 amino acids, at least about 710 amino acids, at least about 720 amino acids, at least about 730 amino acids, at least about 740 amino acids, at least about 750 amino acids, at least about 760 amino acids, at least about 770 amino acids,The second construct has a length of at least about 780 amino acids, at least about 790 amino acids, at least about 800 amino acids, at least about 810 amino acids, or at least about 820 amino acids. In some embodiments, the second construct further comprises a poly(A) sequence.

[0167] In some examples where the number of different constructs in the composition is two, the N-terminal portion encoded by one of the two constructs is encoded by an amino acid sequence from amino acid position 1 to about amino acid position 820 of an inner ear cell target protein (e.g., SEQ ID NO: 6 or 7), or any subrange within this range (e.g., amino acid 1 to at least about amino acid 10, amino acid 1 to at least about amino acid 20, amino acid 1 to at least about amino acid 30, amino acid 1 to at least about amino acid 60, amino acid 1 to at least about amino acid 70, amino acid 1 to at least about amino acid 80, amino acid 1 to at least about amino acid 90, amino acid 1 to at least about amino acid 100, amino acid 1 to at least about amino acid 110, amino acid 1 to at least about amino acid 120, amino acid 1 to at least about amino acid 130, amino acid 1 to at least about amino acid 140, amino acid 1 to at least about amino acid 150, amino acid 1 to at least about amino acid 160, amino acid 1 to at least about amino acid 170, amino acid 1 to at least about amino acid 180, amino acid 1 to at least about amino acid 190, amino acid 1 to at least about amino acid 200, amino acid 1 to at least about amino acid 210, amino acid 1 to at least about amino acid 220, amino acid 1 to at least about amino acid 230, amino acid 1 to at least about amino acid 240, amino acid 1 to at least about amino acid 250, amino acid 1 to at least about amino acid 260, amino acid 1 to at least about amino acid 270, amino acid 1 to at least about amino acid 280, amino acid 1 to at least about amino acid 290, amino acid 1 to at least about amino acid 300, amino acid 1 to at least about amino acid amino acids 1 to at least about 170 amino acids, amino acids 1 to at least about 180 amino acids, amino acids 1 to at least about 190 amino acids, amino acids 1 to at least about 200 amino acids, amino acids 1 to at least about 210 amino acids, amino acids 1 to at least about 220 amino acids, amino acids 1 to at least about 230 amino acids, amino acids 1 to at least about 240 amino acids, amino acids 1 to at least about 250 amino acids, amino acids 1 to at least about 260 amino acids, amino acids 1 to at least about 270 amino acids, amino acids 1 to at least about 280 amino acids, amino acids 1 to at least about 290 amino acids, amino acids 1 to at least about 300 amino acids, amino acids 1 to at least about 310 amino acids, amino acids 1 to at least about 320 amino acids, amino acids 1 to at least about 330 amino acids, amino acids 1 to at least about 340 amino acids, amino acids 1 to at least about 350 amino acids, amino acids 1 to at least about 360 amino acids, amino acids 1 to at least about 370 amino acids, amino acids 1 to at least about 380 amino acids, amino acids 1 to at least about 390 amino acids, amino acids 1 to at least about 400 amino acids, amino acids 1 to at least about 410 amino acids, amino acids 1 to at least about 420 amino acids, amino acids 1 to at least about 430 amino acids, amino acids 1 to at least about 440 amino acids, amino acids 1 to at least about 450 amino acids, amino acids 1 to at least about 460 amino acids, amino acids 1 to at least about 470 amino acids, amino acids 1 to at least about 480 amino acids, amino acids 1 to at least about 490 amino acids, amino acids 1 to at least about 500 amino acids, amino acids 1 to at least about 510 amino acids, amino acids 1 to at least about 520 amino acids, amino acids 1 to at least about 530 amino acids, amino acids 1 to at least about 540 amino acids, amino acids 1 to at least about 550 amino acids, amino acids 1 to at least about 560 amino acids, amino acids 1 to at least about 570 amino acids, amino acids 1 to at least about 580 amino acids, amino acids 1 to at least about 590 amino acids, amino acids 1 to at least about amino acids 700, 1 to at least about amino acids 710, 1 to at least about amino acids 720, 1 to at least about amino acids 730, 1 to at least about amino acids 740, 1 to at least about amino acids 750, 1 to at least about amino acids 760, 1 to at least about amino acids 770, 1 to at least about amino acids 780, 1 to at least about amino acids 790, 1 to at least about amino acids 800, 1 to at least about amino acids 810, or 1 to at least about amino acids 820. In some instances where the number of different constructs in the composition is two, the N-terminal portion of the precursor inner ear cell targeting protein is a portion of the inner ear cell targeting protein (e.g.,1 to about 10, 1 to about 20, 1 to about 30, 1 to about 60, 1 to about 70, 1 to about 80, 1 to about 90, 1 to about 100, 1 to about 110, 1 to about 120, 1 to about 130, 1 to about 140, 1 to about 150, 1 to about 160, 1 to about 170, 1 to about 180, 1 to about 190, 1 to about 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, amino acids 130, amino acids 1 to a maximum of about amino acids 140, amino acids 1 to a maximum of about amino acids 150, amino acids 1 to a maximum of about amino acids 160, amino acids 1 to a maximum of about amino acids 170, amino acids 1 to a maximum of about amino acids 180, amino acids 1 to a maximum of about amino acids 190, amino acids 1 to a maximum of about amino acids 200, amino acids 1 to a maximum of about amino acids 210, amino acids 1 to a maximum of about amino acids 220, amino acids 1 to a maximum of about amino acids 230, amino acids 1 to a maximum of about amino acids 240, amino acids 1 to a maximum of about amino acids 250, amino acids 1 to a maximum of about amino acids 260 amino acids, 1 amino acid to a maximum of about 270 amino acids, 1 amino acid to a maximum of about 280 amino acids, 1 amino acid to a maximum of about 290 amino acids, 1 amino acid to a maximum of about 300 amino acids, 1 amino acid to a maximum of about 310 amino acids, 1 amino acid to a maximum of about 320 amino acids, 1 amino acid to a maximum of about 330 amino acids, 1 amino acid to a maximum of about 340 amino acids, 1 amino acid to a maximum of about 350 amino acids, 1 amino acid to a maximum of about 360 amino acids, 1 amino acid to a maximum of about 370 amino acids, 1 amino acid to a maximum of about 380 amino acids, 1 amino acid to a maximum of about 3 90, from 1 amino acid to a maximum of about 400 amino acids, from 1 amino acid to a maximum of about 410 amino acids, from 1 amino acid to a maximum of about 420 amino acids, from 1 amino acid to a maximum of about 430 amino acids, from 1 amino acid to a maximum of about 440 amino acids, from 1 amino acid to a maximum of about 450 amino acids, from 1 amino acid to a maximum of about 460 amino acids, from 1 amino acid to a maximum of about 470 amino acids, from 1 amino acid to a maximum of about 480 amino acids, from 1 amino acid to a maximum of about 490 amino acids, from 1 amino acid to a maximum of about 500 amino acids, from 1 amino acid to a maximum of about 510 amino acids, from 1 amino acid to a maximum of about 520 amino acids,Amino acids 1, At most about 530 amino acids, 1 amino acid to at most about 540 amino acids, 1 amino acid to at most about 550 amino acids, 1 amino acid to at most about 560 amino acids, 1 amino acid to at most about 570 amino acids, 1 amino acid to at most about 580 amino acids, 1 amino acid to at most about 590 amino acids, 1 amino acid to at most about 600 amino acids, 1 amino acid to at most about 610 amino acids, 1 amino acid to at most about 620 amino acids, 1 amino acid to at most about 630 amino acids, 1 amino acid to at most about 640 amino acids, 1 amino acid to at most about 650 amino acids, 1 amino acid to at most about 660 amino acids, 1 amino acid to at most about 670 amino acids, 1 amino acid to at most about amino acids up to about 780, amino acids 1 up to about 690, amino acids 1 up to about 700, amino acids 1 up to about 710, amino acids 1 up to about 720, amino acids 1 up to about 730, amino acids 1 up to about 740, amino acids 1 up to about 750, amino acids 1 up to about 760, amino acids 1 up to about 770, amino acids 1 up to about 780, amino acids 1 up to about 790, amino acids 1 up to about 800, amino acids 1 up to about 810, or amino acids 1 up to about 820.

[0168] In some examples where the number of different constructs in the composition is two, the C-terminal portion encoded by one of the two constructs is from the last amino acid (e.g., amino acid position 820) to about amino acid position 1 of the inner ear cell target protein (e.g., SEQ ID NO: 6 or 7), or any subrange within this range (e.g., amino acid 820 to at least about amino acid 10, amino acid 820 to at least about amino acid 20, amino acid 820 to at least about amino acid 30, amino acid 820 to at least about amino acid 60, amino acid 820 to at least about amino acid 70, amino acid 820 to at least about amino acid 8 20 to at least about amino acid 80, 820 to at least about amino acid 90, 820 to at least about amino acid 100, 820 to at least about amino acid 110, 820 to at least about amino acid 120, 820 to at least about amino acid 130, 820 to at least about amino acid 140, 820 to at least about amino acid 150, 820 to at least about amino acid 160, 820 to at least about amino acid 170, 820 to at least about amino acid 180, 820 to at least about amino acid 190 amino acids 820 to at least about amino acids 200, amino acids 820 to at least about amino acids 210, amino acids 820 to at least about amino acids 220, amino acids 820 to at least about amino acids 230, amino acids 820 to at least about amino acids 240, amino acids 820 to at least about amino acids 250, amino acids 820 to at least about amino acids 260, amino acids 820 to at least about amino acids 270, amino acids 820 to at least about amino acids 280, amino acids 820 to at least about amino acids 290, amino acids 820 to at least about amino acids 300, amino acids 300, amino acids 820 to at least about amino acids 310, amino acids 820 to at least about amino acids 320, amino acids 820 to at least about amino acids 330, amino acids 820 to at least about amino acids 340, amino acids 820 to at least about amino acids 350, amino acids 820 to at least about amino acids 360, amino acids 820 to at least about amino acids 370, amino acids 820 to at least about amino acids 380, amino acids 820 to at least about amino acids 390, amino acids 820 to at least about amino acids 400, amino acids 820 to at least about amino acids 410,amino acids 820 to at least about amino acids 420, amino acids 820 to at least about amino acids 430, amino acids 820 to at least about amino acids 440, amino acids 820 to at least about amino acids 450, amino acids 820 to at least about amino acids 460, amino acids 820 to at least about amino acids 470, amino acids 820 to at least about amino acids 480, amino acids 820 to at least about amino acids 490, amino acids 820 to at least about amino acids 500, amino acids 820 to at least about amino acids 510, amino acids 820 to at least about amino acids 520, amino acids 820 to at least about amino acids 530, amino acids 820 to at least about amino acids 540, amino acids 820 to at least about amino acids 550, amino acids 820 to at least about amino acids 560, amino acids 820 to at least about amino acids 570, amino acids 820 to at least about amino acids 58, 0, amino acids 820 to at least about amino acids 590, amino acids 820 to at least about amino acids 600, amino acids 820 to at least about amino acids 610, amino acids 820 to at least about amino acids 620, amino acids 820 to at least about amino acids 630, amino acids 820 to at least about amino acids 640, amino acids 820 to at least about amino acids 650, amino acids 820 to at least about amino acids 660, amino acids 820 to at least about amino acids 670, amino acids 820 to at least about amino acids 680, amino acids 820 to at least about amino acids 690, amino acids 820 to at least about amino acids 700, amino acids 820 to at least about amino acids 710, amino acids 820 to at least about amino acids 710, amino acids 820 to at least about amino acids 720, amino acids 820 to at least about amino acids 730, amino acids 820 to at least about amino acids 740, amino acids 820 to at least about amino acids 750, amino acids 820 to at least about amino acids 760, amino acids 820 to at least about amino acids 770, amino acids 820 to at least about amino acids 780, amino acids 820 to at least about amino acids 790, amino acids 820 to at least about amino acids 800, amino acids 820 to at least about amino acids 810, or amino acids 820 to at least about amino acids 820. In some examples where the number of different constructs in the composition is two, the C-terminal portion of the precursor inner ear cell targeting protein is from the last amino acid (e.g., about amino acid position 820) to at most about amino acid position 1 of the inner ear cell targeting protein (e.g., SEQ ID NO: 6 or 7), or any subrange within this range (e.g., amino acid 820 to at most about amino acid 10, amino acid 820 to at most about amino acid 20, amino acid 820 to at most about amino acid 30, amino acid 820 to at most about amino acid 60, amino acid 820 to at most about amino acid 70, amino acid 820 to at most about amino acid 90, amino acid 820 to at most about amino acid 10, amino acid 820 to at most about amino acid 11, amino acid 820 to at most about amino acid 12, amino acid 820 to at most about amino acid 13, amino acid 820 to at most about amino acid 14, amino acid 820 to at most about amino acid 15, amino acid 820 to at most about amino acid 16, amino acid 820 to at most about amino acid 17, amino acid 820 to at most about amino acid 18, amino acid 820 to at most about amino acid 19, amino acid 820 to at most about amino acid 20, amino acid 820 to at most about amino acid 21, amino acid 820 to at most about amino acid 22, amino acid 820 to at most about amino acid 23, amino acid 820 to at most about amino acid 24, amino acid 820 to at most about amino acid 25, amino acid 820 to at most about amino acid 26, amino acid 820 to at most about amino acid 27, amino acid 820 to at most about amino acid 28, amino acid 820 to at most about amino acid ~ about 70 amino acids at most, 820 amino acids ~ about 80 amino acids at most, 820 amino acids ~ about 90 amino acids at most, 820 amino acids ~ about 100 amino acids at most, 820 amino acids ~ about 110 amino acids at most, 820 amino acids ~ about 120 amino acids at most, 820 amino acids ~ about 130 amino acids at most, 820 amino acids ~ about 140 amino acids at most, 820 amino acids ~ about 150 amino acids at most, 820 amino acids ~ about 160 amino acids, 820 amino acids ~ about 170 amino acids at most,820 amino acids to a maximum of about 180 amino acids, 820 amino acids to a maximum of about 190 amino acids, 820 amino acids to a maximum of about 200 amino acids, 820 amino acids to a maximum of about 210 amino acids, 820 amino acids to a maximum of about 220 amino acids, 820 amino acids to a maximum of about 230 amino acids, 820 amino acids to a maximum of about 240 amino acids, 820 amino acids to a maximum of about 250 amino acids, 820 amino acids to a maximum of about 260 amino acids, 820 amino acids to a maximum of about 270 amino acids, 820 amino acids to a maximum of about 280 amino acids, 820 amino acids to a maximum of about 2 290 amino acids, 820 amino acids to a maximum of about 300 amino acids, 820 amino acids to a maximum of about 310 amino acids, 820 amino acids to a maximum of about 320 amino acids, 820 amino acids to a maximum of about 330 amino acids, 820 amino acids to a maximum of about 340 amino acids, 820 amino acids to a maximum of about 350 amino acids, 820 amino acids to a maximum of about 360 amino acids, 820 amino acids to a maximum of about 370 amino acids, 820 amino acids to a maximum of about 380 amino acids, 820 amino acids to a maximum of about 390 amino acids, 820 amino acids to a maximum of about 400 amino acids, 820 amino acids to a maximum of about 3 At most about 410 amino acids, 820 amino acids to a maximum of about 420 amino acids, 820 amino acids to a maximum of about 430 amino acids, 820 amino acids to a maximum of about 440 amino acids, 820 amino acids to a maximum of about 450 amino acids, 820 amino acids to a maximum of about 460 amino acids, 820 amino acids to a maximum of about 470 amino acids, 820 amino acids to a maximum of about 480 amino acids, 820 amino acids to a maximum of about 490 amino acids, 820 amino acids to a maximum of about 500 amino acids, 820 amino acids to a maximum of about 510 amino acids, 820 amino acids to a maximum of about 520 amino acids, 20 to a maximum of about 530 amino acids, 820 to a maximum of about 540 amino acids, 820 to a maximum of about 550 amino acids, 820 to a maximum of about 560 amino acids, 820 to a maximum of about 570 amino acids, 820 to a maximum of about 580 amino acids, 820 to a maximum of about 590 amino acids, 820 to a maximum of about 600 amino acids, 820 to a maximum of about 610 amino acids, 820 to a maximum of about 620 amino acids, 820 to a maximum of about 630 amino acids, 820 to a maximum of about 640 amino acids,820 amino acids ~ Maximum approx. 650 amino acids, 8 amino acids, amino acids 820 to about 660, 820 to about 670, 820 to about 680, 820 to about 690, 820 to about 700, 820 to about 710, 820 to about 720, 820 to about 730, 820 to about 740, 820 to about 750, 820 to about 760, 820 to about 770, 820 to about 780, 820 to about 790, 820 to about 800, 820 to about 810, or 820 to about 820) or a sequence of any length therebetween.

[0169] In some embodiments, the splice site is involved in trans-splicing. In some embodiments, the splice donor site (Trapani et al. EMBO Mol. Med. 6(2):194-211, 2014 (incorporated herein by reference in its entirety)) follows the coding sequence of the N-terminal construct. In the C-terminal construct, the splice acceptor site can be subcloned immediately before the coding sequence of SLC26A4. In some embodiments, silent mutations can be introduced within the coding sequence to generate additional sites for restriction digestion.

[0170] In some embodiments, any of the constructs provided herein can be included in a composition suitable for administration to an animal for the amelioration of symptoms associated with symptomatic and / or non-syndromic hearing loss.

[0171] Pharmaceutical Compositions Among other things, the present disclosure provides pharmaceutical compositions. In some embodiments, the compositions provided herein are suitable for administration to an animal for the relief of symptoms associated with symptomatic and / or non-syndromic hearing loss.

[0172] In some embodiments, the pharmaceutical compositions of the present disclosure can include, for example, one or more polynucleotides described herein, such as one or more constructs. In some embodiments, the pharmaceutical compositions can include one or more AAV particles described herein, such as one or more rAAV constructs encapsidated by one or more AAV serotype capsids.

[0173] In some embodiments, pharmaceutical compositions include one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into any of the compounds described herein. Such compositions may include one or more buffers (e.g., neutral buffered saline, phosphate buffered saline, etc.), one or more carbohydrates (e.g., glucose, mannose, sucrose, and dextran), mannitol, one or more proteins, polypeptides, or amino acids (e.g., glycine), one or more antioxidants, one or more chelating agents (e.g., EDTA or glutathione), and / or one or more preservatives. In some embodiments, the formulation is in the form of an injectable solution, an injectable gel, a drug-release capsule, or the like.

[0174] In some embodiments, the compositions of the present disclosure are formulated for intravenous administration. In some embodiments, the compositions of the present disclosure are formulated for intracochlear administration. In some embodiments, the therapeutic composition is formulated to include lipid nanoparticles, polymeric nanoparticles, minicircle DNA, and / or CELiD DNA.

[0175] In some embodiments, a therapeutic composition is formulated to contain a synthetic perilymph solution. For example, in some embodiments, the synthetic perilymph solution contains 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl, 1-10 mM glucose, and 2-50 mM HEPES, and has a pH of about 6 to about 9. In some embodiments, a therapeutic composition is formulated to contain a physiologically suitable solution. For example, in some embodiments, the physiologically suitable solution includes commercially available 1x PBS containing 8.10 mM disodium phosphate, 1.5 mM monopotassium phosphate, 2.7 mM potassium chloride, 172 mM sodium chloride, and pluronic acid F68 prepared to a final concentration of 0.001% pluronic acid F68. In some embodiments, an alternative pluronic acid is utilized. In some embodiments, an alternative ion concentration is utilized.

[0176] In some embodiments, any of the pharmaceutical compositions described herein may further comprise one or more agents that facilitate entry of the nucleic acid or any of the constructs described herein into mammalian cells (e.g., liposomes or cationic lipids). In some embodiments, any of the constructs described herein may be formulated using natural and / or synthetic polymers. Non-limiting examples of polymers that may be included in any of the compositions described herein include DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.), formulations from Mirus Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PhaseRX polymer formulations (e.g., SMARTT POLYMER TECHNOLOGY® (PhaseRX, Seattle, Wash.), DMRI / DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrins, dendrimers, and poly(lactic-co-glycolic acid) (PLGA) polymers from Calando Pharmaceuticals (Pasadena, Calif.), RONDEL™ (RNAi / oligonucleotide nanoparticle delivery) polymers (Arrowhead Research Corp., Pasadena, Calif.), and SMARTT POLYMER TECHNOLOGY® (PhaseRX, Seattle, Wash.). Corporation, Pasadena, Calif.), and pH-responsive coblock polymers (such as, but not limited to, those manufactured by PhaseRX (Seattle, Wash.)).Many of these polymers have demonstrated efficacy in the in vivo delivery of oligonucleotides to mammalian cells (see, e.g., deFougerolles, Human Gene Ther. 19:125-132, 2008; Rozema et al., Proc. Natl. Acad. Sci. USA 104:12982-12887, 2007; Rozema et al., Proc. Natl. Acad. Sci. USA 104:12982-12887, 2007; Hu-Lieskovan et al., Cancer Res. 65:8984-8982, 2005; Heidel et al., Proc. Natl. Acad. Sci. USA 104:5715-5721, 2007, each of which is incorporated by reference in its entirety).

[0177] In some embodiments, the composition comprises a pharmaceutically acceptable carrier (e.g., phosphate buffered saline, saline, or bacteriostatic water). Once formulated, the solution is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, such as injectable solutions, injectable gels, drug-release capsules, etc.

[0178] In some embodiments, the compositions provided herein can be formulated to be compatible with, for example, their intended route of administration, a non-limiting example of which is topical administration (e.g., intracochlear administration).

[0179] In some embodiments, the compositions provided comprise one nucleic acid construct. In embodiments, provided compositions comprise two or more different constructs. In some embodiments, a composition comprises a single nucleic acid construct comprising a coding sequence encoding a pendrin protein and / or functional characteristic portions thereof. In some embodiments, a composition comprises a single nucleic acid construct comprising a coding sequence encoding a pendrin protein and / or functional characteristic portions thereof, wherein, upon introduction into a mammalian cell, the coding sequence is integrated into the genome of the mammalian cell. In some embodiments, a composition comprising at least two different constructs, e.g., constructs, comprises coding sequences encoding different portions of a pendrin protein, wherein the constructs combine to generate a sequence encoding an active pendrin protein (e.g., a full-length pendrin protein) in a mammalian cell, thereby treating associated symptomatic or non-syndromic sensorineural hearing loss in a subject in need thereof.

[0180] Kits containing any of the compositions described herein are also provided. In some embodiments, the kits may include a solid composition (e.g., a lyophilized composition containing at least two different constructs described herein) and a liquid for solubilizing the lyophilized composition. In some embodiments, the kits may include a pre-filled syringe containing any of the compositions described herein.

[0181] In some embodiments, the kit includes a vial containing any of the compositions described herein (e.g., an aqueous composition, e.g., formulated as an aqueous pharmaceutical composition).

[0182] In some embodiments, the kit may include instructions for performing any of the methods described herein.

[0183] Medication and dosage In some embodiments, the compositions disclosed herein, e.g., one or more AAV vectors disclosed herein, are administered as a single dose or as multiple doses.

[0184] 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.

[0185] 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 0.09 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.

[0186] 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 to 2.00 mL, about 0.02 to 1.90 mL, about 0.03 to 1.8 mL, about 0.04 to 1.70 mL, about 0.05 to 1.60 mL, about 0.06 to 1.50 mL, about 0.06 to 1.40 mL, about 0.07 to 1.30 mL, about 0.08 to 1.20 mL, or about 0.09 to 1.10 mL. In some embodiments, a composition disclosed herein (e.g., a composition comprising one or more rAAV constructs disclosed herein) comprises a volume of about 0.01 to 2.00 mL, about 0.02 to 2.00 mL, about 0.03 to 2.00 mL, about 0.04 to 2.00 mL, about 0.05 to 2.00 mL, about 0.06 to 2.00 mL, about 0.07 to 2.00 mL, about 0.08 to 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.

[0187] Genetically modified cells The present disclosure also provides cells (e.g., animal cells, e.g., mammalian cells, e.g., primate cells, e.g., human cells) comprising any of the nucleic acids, constructs, or compositions described herein. In some embodiments, the animal cells are human cells (e.g., human supporting cells or human hair cells). In other embodiments, the animal cells are non-human mammalian cells (e.g., simian cells, feline cells, canine cells, etc.). One of skill in the art will understand that the nucleic acids and constructs described herein can be introduced into any animal cell (e.g., supporting cells or hair cells of any animal suitable for veterinary intervention). Non-limiting examples of constructs and methods for introducing constructs into animal cells are described herein.

[0188] In some embodiments, the animal cell can be any cell of the inner ear, including hair cells and / or supporting cells, including, but not limited to, Hensen's cells, Deiters' cells, cells of the endolymphatic sac and duct, transitional cells within the saccule, utricle, and ampulla, inner and outer hair cells, spiral ligament cells, spiral ganglion cells, spiral eminence cells, outer capsule cells, marginal cells, intermediate cells, basal cells, inner pillar cells, outer pillar cells, cells of Claudius, inner border cells, inner phalangeal cells, or cells of the stria vascularis.

[0189] In some embodiments, the animal cell is a specialized cell of the cochlea. In some embodiments, the animal cell is a hair cell. In some embodiments, the animal cell is an inner cochlear hair cell or an outer cochlear hair cell. In some embodiments, the animal cell is an inner cochlear hair cell. In some embodiments, the animal cell is an outer cochlear hair cell.

[0190] In some embodiments, the animal cells are in vitro. In some embodiments, the animal cells are cell types that are endogenously present in an animal, e.g., a primate and / or a human. In some embodiments, the animal cells are autologous cells obtained from an animal and cultured ex vivo.

[0191] Genetically modified animal models The present disclosure also provides animals (e.g., mammals, e.g., rodents, e.g., mice or rats) suitable for testing any of the nucleic acids, constructs, or compositions described herein. In some embodiments, the nucleic acids and / or constructs described herein can be introduced into any animal cells (e.g., supporting cells or hair cells of any animal suitable for veterinary intervention). However, some animals are suitable for controlled hearing analysis experiments. Non-limiting examples of animals suitable for introduction of constructs and methods for analyzing the constructs are described herein.

[0192] In some embodiments, the present disclosure provides a method for producing an animal, comprising genetically modifying the animal to contain a mutant Slc26a4 gene. In some embodiments, the endogenous Slc26a4 gene is modified so that the polypeptide encoded by the Slc26a4 gene contains an L236P mutation when compared to SEQ ID NO: 56. In some embodiments, the endogenous Slc26a4 gene is modified so that the polypeptide encoded by the Slc26a4 gene comprises or consists of the sequence according to SEQ ID NO: 57.

[0193] In some embodiments, the endogenous Slc26a4 gene is knocked out or inhibited in the genetically modified animal. In some embodiments, a mutant Slc26a4 gene is knocked in or added to the genetically modified animal. In some embodiments, the mutant Slc26a4 gene encodes a polypeptide comprising an L236P mutation when compared to SEQ ID NO: 56. In some embodiments, the mutant Slc26a4 gene encodes a polypeptide comprising or consisting of the sequence set forth in SEQ ID NO: 57.

[0194] In some embodiments, the mutant Slc26a4 gene is present at the endogenous locus of the Slc26a4 gene. In some embodiments, the genetically modified animal is homozygous for the mutant Slc26a4 gene. In some embodiments, the genetically modified animal is heterozygous for the mutant Slc26a4 gene.

[0195] In particular, in some embodiments, the present disclosure provides a genetically modified mouse whose genome comprises a recombinant Slc26a4 gene that recapitulates one or more known human disease genotypes. In some embodiments, the mutant Slc26a4 gene encodes a polypeptide having a L236P mutation when compared to SEQ ID NO: 56. In some embodiments, the mutant Slc26a4 gene mutation encodes the polypeptide set forth in SEQ ID NO: 57.

[0196] In some embodiments, the present disclosure provides genetically modified animals suitable for use in auditory analysis experiments. In some embodiments, the genetically modified animals are genetically modified mice. In some embodiments, genetically modified mice suitable for use in auditory analysis experiments are the FVB strain. In some embodiments, genetically modified mice suitable for use in auditory analysis experiments include FVB, 129 / Sv-+p+Tyr-c+Mgf-SIJ / J, A / HeJ, AKR / J, BALB / cByJ, BALB / cJ, BDP / J, BXSB / MpJ, C3H / HeJ, C3H / HeOuJ, C3HeB / FeJ, C57BL / 10J, C57BL / 10SnJ, C57BL / 6ByJ, CASA / RK, CAST / Ei, CBA / J, CZEC H II / Ei, DBA / 2HaSmn, FVB / NJ, HRS / J hrl+, MOLD / Rk, MOLF / Ei, MOLG / Dn, NON / LtJ, NZB / B1NJ, NZO / NIJ, NZW / LacJ, PERA / camEi, PERC / Ei, PL / J, RBA / Dn, RBF / DnJ, RF / J, RHJ / Le hrrh-J / +, RIIIS / J, SEC / 1ReJ, SENCARC / PtJ, SF / CamEi, SHR / GnEi, SJL / J, SM / J, SPRET / Ei, ST / bJ, or SWR / J strains (e.g., Zheng et al., Assessment of hearing in 80 inbred strains of mice by ABR threshold analysis. Hear Res. 1999 (incorporated herein by reference in its entirety). In some embodiments, the mutations contained in the genetically modified mice are generated in a background suitable for hearing analysis experiments. In some embodiments, the genetically modified mice are of a mouse strain suitable for use in orientation analysis experiments. In some embodiments, the genetically modified mice are not of the CBA / CaJ or CBA / J strain.

[0197] In some embodiments, genetically modified animals are treated with AAV particles, constructs, or compositions described herein.In some embodiments, genetically modified animals are injected with AAV particles described herein.In some embodiments, genetically modified animals are injected with compositions described herein.In some embodiments, injection is carried out through puncture of the round window membrane in email.

[0198] The present disclosure provides for the use of the genetically modified animals described herein to evaluate and / or characterize the AAV particles described herein.

[0199] The present disclosure provides for the use of the genetically modified animals described herein to evaluate and / or characterize the compositions described herein.

[0200] In some embodiments, the uses provided herein can be part of a release test.

[0201] method Among other things, the present disclosure provides methods. In some embodiments, the methods include introducing a composition described herein into the inner ear (e.g., cochlea) of a subject. For example, in some embodiments, methods are provided herein that include administering a therapeutically effective amount of any of the compositions described herein to the inner ear (e.g., cochlea) of a subject (e.g., an animal, e.g., a mammal, e.g., a primate, e.g., a human). In some embodiments of any of these methods, the subject has previously been identified as having a defective inner ear cell target gene (e.g., a supporting cell and / or auditory cell target gene having a mutation that results in decreased expression and / or activity of a supporting cell and / or auditory cell target protein encoded by the gene). Some embodiments of any of these methods further include determining that the subject has a defective inner ear cell target gene prior to the introducing or administering step. Some embodiments of any of these methods can further include detecting a mutation in the inner ear cell target gene in the subject. Some embodiments of any of the present methods can further include identifying or diagnosing the subject as having non-syndromic or syndromic sensorineural hearing loss.

[0202] In some embodiments, provided herein are methods for correcting an inner ear cell target gene defect (e.g., a defect in the SLC26A4 gene) in the inner ear of a subject (e.g., an animal, e.g., a mammal, e.g., a primate, e.g., a human). In some embodiments, the method comprises administering to the inner ear of the subject a therapeutically effective amount of any of the compositions described herein. and administering repairs and / or ameliorates an inner ear cell target gene defect in any cell subset of the subject's inner ear. In some embodiments, the inner ear target cells can be sensory cells, e.g., hair cells, and / or non-sensory cells, e.g., supporting cells, and / or all or any subset of inner ear cells.

[0203] Also provided herein are methods for increasing the expression level of an inner ear cell target protein in any subset of inner ear cells of a subject (e.g., an animal, e.g., a mammal, e.g., a primate, e.g., a human), comprising administering to the inner ear of the subject a therapeutically effective amount of any of the compositions described herein, wherein the administration results in an increase in the expression level of an inner ear cell target protein (e.g., pendrin protein) in any subset of cells of the subject's inner ear. In some embodiments, the inner ear target cells can be sensory cells, e.g., hair cells, and / or non-sensory cells, e.g., supporting cells, and / or all or any subset of inner ear cells.

[0204] Also provided herein are methods for treating hearing loss, e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss, in a subject (e.g., an animal, e.g., a mammal, e.g., a primate, e.g., a human) identified as having a defective inner ear cell target gene, the method comprising administering to the inner ear of the subject a therapeutically effective amount of any of the compositions described herein.

[0205] Also provided herein are methods for restoring synapses and / or preserving spiral ganglion neurons in a subject identified or diagnosed with an inner ear disorder, comprising administering to the subject's inner ear a therapeutically effective amount of any of the compositions described herein.

[0206] Also provided herein are methods for reducing the size of the vestibular aqueduct and / or restoring the vestibular aqueduct to an appropriate size. Also provided herein are methods for restoring endolymphatic pH to an appropriate and / or acceptable level in a subject identified or diagnosed with an inner ear disorder, comprising administering to the subject's inner ear a therapeutically effective amount of any of the compositions described herein.

[0207] Also provided herein are methods that include administering to the inner ear of a subject a therapeutically effective amount of any of the compositions described herein.

[0208] Also provided herein are surgical methods for treating hearing loss (e.g., non-syndromic sensorineural hearing loss or symptomatic sensorineural hearing loss). In some embodiments, the method includes introducing a first incision into a subject's cochlea at a first incision point and intracochlearly administering a therapeutically effective amount of any of the compositions provided herein. In some embodiments, the composition is administered to the subject at the first incision point. In some embodiments, the composition is administered to the subject within or through the first incision.

[0209] In some embodiments of any of the methods described herein, any of the compositions described herein are administered to a subject into or through the cochlear round window membrane. In some embodiments of any of the methods described herein, any of the compositions described herein are administered to a subject into or through the cochlear round window membrane. In some embodiments of any of the methods described herein, the composition is administered using a medical device capable of making multiple incisions in the round window membrane. In some embodiments, the medical device comprises a plurality of microneedles. In some embodiments, the medical device comprises a plurality of microneedles comprising a generally circular first side, each microneedle having a diameter of at least about 10 microns. In some embodiments, the medical device comprises a base and / or reservoir capable of holding the composition. In some embodiments, In some embodiments, the medical device comprises a plurality of hollow microneedles each comprising a lumen through which the composition can be transported. In some embodiments, the medical device comprises a means for generating at least a partial vacuum.

[0210] In some embodiments, the technology of the present disclosure is used to treat subjects with hearing loss or at risk of hearing loss.For example, in some embodiments, the subject has autosomal recessive hearing loss caused by at least one pathogenic variant in SLC26A4.Those skilled in the art will understand that many different mutations in SLC26A4 can cause pathogenic variants.In some such embodiments, the pathogenic variant causes or is at risk of causing hearing loss.

[0211] In some embodiments, a subject experiencing hearing loss is evaluated to determine whether and where one or more mutations that may cause hearing loss may be present. In some such embodiments, the status of the SLC26A4 gene product or function is evaluated (e.g., by protein or sequencing analysis). In some embodiments of any of the methods described herein, the subject or animal is a mammal, and in some embodiments, the mammal is a livestock animal, a farm animal, a zoo animal, a non-human primate, or a human. In some embodiments of any of the methods described herein, the animal, subject, or mammal is an adult, a teenager, a juvenile, a child, a toddler, an infant, or a newborn. In some embodiments of any of the methods described herein, the animal, subject, or mammal is 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 1 to 110, 2 to 5, 2 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 1 0-100, 10-110, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-110, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 40-60, 40-70, 40-80, 40-90, 40-100, 50-70, 50-80, 50-90, 50-100, 60-80, 60-90, 60-100, 70-90, 70-100, 70-110, 80-100, 80-110, or 90-110 years old. In some embodiments of any of the methods described herein, the subject or mammal is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months old.

[0212] In some embodiments of any of the methods described herein, the method provides hearing improvement (e.g., any of the metrics for determining hearing improvement described herein) in a subject in need thereof for at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 100 days, at least 105 days, at least 110 days, at least 115 days, at least 120 days, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0213] In some embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) has or is at risk of developing syndromic or non-syndromic sensorineural hearing loss. In some embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) has previously been identified as having a mutation in the SLC26A4 gene ... a mutation in the SLC26A4 gene, as described herein or in the art, associated with syndromic or non-syndromic sensorineural hearing loss. They have any of the mutations in the SLC26A4 gene known to cause glaucoma.

[0214] In some embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) has been identified (e.g., by genetic testing) as a carrier of a mutation in the SLC26A4 gene. In some embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) has been identified as having a mutation in the SLC26A4 gene and has been diagnosed with syndromic or non-syndromic sensorineural hearing loss. In some embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) has been identified as having syndromic or non-syndromic sensorineural hearing loss.

[0215] In some embodiments, a subject (e.g., an animal, e.g., a mammal, e.g., a human) has been identified as being at risk for hearing loss (e.g., at risk of being a carrier of a genetic mutation, e.g., an SLC26A4 mutation). In some such embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) may have certain risk factors for hearing loss, or may be at risk for hearing loss (e.g., a parent known to be a carrier, a sibling affected, or symptoms of hearing loss). In some such embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) has been identified as being a carrier of a mutation in the SLC26A4 gene (e.g., by genetic testing) that has not been previously identified (i.e., is not a published or otherwise known variant of SLC26A4). In some such embodiments, the identified mutation may be novel (i.e., not previously described in the literature), and methods of treating a subject suffering from or susceptible to hearing loss are individualized to the mutation(s) of the particular patient.

[0216] In some embodiments, successful treatment of symptomatic or non-syndromic sensorineural hearing loss can be determined in a subject using any of the conventional functional hearing tests known in the art, including, but not limited to, various types of audiometric assays (e.g., pure tone tests, speech tests, middle ear tests, auditory brainstem responses, and otoacoustic emissions).

[0217] In some embodiments of any of the methods provided herein, two or more doses of any of the compositions described herein are introduced or administered to the cochlea of ​​a subject.Some embodiments of any of these methods can include: introducing or administering a first dose of the composition to the cochlea of ​​the subject; evaluating the hearing function of the subject after the introduction or administration of the first dose; and administering an additional dose of the composition to the cochlea of ​​a subject who has been found not to have hearing function within normal ranges (e.g., as determined using any test for hearing acuity known in the art).

[0218] In some embodiments of any of the methods provided herein, the composition can be formulated for intracochlear administration. In some embodiments of any of the methods described herein, the composition can be administered via intracochlear administration or local administration. In some embodiments of any of the methods described herein, the composition is administered through the use of a medical device (e.g., any of the exemplary medical devices described herein).

[0219] In some embodiments, intracochlear administration can be performed using any of the methods described herein or known in the art. For example, in some embodiments, the composition can be administered or introduced into the cochlea using the following surgical technique: First, using visualization with a 0-degree, 2.5 mm rigid endoscope, the ear canal is cleared and a round knife is used to clearly delineate an approximately 5 mm ear canal flap. The ear canal flap is then elevated and the middle ear is approached posteriorly. The chorda tympani nerve is identified and separated. The tympanic bone is then removed using a curette to expose the round window membrane. To enhance apical distribution of the administered or introduced composition, a small 2 mm fenestration can be created within the oval window using a surgical laser to allow for perilymphatic displacement during transround window membrane injection of the composition. The microinfusion device is then primed and brought into the surgical field. The device is maneuvered toward the round window, with its tip positioned within the bony prominence of the round window, allowing penetration of the membrane by the microneedle(s). The foot pedal is engaged to allow for a measured, consistent infusion of the composition. The device is then withdrawn, and the round window and base of the stapes are sealed with a gelfoam patch.

[0220] In some embodiments of any of the methods provided herein, the subject has or is at risk of developing syndromic or non-syndromic sensorineural hearing loss. In some embodiments of any of the methods provided herein, the subject has previously been identified as having a mutation in an inner ear cell target gene, a gene that can be expressed in supporting cells and / or hair cells.

[0221] In some embodiments of any of the methods provided herein, the subject has been identified (e.g., by genetic testing) as a carrier of a mutation in an inner ear cell target gene. In some embodiments of any of the methods provided herein, the subject has been identified as having a mutation in an inner ear cell target gene and has been diagnosed with hearing loss (e.g., nonsyndromic sensorineural hearing loss or syndromic sensorineural hearing loss, e.g., Pendred syndrome or DFNB4). In some embodiments of any of the methods described herein, the subject has been identified as having hearing loss (e.g., nonsyndromic sensorineural hearing loss or syndromic sensorineural hearing loss). In some embodiments, successful treatment of hearing loss (e.g., nonsyndromic sensorineural hearing loss or syndromic sensorineural hearing loss) can be determined in the subject using any of the conventional functional hearing tests known in the art. Non-limiting examples of functional hearing tests include various types of audiometric assays (e.g., pure tone tests, speech tests, middle ear tests, auditory brainstem responses, and otoacoustic emissions).

[0222] In some embodiments, the subject cells are in vitro. In some embodiments, the subject cells are first obtained from a subject and cultured ex vivo. In some embodiments, the subject cells have previously been determined to have a defective inner ear cell target gene. In some embodiments, the subject cells have previously been determined to have a defective hair cell target gene. In some embodiments, the subject cells have previously been determined to have a defective supporting cell target gene.

[0223] In some embodiments of these methods, expression of an active inner ear cell target protein (e.g., pendrin protein) increases after treatment, e.g., after administration of one or more of the compositions described herein. In some embodiments, the increase in expression of an active inner ear cell target protein (e.g., pendrin protein) described herein is relative to a control level, e.g., compared to the expression level of the inner ear cell target protein before introduction of a composition comprising any of the construct(s) described herein.

[0224] Methods for detecting the expression and / or activity of a target protein (e.g., pendrin protein) are known in the art. In some embodiments, the expression level of an inner ear cell target protein can be detected directly (e.g., detecting the inner ear cell target protein or target mRNA). Non-limiting examples of techniques that can be used to directly detect the expression and / or activity of a target RNA or protein (e.g., the SLC26A4 gene product and / or pendrin protein or a functional characteristic portion thereof) include real-time PCR, Western blotting, immunoprecipitation, immunohistochemistry, mass spectrometry, or immunofluorescence. In some embodiments, the expression of an inner ear cell target protein can be detected indirectly. The hearing loss may be detected clinically (e.g., through functional hearing testing).

[0225] Devices, Administration, and Surgical Methods Provided herein is a therapeutic delivery system for treating hearing loss (e.g., non-syndromic sensorineural hearing loss or symptomatic sensorineural hearing loss). In one aspect, the therapeutic delivery system comprises: i) a medical device capable of creating one or more incisions in the round window membrane of the inner ear of a subject in need thereof; and ii) an effective dose of a composition (e.g., any of the compositions described herein). In some embodiments, the medical device comprises a plurality of microneedles.

[0226] Also provided herein are surgical methods for the treatment of hearing loss (e.g., non-syndromic sensorineural hearing loss or symptomatic sensorineural hearing loss). In some embodiments, the method includes introducing a first incision into a subject's cochlea at a first incision point and administering a therapeutically effective amount of any of the compositions provided herein into the cochlea. In some embodiments, the composition is administered to the subject at the first incision point. In some embodiments, the composition is administered to the subject into or through the first incision.

[0227] In some embodiments of any of the methods provided herein, any of the compositions described herein are administered to a subject into or through the cochlear round window membrane. In some embodiments of any of the methods provided herein, any of the compositions described herein are administered to a subject into or through the cochlear round window membrane. In some embodiments of any of the methods provided herein, the composition is administered using a medical device capable of making multiple incisions in the round window membrane. In some embodiments, the medical device comprises a plurality of microneedles. In some embodiments, the medical device comprises a plurality of microneedles comprising a generally circular first aspect, each microneedle having a diameter of at least about 10 microns. In some embodiments, the medical device comprises a base and / or reservoir capable of holding the composition. In some embodiments, the medical device comprises a plurality of hollow microneedles each comprising a lumen through which the composition can be transported. In some embodiments, the medical device comprises a means for generating at least a partial vacuum.

[0228] In some embodiments, the compositions disclosed herein are formulated as sterile suspensions for intracochlear administration.In some embodiments, the compositions comprise constructs in an amount of at least 1E11, at least 5E11, at least 1E12, at least 5E12, at least 1E13, at least 2E13, at least 3E13, at least 4E13, at least 5E13, at least 6E13, at least 7E13, at least 8E13, at least 9E13 or at least 1E14 vector genome (vg) per milliliter (mL). In some embodiments, the composition comprises the construct in an amount of at most 1E15, at most 5E14, at most 1E14, at most 5E13, at most 1E13, at most 9E12, at most 8E12, at most 7E12, at most 6E12, at most 5E12, at most 4E12, at most 3E12, at most 2E12, or at most 1E12 vector genomes (vg) per milliliter (mL). In some embodiments, the composition comprises the construct in an amount of 1E12 to 1E13, 5E12 to 5E13, or 1E13 to 2E13 vector genomes (vg) per milliliter (mL).

[0229] In some embodiments, the compositions disclosed herein are administered in an operating room under controlled, sterile conditions by a board-certified surgeon experienced in performing ear surgery. In some embodiments, the administration procedure is a microscopic or endoscopically assisted transmetal exploratory tympanotomy and a laser-assisted microstapes procedure, followed by a round window injection delivering 0.09 mL of a solution containing a composition disclosed herein. is a common procedure used to expose middle ear structures. Transmetal exploratory tympanotomy is often accompanied by laser-assisted stapedectomy (removal of the stapes footplate) or stapedotomy (drilling a hole in the stapes footplate), for example, for patients with otosclerosis. In some embodiments, an approximately 0.25 mm vent in the stapes footplate (created using an otologic laser) serves to prevent potentially harmful increases in intralabyrinthic pressure.

[0230] In some embodiments, disclosed herein is a sterile, single-use delivery device for administering a composition disclosed herein through the round window membrane into the perilymphatic fluid of the inner ear using a vent located in the stapes footplate. In some embodiments, this custom device is specifically designed for the intracochlear route of administration, offering advantages over available materials in terms of both potential safety and efficacy of the therapeutic agent. In some embodiments, design elements of the delivery device include maintaining sterility of the infused fluid, minimizing air bubbles introduced into the inner ear, the ability to precisely deliver small volumes at controlled flow rates (with the use of standard pumps), visualization of the round window membrane and tolerance for delivery through the ear canal by the surgeon, minimizing damage to the round window membrane or to cochlear structures beyond the round window membrane, and / or minimizing reflux from the round window membrane.

[0231] In some embodiments, any of the methods disclosed herein includes a dose escalation study to evaluate safety and tolerability in a subject, e.g., a mammal, e.g., a human, e.g., a patient, e.g., a patient with DFNB4 or Pendred syndrome symptoms. In some embodiments, a composition disclosed herein is administered in a dosing regimen disclosed herein. In some embodiments, the dosing regimen includes either unilateral or bilateral intracochlear administration of a dose of a composition disclosed herein, e.g., as described herein. In some embodiments, the dosing regimen comprises delivering 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 comprises 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. In some embodiments, the dosing regimen comprises 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.

[0232] In some embodiments of any of the methods provided herein, two or more doses of any of the compositions described herein are introduced or administered to the cochlea of ​​a subject.Some embodiments of any of these methods can include: introducing or administering a first dose of the composition to the cochlea of ​​the subject; evaluating the hearing function of the subject after the introduction or administration of the first dose; and administering an additional dose of the composition to the cochlea of ​​a subject who has been found not to have hearing function within normal ranges (e.g., as determined using any test for hearing acuity known in the art).

[0233] In some embodiments of any of the methods provided herein, the composition may be formulated for intracochlear administration. In embodiments, the compositions described herein may be administered via intracochlear or topical administration. In some embodiments of any of the methods described herein, the composition is administered through the use of a medical device (e.g., any of the exemplary medical devices described herein).

[0234] In some embodiments, the target cells are in vitro. In some embodiments, the target cells are first obtained from a subject and cultured ex vivo. In some embodiments, the target cells are otherwise considered healthy and are cultured and expanded ex vivo. In some embodiments, the target cells have previously been determined to have a defective inner ear cell target gene. In some embodiments, the target cells have previously been determined to have a defective hair cell target gene. In some embodiments, the target cells have previously been determined to have a defective supporting cell target gene.

[0235] In some embodiments, when the subject is a rodent, e.g., a mouse, the surgical approach described in Yoshimura et al., 2018, is used, which involves delivery through the round window membrane with fenestration of the posterior semicircular canal, which has demonstrated robust and reliable transduction regardless of the animal's age at the time of injection (Yoshimura 2018, incorporated herein by reference in its entirety). Briefly, a postauricular incision is made to access the temporal bone. A portion of the sternocleidomastoid muscle is divided to expose the otic vesicle. A 0.5-0.6 mm diameter otologic drill is used to drill a small hole in the otologic vesicle, which is then widened to visualize the stapedial artery and round window membrane. A fenestration of the posterior semicircular canal is performed using an otologic drill (0.5-0.6 mm diameter) to serve as a vent for the inner ear during cochlear administration. The round window membrane is pierced with a mouse delivery device consisting of a borosilicate capillary pipette and a 10 µL Hamilton syringe, and 1 µL of solution containing viral particles (approximately 40-50% of the total inner ear volume) is delivered into the scala tympani through the round window membrane at a rate of 300 nL / min.

[0236] In some embodiments, when the subject is an NHP, a postauricular incision is made and soft tissue dissection is performed down to the level of the periosteum. In some embodiments, the periosteum is incised and elevated to expose the mastoid bone. A cortical mastoidectomy is performed using a combination of high-speed cutting and a diamond drill burr. The facial nerve cavity is then opened, allowing proper visualization of the round window and oval window (OW). A fenestration of the stapes footplate within the OW is performed using a Rosen needle. As with other models, the fenestration allows for a larger volume to be injected without damaging the inner ear. Furthermore, aeration allows the solution containing rAAV particles to flow toward the apex of the cochlea. 30 μL of the solution containing rAAV particles (approximately 40-50% of the total inner ear volume) is delivered through the round window membrane at a rate of 30 μL / min.

[0237] In some embodiments, when the subject is a mammal, e.g., a human, a minimally invasive approach through the ear canal is used, for example, because the relevant structures are relatively large even at birth. In some embodiments, the clinical administration procedure involves transmetal exploratory tympanotomy and laser-assisted stapedectomy (using potassium titanyl phosphate [KTP] or a CO2 otologic laser to place a small vent hole (approximately 0.25 mm) in the stapes footplate), followed by round window infusion to deliver approximately 0.09 mL (or 90 μL, approximately 40-50% of the total inner ear volume) of a solution containing a composition disclosed herein (e.g., rAAV-SLC26A4 particles) through the round window membrane within a 3-minute period. In some embodiments, the insufflation serves to prevent potentially harmful increases in intralabyrinthine pressure. Transmetal exploratory tympanotomy is a common procedure used to expose middle ear structures. Transcanal exploratory tympanotomy is often accompanied by laser-assisted stapedectomy (removal of the stapes footplate) or stapedotomy (drilling a hole in the stapes footplate), for example, for patients with otosclerosis.

[0238] In some embodiments, the present disclosure provides a method for accessing the middle and / or inner ear via the ear canal. This paper describes a delivery approach that utilizes minimally invasive, well-accepted surgical techniques to treat rheumatoid arthritis. The procedure involves opening one of the physical barriers between the middle and inner ear at the oval window, followed by delivery of a composition disclosed herein at a controlled flow rate and fixed volume through the round window membrane using a device disclosed herein, as shown, for example, in Figures 15-18.

[0239] In some embodiments, surgical procedures on mammals (e.g., rodents (e.g., mice, rats, hamsters, or rabbits), primates (e.g., NHPs (e.g., macaques, chimpanzees, monkeys, or apes), or humans) may include insufflation to increase AAV vector transduction rates along the length of the cochlea. In some embodiments, the absence of an insufflation during surgery results in lower AAV vector cochlear cell transduction rates compared to AAV vectors following surgery performed with an insufflation. In some embodiments, aeration can result in a cochlear cell transduction rate of approximately 75-100% throughout the cochlea. In some embodiments, aeration allows for an IHC transduction rate of approximately 50-70%, approximately 60-80%, approximately 70-90%, or approximately 80-100% at the base of the cochlea. In some embodiments, aeration allows for an IHC transduction rate of approximately 50-70%, approximately 60-80%, approximately 70-90%, or approximately 80-100% at the apex of the cochlea.

[0240] The delivery device described herein may be placed in a sterile field in an operating room, and the end of the tubing may be removed from the sterile field, loaded with a composition disclosed herein (e.g., one or more AAV vectors), and connected to a syringe attached to a pump. After properly priming the system to remove air, a needle may be passed through the middle ear under visualization (surgical microscope, endoscope, and / or distal tip camera). A needle (or microneedle) may be used to puncture the RWM. The needle may be inserted until the stopper contacts the RWM. The device may then be held in place while a composition disclosed herein is delivered to the inner ear at a controlled flow rate for a selected period of time. In some embodiments, the flow rate (or infusion rate) may include a rate of about 30 μL / min, or about 25 μL / min to about 35 μL / min, or about 20 μL / min to about 40 μL / min, or about 20 μL / min to about 70 μL / min, or about 20 μL / min to about 90 μL / min, or about 20 μL / min to about 100 μL / min. In some embodiments, the flow rate is about 20 μL / min, about 30 μL / min, about 40 μL / min, about 50 μL / min, about 60 μL / min, about 70 μL / min, about 80 μL / min, about 90 μL / min, or about 100 μL / min. In some embodiments, the selected period (i.e., the period during which the compositions disclosed herein are delivered) may be about 3 minutes, or about 2.5 to about 3.5 minutes, or about 2 to about 4 minutes, or about 1.5 to about 4.5 minutes, or about 1 to about 5 minutes. In some embodiments, the total volume of the compositions disclosed herein delivered to the inner ear may be about 0.09 mL, or about 0.08 to about 0.10 mL, or about 0.07 to about 0.11 mL. In some embodiments, the total volume of the compositions disclosed herein is equal to about 40% to about 50% of the volume of the inner ear.

[0241] Once delivery is complete, the device can be removed. In some embodiments, the devices described herein can be configured as single-use, disposable products. In other embodiments, the devices described herein can be configured as multi-use, sterilizable products, e.g., with a replaceable and / or sterilizable needle subassembly. The single-use device can be properly disposed of (e.g., in a biohazard sharps container) after administration is complete.

[0242] In some embodiments, the compositions disclosed herein can be administered to a subject via a surgical procedure. In some embodiments, administration, for example, via a surgical procedure, includes injecting a composition disclosed herein into the inner ear via a delivery device described herein. In some embodiments, the surgical procedure disclosed herein comprises performing a trans-metal decompression and injecting a composition disclosed herein into the inner ear via a delivery device described herein.

[0243] In some embodiments, the surgical procedure comprises performing a transcanalicular dehiscence, performing laser-assisted microstapes surgery, injecting a composition disclosed herein into the inner ear via a delivery device described herein, applying a sealant around the subject's round window and / or oval window, and lowering the subject's canal tympanic flap into an anatomical position.

[0244] In some embodiments, the surgical procedure comprises performing a transcanal de-tympanotomy, preparing the subject's round window, performing laser-assisted microstapes surgery, preparing both a delivery device described herein and a composition disclosed herein for delivery to the inner ear, injecting the composition disclosed herein via the delivery device into the inner ear, applying a sealant around the subject's round window and / or oval window, and lowering the subject's canal tympanic flap into an anatomical position.

[0245] In some embodiments, performing laser-assisted stapes surgery includes using a KTP otic laser and / or a CO2 otic laser.

[0246] As another example, the compositions disclosed herein are administered using devices and / or systems specifically designed for the intracochlear route of administration. In some embodiments, design elements of the devices described herein may include maintaining sterility of the infused fluid, minimizing air bubbles introduced into the inner ear, the ability to precisely deliver low volumes at a controlled rate, delivery through the ear canal by the surgeon, minimizing damage to the round window membrane (RWM) or inner ear, e.g., minimizing damage to cochlear structures beyond the RWM, and / or minimizing back leakage of the infused fluid through the RWM.

[0247] The devices, systems, and methods provided herein also demonstrate the potential for safely and efficiently delivering compositions disclosed herein to the inner ear to treat conditions and disorders that would benefit from delivery of the compositions disclosed herein to the inner ear, including, but not limited to, hearing impairment, as described herein. As another example, by placing a vent in the stapes footplate and injecting through the RWM, the compositions disclosed herein are dispersed throughout the cochlea with minimal dilution at the site of action. Development of the described devices allows surgical administration procedures to be performed through the human ear canal. The described devices can inject a volume of fluid into the perilymph of the cochlea and then be removed from the ear. In a subject, the device may be advanced through the ear canal under surgical microscopic control or with an endoscope.

[0248] Exemplary devices for use in any of the methods disclosed herein are depicted in Figures 15-18. Figure 15 shows an exemplary device 10 for delivering fluid to the inner ear. The device 10 includes a knurled handle 12 and a distal handle adhesive 14 (e.g., an epoxy such as Loctite 4014) that couples to a telescoping hypotube needle support 24. The knurled handle 12 (or handle portion) may include knurling features and / or grooves for enhanced grip. The knurled handle 12 (or handle portion) may be about 5 mm to about 15 mm thick, or about 5 mm to about 12 mm thick, or about 6 mm to about 10 mm thick, or about 6 mm to about 9 mm thick, or about 7 mm to about 8 mm thick. The knurled handle 12 (or handle portion) may be hollow to allow fluid to pass through the device 10 during use. The device 10 also includes a proximal handle connector 14 at the proximal end 18 of the knurled handle 12. The device 10 may include an adhesive 16, a needle subassembly 26 (shown in FIG. 16) having a stopper 28 (shown in FIG. 16) at the distal end 20 of the device 10, and a strain relief mechanism 22. The strain relief mechanism 22 may be constructed from Santoprene, Pebax, polyurethane, silicone, nylon, and / or thermoplastic elastomers. The telescoping hypotube needle support 24 surrounds and supports a bending needle 38 (shown in FIG. 16) disposed therein.

[0249] Still referring to FIG. 15 , stopper 28 may be constructed of a thermoplastic material or a plastic polymer (e.g., a UV-curable polymer), as well as other suitable materials, and may be used to prevent bending needle 38 from being inserted too far into the ear canal (e.g., to prevent insertion of bending needle 38 into the side wall or other inner ear structure). Device 10 may also include a tapered portion 23 disposed between knurled handle 12 and distal handle adhesive 14, which is coupled to telescoping hypotube needle support 24. Knurled handle 12 (or handle portion) may include tapered portion 23 at the distal end of handle portion 12. Device 10 may also include tubing 36 fluidly connected to proximal end 16 of device 10, which may serve as a fluid inlet line connecting the device to upstream components (e.g., a pump, a syringe, and / or upstream components, which in some embodiments may be coupled to a control system and / or power source (not shown)). In some embodiments, the bending needle 38 (shown in FIG. 16 ) extends from the distal end 20, through the telescoping hypotube needle support 24, through the tapered portion 23, through the knurled handle 12, and through the strain relief mechanism 22, and is directly fluidly connected to the tubing 36. In other embodiments, the bending needle 38 is fluidly connected to the hollow interior of the knurled handle (e.g., via the telescoping hypotube needle support 24) and then fluidly connected to the tubing 36 at the proximal end 16. In embodiments in which the bending needle 38 does not extend through the interior of the device 10 at all, the contact areas (e.g., between the overlapping, nested hypotubes 42), tolerances, and / or sealants between interface components must be sufficient to prevent therapeutic fluid from leaking from the device 10, which operates at relatively low pressures (e.g., from about 1 Pascal to about 50 Pa, or from about 2 Pa to about 20 Pa, or from about 3 Pa to about 10 Pa).

[0250] FIG. 16 shows a side view of a bent needle subassembly 26, according to aspects of an embodiment of the present disclosure. The bent needle subassembly 26 includes a needle 38 having a bent portion 32. The bent needle subassembly 26 may also include a stopper 28 coupled to the bent portion 32. The bent portion 32 includes an angled tip 34 at the distal end 20 of the device 10 for piercing an ear membrane (e.g., the RWM). The needle 38, the bent portion 32, and the angled top 34 are hollow to allow fluid to flow therethrough. The angle 46 (as shown in FIG. 18 ) of the bent portion 32 may vary. The geometry of the stopper 28 may be cylindrical, disc-shaped, annular, dome-shaped, and / or other suitable shapes. The stopper 28 may be molded in place on the bent portion 32. For example, the stopper 28 may be concentrically disposed around the bent portion 32 using adhesive or a compression fitting. Examples of adhesives include ultraviolet curable adhesives (e.g., Dymax 203A-CTH-FT), elastomeric adhesives, thermosetting adhesives (e.g., epoxy or polyurethane), or emulsion adhesives (e.g., polyvinyl acetate). The stopper 28 fits concentrically around the bent portion 32 so that the angled tip 34 is inserted into the ear at the desired insertion depth. The bent needle 38 can be formed from a straight needle using incremental forming and other suitable techniques.

[0251] 17 shows a perspective view of an exemplary device 10 for delivering fluid to the inner ear. The tube 36 can be about 1300 mm (dimension 11 in FIG. 17) to about 1600 mm in length, or about 1400 mm to about 1500 mm, or about 1430 mm to about 1450 mm in length. The release mechanism 22 may be about 25 mm to about 30 mm in length (dimension 15 in FIG. 17), or about 20 mm to about 35 mm in length. The handle 12 may be about 155.4 mm in length (dimension 13 in FIG. 17), or about 150 mm to about 160 mm, or about 140 mm to about 170 mm in length. The telescoping hypotube needle support 24 may have two or more nested hypotubes, for example, three nested hypotubes 42A, 42B, and 42C, or four nested hypotubes 42A, 42B, 42C, and 42D. The overall length of the hypotubes 42A, 42B, 42C and tip assembly 26 (dimension 17 in FIG. 31) may be about 25 mm to about 45 mm, or about 30 mm to about 40 mm, or about 35 mm. Additionally, the telescoping hypotube needle support 24 may be approximately 36 mm in length, or approximately 25 mm to approximately 45 mm, or approximately 30 mm to approximately 40 mm. The three nested hypotubes 42A, 42B, and 42C may have lengths of 3.5 mm, 8.0 mm, and 19.8 mm, respectively, plus or minus approximately 20%. The innermost nested hypotube (or narrowest portion) of the telescoping hypotube support 24 may be concentrically disposed around the needle 38.

[0252] FIG. 18 illustrates a perspective view of a bent needle subassembly 26 coupled to the distal end 20 of the device 10, according to aspects of an embodiment of the present disclosure. As shown in FIG. 18, the bent needle subassembly 26 may include a needle 38 coupled to a bent portion 32. In other embodiments, the bent needle 38 may be a single needle (e.g., a straight needle that is later bent to include the desired angle 46). The needle 38 may be a 33-gauge needle, or may include a gauge of about 32 to about 34, or about 31 to 35. With finer gauges, care must be taken to prevent kinking or damage to the tubing 36. The needle 38 can be attached to the handle 12 to safely and accurately position the needle 38 in the inner ear. As shown in FIG. 18, the bent needle subassembly 26 may include a stopper 28 disposed around the bent portion 32. FIG. 18 also illustrates that the bent portion 32 may include an angled tip 34 for piercing the ear membrane (e.g., the RWM). Stopper 28 may have a height 48 of about 0.5 mm, or about 0.4 mm to about 0.6 mm, or about 0.3 mm to about 0.7 mm. Bent portion 32 may have a length 52 of about 1.45 mm, or about 1.35 mm to about 1.55 mm, or about 1.2 mm to about 1.7 mm. In other embodiments, bent portion 32 may have a length greater than 2.0 mm, such that the distance between the distal end of stopper 28 and the distal end of angled tip 34 is about 0.5 mm to about 1.7 mm, or about 0.6 mm to about 1.5 mm, or about 0.7 mm to about 1.3 mm, or about 0.8 mm to about 1.2 mm. FIG. 18 illustrates that stopper 28 may have a geometric shape that is cylindrical, disc-shaped, and / or dome-shaped. Those skilled in the art will understand that other geometric shapes may be used.

[0253] Assessment of hearing loss and recovery In some embodiments, hearing function is determined using auditory brainstem response measurements (ABR). In some embodiments, hearing is tested by measuring distortion product otoacoustic emissions (DPOAEs). In some such embodiments, measurements are taken from one or both ears of the subject. In some such embodiments, recordings are compared to previous recordings for the same subject and / or known thresholds for such response measurements used to define, for example, hearing loss versus acceptable hearing ranges to be defined as normal hearing. In some embodiments, the subject has ABR and / or DPOAE measurements recorded before receiving any treatment. In some embodiments, subjects treated with one or more techniques described herein will have improved ABR and / or DPOAE measurements after treatment compared to before treatment. In some embodiments, ABR and / or DPOAE measurements are taken after treatment is administered and at regular follow-up intervals after treatment.

[0254] In some embodiments, auditory function is determined using speech pattern recognition or by a speech therapist. In some embodiments, auditory function is determined by pure tone testing. In some embodiments, auditory function is determined by bone conduction testing. In some embodiments, auditory function is determined by acoustic reflex testing. In some embodiments, auditory function is determined by tympanometry. In some embodiments, auditory function is determined by any combination of auditory analyses known in the art. In some such embodiments, measurements are taken globally and / or from one or both ears of the subject. In some such embodiments, recordings and / or expert analyses are compared to previous recordings / analyses of the same subject and / or known thresholds for such response measurements used, for example, to define hearing loss versus acceptable hearing ranges to be defined as normal hearing. In some embodiments, a subject has speech pattern recognition, pure tone testing, bone conduction testing, acoustic reflex testing, and / or tympanometry measurements and / or analyses performed before undergoing any treatment. In some embodiments, a subject treated with one or more techniques described herein has improved speech pattern recognition, pure tone testing, bone conduction testing, acoustic reflex testing, and / or tympanometry measurements after treatment compared to before treatment, hi some embodiments, speech pattern recognition, pure tone testing, bone conduction testing, acoustic reflex testing, and / or tympanometry measurements are performed after treatment is administered and at regular follow-up intervals after treatment.

[0255] In some embodiments, hearing function in both the treated ear and the contralateral control ear can be significantly altered as a function of transduction with an rAAV gene therapy product, as described herein. In some embodiments, rAAV particles can cross between the treated ear and the contralateral ear in neonatal and / or adult animals. In some embodiments, such crossing can be due to the patency of the rodent cochlea (e.g., fluid communication between the perilymph, CSF, and perilymph of the contralateral ear).

[0256] Characterization Method The term "mutation of the SLC26A4 gene" refers to a modification in a known consensus functional SLC26A4 gene that results in the production of a pendrin protein having one or more of the following: one or more amino acid deletions, one or more amino acid substitutions, and one or more amino acid insertions, compared to the consensus functional pendrin protein, and / or results in a reduced expression level of the encoded pendrin protein in a mammalian cell, compared to the expression level of the encoded pendrin protein in a mammalian cell that does not have the mutation. In some embodiments, the mutation may result in the production of a pendrin protein having a deletion of one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids). In some embodiments, the mutation may result in a frameshift in the SLC26A4 gene. The term "frameshift" is known in the art to encompass any mutation in a coding sequence that results in a shift in the reading frame of the coding sequence. In some embodiments, the frameshift can result in a non-functional protein. In some embodiments, the point mutation can be a nonsense mutation (i.e., resulting in a premature stop codon in an exon of a gene). The nonsense mutation can result in the production of a truncated protein that may or may not be functional (compared to the corresponding consensus functional protein). In some embodiments, the mutation can result in the loss (or reduced levels) of expression of SLC26A4 mRNA or pendrin protein, or both mRNA and protein. In some embodiments, the mutation can result in the production of a modified pendrin protein that has a loss or reduction in one or more biological activities (functions) compared to the consensus functional pendrin protein.

[0257] In some embodiments, the mutation is an insertion of one or more nucleotides into the SLC26A4 gene. In some embodiments, the mutation is in the regulatory and / or control sequences of the pendrin gene, i.e., in a portion of the gene that is not the coding sequence. In some embodiments, the mutation in the regulatory and / or control sequence may be in a promoter or enhancer region and may prevent or reduce proper transcription of the SLC26A4 gene. In some embodiments, the mutation is in a known heterologous gene known to interact with the pendrin protein or the SLC26A4 gene (e.g., FOXI1 or KCNJ10).

[0258] Methods for genotyping and / or detecting expression or activity of SLC26A4 mRNA and / or pendrin protein are known in the art (e.g., Ito et al., J. Immunol. 1999, 10:131-132). See, e.g., Roesch et al., World J Otorhinolaryngol. 2013 May 28;3(2):26-34, and Roesch et al., Int J Mol Sci. 2018 Jan;19(1):209 (each of which is incorporated herein by reference in its entirety). In some embodiments, the expression level of SLC26A4 mRNA or pendrin protein can be detected directly (e.g., detection of pendrin protein, detection of SLC26A4 mRNA, etc.). Non-limiting examples of techniques that can be used to directly detect SLC26A4 expression and / or activity include, for example, real-time PCR, quantitative real-time PCR, Western blotting, immunoprecipitation, immunohistochemistry, mass spectrometry, or immunofluorescence. In some embodiments, the expression of SLC26A4 and / or pendrin protein can be detected indirectly (e.g., through functional hearing testing, ABR, DPOAE, etc.).

[0259] In some embodiments, a tissue sample (e.g., comprising one or more inner ear cells, e.g., comprising one or more hair cells and / or one or more supporting cells) may be evaluated by morphological analysis to determine the morphology of the hair cells and / or supporting cells before and after administration of any agent described herein (e.g., a composition, e.g., a composition comprising a construct and / or particle, etc.). In some such embodiments, standard immunohistochemical or histological analysis may be performed. In some embodiments, when cells are used in vitro or ex vivo, additional immunocytochemical or immunohistochemical analysis may be performed. In some embodiments, one or more assays of one or more proteins or transcripts (e.g., Western blot, ELISA, polymerase chain reaction) may be performed on one or more samples from a subject or in vitro cell population.

[0260] Target treatment method The present disclosure provides, inter alia, that the technology described herein may be used to treat underlying diseases and / or symptoms in subjects suffering from or at risk for ear diseases characterized by mutations in the SLC26A4 gene (e.g., DFNB4 and / or Pendred syndrome).

[0261] In some embodiments, the method includes administering to a subject a construct (e.g., an rAAV construct), particle (e.g., an rAAV particle), or composition described herein. In some embodiments, the method is a therapeutic method. In some embodiments, the subject is suffering from or at risk for an ear disease characterized by a mutation in the SLC26A4 gene (e.g., DFNB4 and / or Pendred syndrome).

[0262] In some embodiments, administering to a subject a construct (e.g., rAAV construct), particle (e.g., rAAV particle), or composition described herein may alleviate and / or ameliorate one or more symptoms associated with an ear disease characterized by a mutation in the SLC26A4 gene (e.g., DFNB4 and / or Pendred syndrome). Symptoms include, for example, sensorineural hearing loss, enlarged vestibular aqueduct, cochlear hypoplasia (e.g., reduced cochlear turnover), and / or other conditions associated with an ear disease characterized by a mutation in the SLC26A4 gene (e.g., DFNB4 and / or Pendred syndrome). Symptoms may include: too little), ataxia / incoordination, neurological speech disorder, and / or dizziness.

[0263] In some embodiments, subjects are genetically and / or symptomatically characterized as described herein before, during, and / or after treatment with techniques described herein (e.g., real-time PCR, quantitative real-time PCR, Western blotting, immunoprecipitation, immunohistochemistry, mass spectrometry, or immunofluorescence, indirect phenotyping of gene and / or protein expression (e.g., through functional hearing testing, ABR, DPOAE, etc.), etc.). In some embodiments, subjects suffering from or at risk for an ear disease characterized by a mutation in the SLC26A4 gene (e.g., DFNB4 and / or Pendred syndrome) may have their associated disease state characterized by tissue sampling (e.g., comprising one or more inner ear cells, e.g., comprising one or more hair cells and / or one or more supporting cells). In some embodiments, tissue is assessed by morphological analysis to determine hair cell and / or supporting cell morphology before, during, and / or after administration of any technique (e.g., methodology, e.g., composition, e.g., composition comprising constructs and / or particles, etc.) described herein. In some such embodiments, standard immunohistochemical or histological analysis may be performed. In some embodiments, when cells are used in vitro or ex vivo, additional immunocytochemical or immunohistochemical analysis may be performed. In some embodiments, one or more assays (e.g., Western blot, ELISA, polymerase chain reaction) of one or more proteins or transcripts may be performed on one or more samples from a subject or in vitro cell population.

[0264] In some embodiments, administering to a subject a construct (e.g., an rAAV construct), particle (e.g., an rAAV particle), or composition described herein improves the patient's immunohistochemistry assessment (e.g., a test as described above) when compared to immunohistochemistry tests performed before treatment with the techniques described herein or when compared to a control population.

[0265] Production method AAV systems are generally well known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994); Cotten et al., PNASUSA, 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3):141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(4):699-708 (2012), each of which is incorporated herein by reference in its entirety). Methods for producing and using AAV constructs are described, for example, in U.S. Pat. Nos. 5,139,941, 4,797,368, and PCT Application No. US2019 / 060328, each of which is incorporated by reference in its entirety.

[0266] Methods for obtaining viral constructs are known in the art. For example, to produce an AAV construct, the methods typically involve culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein or fragment thereof, a recombinant AAV construct comprised of a functional rep gene, AAV inverted terminal repeats (ITRs) and coding sequence, and / or sufficient helper functions to allow packaging of the recombinant AAV construct into an AAV capsid protein.

[0267] In some embodiments, the AAV construct is packaged into an AAV capsid within a host cell. Components cultured in a medium may be provided to the host cell in trans. Alternatively, any one or more components (e.g., recombinant AAV construct, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell engineered to contain one or more such components using methods known to those of skill in the art. In some embodiments, such stable host cells contain such component(s) under the control of an inducible promoter. In some embodiments, such component(s) may be under the control of a constitutive promoter. In some embodiments, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, stable host cells may be generated that are derived from HEK293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inducible promoter. Other stable host cells may be generated by those of skill in the art using routine methods.

[0268] The recombinant AAV construct, rep sequence, cap sequence, and helper functions necessary for producing the AAV of the present disclosure may be delivered to the packaging host cell using any suitable genetic elements (e.g., constructs). The selected genetic elements may be delivered by any suitable method known in the art to those skilled in the art of nucleic acid manipulation, including genetic engineering, recombinant engineering, and synthetic techniques (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, incorporated herein by reference in its entirety). Similarly, methods for generating AAV particles are well known, and any suitable method can be used with the present disclosure (see, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745, incorporated herein by reference in their entirety).

[0269] In some embodiments, recombinant AAV can be produced using a triple transfection method (e.g., as described in U.S. Pat. No. 6,001,650, incorporated herein by reference in its entirety). In some embodiments, recombinant AAV is produced by transfecting a host cell with a recombinant AAV construct (including coding sequences) that is packaged into an AAV particle, an AAV helper function construct, and an accessory function construct. The AAV helper function construct encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans for productive AAV replication and encapsidation. In some embodiments, the AAV helper function construct supports efficient AAV construct production without producing any detectable wild-type AAV particles (i.e., AAV particles containing functional rep and cap genes). Non-limiting examples of constructs suitable for use in the present disclosure include pHLP19 (see, e.g., U.S. Pat. No. 6,001,650, incorporated herein by reference in its entirety) and pRep6cap6 constructs (see, e.g., U.S. Pat. No. 6,156,303, incorporated herein by reference in its entirety). Accessory function constructs encode nucleotide sequences for non-AAV-derived viral and / or cellular functions (i.e., "accessory functions") on which AAV depends for replication. Accessory functions may include functions required for AAV replication, including, but not limited to, moieties involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0270] Further methods for generating and isolating AAV viral constructs suitable for delivery to a subject include: For example, U.S. Patent No. 7,790,449, U.S. Patent No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and U.S. Patent No. 7,588,772 (each of which is incorporated herein by reference in its entirety). In one system, a producer cell line is transiently transfected with a construct encoding a coding sequence flanked by ITRs and a construct(s) encoding rep and cap. In another system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a coding sequence flanked by ITRs. In each of these systems, AAV particles are produced in response to infection with a helper adenovirus or herpesvirus, and AAV is separated from contaminating virus. Other systems do not require infection with a helper virus to restore AAV-helper functions (i.e., adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase). In such systems, helper functions may be supplied by transient transfection of cells with constructs encoding the helper functions, or cells may be engineered to stably contain genes encoding the helper functions, the expression of which may be controlled at the transcriptional or post-transcriptional level.

[0271] In some embodiments, the viral construct titer after purification is determined. In some embodiments, the titer is determined using quantitative PCR. In certain embodiments, a construct-specific TaqMan probe is utilized to determine the construct level. In certain embodiments, the TaqMan probe is represented by SEQ ID NO: 49, while the forward and reverse amplification primers are exemplified by SEQ ID NOs: 54 and 55, respectively. Exemplary TaqMan probe for quantification of constructs (SEQ ID NO: 49) / 56-FAM / TAATTCCAA / ZEN / CCAGCAGAGTCAGGGC / 3IABkFQ / Exemplary forward qPCR primers for quantification of the construct (SEQ ID NO: 54) GATACAGCTAGAGTCCTGATTGC Exemplary reverse qPCR primers for quantification of the construct (SEQ ID NO: 55) GATCTGCCAAGTACCTCACTATG

[0272] As described herein, in some embodiments, the viral construct of the present disclosure is an adeno-associated virus (AAV) construct. Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV Anc80, as well as variants thereof. In some embodiments, the AAV particle is an AAV2 / 6, AAV2 / 8, AAV2 / 9, or AAV2 / Anc80 particle (e.g., a construct with AAV2 ITRs and an AAV6, AAV8, AAV9, or Anc80 capsid). For other AAV particles and constructs, see, for example, Sharma et al., Brain Res Bull. 2010 Feb. 15;81(2-3):273 (incorporated herein by reference in its entirety). Generally, any AAV particle can be used to deliver the coding sequence described herein.However, different serotypes have different tropisms, such as preferentially infecting different tissues.In some embodiments, AAV construct is a self-complementary AAV construct.

[0273] The present disclosure provides, among other things, methods for producing AAV-based constructs. In some embodiments, such methods involve the use of host cells. In some embodiments, the host cells are mammalian cells. Host cells can be used as recipients of AAV helper constructs, AAV minigene plasmids, accessory function constructs, and / or other transferred DNA associated with the production of recombinant AAV. The term includes the progeny of the original transfected cell. Thus, as used herein, "host cell" refers to a cell that contains an exogenous DNA sequence. The sequence may refer to a transfected cell. It is understood that the progeny of a single parental cell may not be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.

[0274] Additional methods for producing and isolating AAV particles suitable for delivery to a subject are described, for example, in U.S. Pat. No. 7,790,449, U.S. Pat. No. 7,282,199, WO 2003 / 042397, WO 2005 / 033321, WO 2006 / 110689, and U.S. Pat. No. 7,588,772 (each of which is incorporated herein by reference in its entirety). In one system, a producer cell line is transiently transfected with a construct encoding a coding sequence flanked by ITRs and a construct(s) encoding rep and cap. In another system, a packaging cell line that stably supplies rep and cap is transiently transfected with a...

Claims

1. A construct comprising a coding sequence operably linked to a promoter, said coding sequence encoding a pendrin protein.

2. The construct of claim 1 , wherein the coding sequence is the SLC26A4 gene.

3. The construct of claim 2 , wherein the SLC26A4 gene is a primate SLC26A4 gene.

4. The construct of claim 2 or 3, wherein the SLC26A4 gene is a human SLC26A4 gene.

5. The construct of claim 4, wherein the human SLC26A4 gene comprises the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

6. The construct of claim 4 or 5, wherein the human SLC26A4 gene comprises the nucleic acid sequence set forth in SEQ ID NO:

1.

7. The construct of claim 1 , wherein the pendrin protein is a primate pendrin protein.

8. The construct of claim 1 or 7, wherein the pendrin protein is a human pendrin protein.

9. The construct of claim 8 , wherein the pendrin protein comprises the amino acid sequence set forth in SEQ ID NO:

6.

10. The construct of any one of claims 1 to 9, wherein the promoter is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.

11. The construct of any one of claims 1 to 10, wherein the promoter is an inner ear cell-specific promoter.

12. 12. The construct of claim 11, wherein the inner ear cell-specific promoter is a GJB2 promoter, a GJB6 promoter, a SLC26A4 promoter, a TECTA promoter, a DFNA5 promoter, a COCH promoter, a NDP promoter, a SYN1 promoter, a GFAP promoter, a PLP promoter, a TAK1 promoter, a SOX21 promoter, a SOX2 promoter, a FGFR3 promoter, a PROX1 promoter, a GLAST1 promoter, a LGR5 promoter, a HES1 promoter, a HES5 promoter, a NOTCH1 promoter, a JAG1 promoter, a CDKN1A promoter, a CDKN1B promoter, a SOX10 promoter, a P75 promoter, a CD44 promoter, a HEY2 promoter, a LFNG promoter, or an S100b promoter.

13. The construct according to any one of claims 1 to 10, wherein the promoter is a CAG promoter, a CBA promoter, a CMV promoter, or a CB7 promoter.

14. The construct of claim 13 , wherein the promoter comprises the nucleic acid sequence set forth in SEQ ID NO:

43.

15. 15. The construct of any one of claims 1 to 14, further comprising two AAV inverted terminal repeats (ITRs), said two AAV ITRs flanking said coding sequence and promoter.

16. 16. The construct of claim 15, wherein the two AAV ITRs are or are derived from AAV2 ITRs.

17. the two AAV ITRs are (i) a 5′ ITR comprising the nucleic acid sequence set forth in SEQ ID NO: 10 and a 3′ ITR comprising the nucleic acid sequence set forth in SEQ ID NO: 11; or (ii) a 5' ITR comprising the nucleic acid sequence set forth in SEQ ID NO: 12 and a 3' ITR comprising the nucleic acid sequence set forth in SEQ ID NO:

13.

18. The construct of claim 1 , wherein the construct comprises the nucleic acid sequence set forth in SEQ ID NO:

39.

19. The construct of claim 1 , wherein the construct comprises the nucleic acid sequence set forth in SEQ ID NO:

40.

20. An AAV particle comprising the construct of any one of claims 1 to 19.

21. 21. The AAV particle of claim 20, further 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.

22. The AAV particle of claim 21 , wherein the AAV capsid is an AAV Anc80 capsid.

23. A composition comprising the construct of any one of claims 1 to 19.

24. A composition comprising the AAV particles of any one of claims 20 to 22.

25. 25. The composition of claim 23 or 24, wherein the composition is a pharmaceutical composition.

26. 26. The composition of claim 25, further comprising a pharmaceutically acceptable carrier.

27. An ex vivo cell comprising the composition of any one of claims 23 to 26.

28. To ex vivo cells, (i) a construct according to any one of claims 15 to 19; and (ii) AAV Rep gene, AAV Cap gene, AAV VA gene, AAV and one or more helper plasmids collectively containing the AAV E2a gene and the AAV E4 gene.

29. 27. A method comprising introducing into the inner ear of a subject the composition of claim 25 or 26.

30. 27. A method of treatment comprising introducing into the inner ear of a subject a composition according to claim 25 or 26.

31. 29 or 21, wherein the composition of claim 25 or 26 is introduced into the cochlea of ​​the subject.

31. The method according to claim 30.

32. The method of claims 29 to 31, wherein the composition of claim 25 or 26 is introduced by round window membrane injection.

33. 33. The method of any one of claims 29 to 32, further comprising measuring the hearing level of the subject.

34. 34. The method of claim 33, wherein the hearing level is measured by performing an auditory brainstem response (ABR) test.

35. 35. The method of claim 33 or 34, further comprising comparing the hearing level of the subject to a reference hearing level.

36. 36. The method of claim 35, wherein the reference hearing level is a published or historical reference hearing level.

37. 36. The method of claim 35, wherein the hearing level of the subject is measured after the composition of claim 25 or 26 is introduced, and the reference hearing level is the hearing level of the subject measured before the composition of claim 25 or 26 is introduced.

38. The method of any one of claims 29 to 37, further comprising measuring the level of pendrin protein in the subject.

39. 39. The method of claim 38, wherein the level of the pendrin protein is measured in the subject's inner ear.

40. 40. The method of claim 38 or 39, wherein the level of the pendrin protein is measured in the cochlea of ​​the subject.

41. The method of any one of claims 38 to 40, further comprising comparing the level of pendrin protein in the subject with a reference pendrin protein level.

42. 42. The method of claim 41, wherein the reference pendrin protein level is a published or historical pendrin protein level.

43. The method of claim 41, wherein the level of the pendrin protein in the subject is measured after the composition described in claim 25 or 26 is introduced, and the reference pendrin protein level is the pendrin protein level of the subject measured before the composition described in claim 25 or 26 is introduced.

44. 28. Use of a construct according to any one of claims 1 to 19, an AAV particle according to any one of claims 20 to 22, or a composition according to any one of claims 23 to 27 for the treatment of hearing loss in a subject suffering from or at risk of hearing loss.

45. Use of a construct according to any one of claims 1 to 19, an AAV particle according to any one of claims 20 to 22, or a composition according to any one of claims 23 to 27 in the manufacture of a medicament for the treatment of hearing loss.

46. A construct according to any one of claims 1 to 19 for use as a medicament, claim 2 28. An AAV particle according to any one of claims 0 to 22, or a composition according to any one of claims 23 to 27.

47. A construct according to any one of claims 1 to 19, an AAV particle according to any one of claims 20 to 22, or a composition according to any one of claims 23 to 27 for use in the treatment of hearing loss.

48. A genetically modified mouse, the genome of which comprises a modified Slc26a4 gene encoding the polypeptide set forth in SEQ ID NO: 57, wherein the genetically modified mouse is a genetically modified version of a mouse strain suitable for use in auditory analysis experiments.

49. 49. The genetically modified mouse of claim 48, wherein the mouse strain suitable for use in the auditory analysis experiment is not CBA / CaJ or CBA / J.

50. Mouse strains suitable for use in the auditory analysis experiments are FVB, 129 / Sv-+p+Tyr-c+Mgf-SIJ / J, A / HeJ, AKR / J, BALB / cByJ, BALB / cJ, BDP / J, BXSB. / MpJ, C3H / HeJ, C3H / HeOuJ, C3HeB / FeJ, C57BL / 10J, C57BL / 10SnJ, C57BL / 6ByJ, CASA / RK, CAST / Ei, CBA / J, CZECH II / Ei, DBA / 2HaSmn, FVB / NJ, HRS / J hrl+, MOLD / Rk, MOLF / Ei, MOLG / Dn, NON / LtJ, NZB / B1NJ, NZO / NIJ, NZ W / LacJ, PERA / camEi, PERC / Ei, PL / J, RBA / Dn, RBF / DnJ, RF / J, RHJ / Le 49. The genetically modified mouse according to claim 48, which is an hrrh-J / +, RIIIS / J, SEC / 1ReJ, SENCARC / PtJ, SF / CamEi, SHR / GnEi, SJL / J, SM / J, SPRET / Ei, ST / bJ, or SWR / J strain.

51. A method comprising injecting a composition according to any one of claims 1 to 19, an AAV particle according to any one of claims 20 to 22, or a composition according to any one of claims 23 to 26 through a perforation in the round window membrane of a mouse according to any one of claims 48 to 50.

52. 1. A method of treating hearing loss, comprising: The method comprises introducing a composition described in any one of claims 1 to 19, an AAV particle described in any one of claims 20 to 22, or a composition described in any one of claims 23 to 26 into the inner ear of a subject.

53. 53. The method of claim 52, wherein the composition is introduced by round window membrane injection.

54. 54. The method of claim 52 or 53, wherein the hearing loss is associated with a mutation in the SLC26A4 gene.

55. 55. The method of any one of claims 52 to 54, wherein the hearing loss and treatment of hearing loss is characterized as a function of ABR and / or distortion product otoacoustic emissions (DPOAE) measurements recorded before receiving any treatment and compared with ABR and / or DPOAE measurements after treatment.

56. A kit comprising a composition comprising the construct of any one of claims 1 to 19, a composition comprising the AAV particles of any one of claims 20 to 22, or a composition of any one of claims 23 to 27.

57. 57. The kit of claim 56, wherein the composition is pre-loaded into the device.

58. 58. The kit of claim 57, wherein the device is a microcatheter.

59. 59. The kit of claim 58, wherein the microcatheter is shaped to allow entry into the middle ear cavity via the ear canal and to allow an end of the microcatheter to contact the RWM.

60. 59. The kit of claim 57 or 58, wherein the distal end of the microcatheter comprises at least one microneedle having a diameter of 10 to 1,000 microns.

61. 57. The kit of claim 56, further comprising a device.

62. 62. The kit of claim 61, wherein the device is the device of Figures 15-18 or a device described herein.

63. 63. The kit of claim 62, wherein the device comprises a needle comprising a bent portion and an angled tip.

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