Compositions and methods for treating CLRN1-associated hearing loss and / or vision loss

Abstract

The present disclosure provides a construct comprising a coding sequence operably linked to a promoter, the coding sequence encoding a clarin 1 protein. Exemplary constructs include AAV constructs. Also provided are methods of using the disclosed constructs for the treatment of hearing loss and / or profound hearing loss. Also provided are methods of using the disclosed constructs for the treatment of vision loss.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 131,413, filed December 29, 2020, the entire contents of which are incorporated herein by reference. [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 non-syndromic profound hearing loss are associated with permanent hearing loss caused by damage to structures in the inner ear (profound sensorineural hearing loss), but some forms may involve changes in the middle ear (conductive hearing loss). The majority of sensorineural hearing loss in humans is caused by abnormalities in the hair cells of the organ of Corti, located in the cochlea (malfunction of hair cells). 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).

[0003] Vision loss, also known as visual impairment or visual impairment, is a decrease in the ability to see to a degree that cannot be corrected by means such as glasses. The causes of vision loss are very diverse, ranging from conditions that affect the eyes to conditions that affect the visual processing centers in the brain. For example, vision loss in patients suffering from Usher III syndrome or retinitis pigmentosa occurs as the light-sensing cells of the retina gradually deteriorate and eventually atrophy. Other examples of vision loss include diabetic retinopathy, glaucoma, age-related macular degeneration, and cataracts. Summary of the Invention

[0004] The present disclosure provides the recognition that diseases or conditions associated with hearing loss may be treated, for example, through the replacement or addition of certain gene products. The present disclosure further provides that gene products involved in the development, function, and / or maintenance of inner ear cells may be useful in treating diseases or conditions associated with the loss of hair cells and / or supporting cells (e.g., supporting hair cells). Thus, the present disclosure provides for administering compositions that result in the expression of gene products involved in the development, function, and / or maintenance of inner ear cells, such as supporting cells and hair cells, and / or for using such compositions in the treatment of hearing loss or diseases or conditions associated with hearing loss. In some embodiments, the gene product may be encoded by the CLRN1 gene or a characteristic portion thereof. In some embodiments, the gene product may be the clarin 1 protein or a characteristic portion thereof.

[0005] The present disclosure further provides the recognition that diseases or conditions associated with vision loss may be treated, for example, via replacement or addition of a particular gene product. The present disclosure further provides that gene products involved in the development, function, and / or maintenance of ocular cells may be useful in treating diseases or conditions associated with loss of ocular cells and / or supporting cells (e.g., supporting hair cells). Thus, the present disclosure provides for administering compositions that result in expression of gene products involved in the development, function, and / or maintenance of ocular cells, such as supporting cells, and / or for using such compositions in treating vision loss or diseases or conditions associated with vision loss. In some embodiments, the gene product may be encoded by the CLRN1 gene or a characteristic portion thereof. In some embodiments, the gene product may be a clarin 1 protein or a characteristic portion thereof.

[0006] The present disclosure further provides that the AAV particles may 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, diseases or conditions associated with hearing loss. The present disclosure further provides that the AAV particles may be useful for administering compositions that result in expression of gene products involved in the development, function, and / or maintenance of ocular cells, and / or for treating vision loss, diseases or conditions associated with vision loss. As described herein, the AAV particles include (i) an AAV polynucleotide construct (e.g., a recombinant AAV polynucleotide construct), and (ii) a capsid that includes a capsid protein. In some embodiments, the AAV polynucleotide construct includes a CLRN1 gene or a characteristic portion thereof. In some embodiments, the AAV particles described herein are referred to as rAAV-CLRN1 or rAAV-CLRN1 particles. In some embodiments, the AAV particles described herein are referred to as rAAV Anc80-CLRN1 or rAAV Anc80-CLRN1 particles.

[0007] The present disclosure further provides a composition comprising a polynucleotide construct comprising CLRN1 gene or a characteristic portion thereof.In some embodiments, the construct may further comprise a regulatory element operably attached to the coding sequence.In certain embodiments, the regulatory element included facilitates tissue-specific expression at physiologically suitable levels.

[0008] Also provided herein are methods of administering the constructs and compositions described herein. In certain embodiments, administration involves surgical intervention and delivery of rAAV particles comprising the therapeutic construct. In certain embodiments, 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 effectiveness of the intervention is determined through established tests, and measurements are compared to control or reference measurements.

[0009] definition The scope of the present disclosure is defined by the appended claims and is not limited by the specific embodiments described herein. Those skilled in the art will recognize, upon reading this specification, various modifications that may be equivalent to such described embodiments or that may otherwise be within the scope of the claims. Generally, the terms used in this specification follow the meanings understood in the art unless expressly indicated otherwise. Explicit definitions of certain terms are provided below. The meaning of these and other terms in specific instances throughout this specification will be clear to those skilled in the art from the context.

[0010] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in and of itself, imply that one claim element has any priority, precedence, or order of performance of the method over another claim element, but is used solely as a marker to distinguish one claim element having a particular name from another element having the same name (but because of the use of the ordinal terminology).

[0011] The articles "a" and "an" as used herein should be understood to include plural referents unless clearly indicated to the contrary. A claim or description including "or" between one or more elements of a group is considered 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 the contrary is indicated or otherwise clear from the context. In some embodiments, the invention provides that exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. In some embodiments, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process. It is understood that the disclosure encompasses all variations, combinations, permutations of one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims that are introduced into another claim that relies on the same base claim (or any other claim that is related), unless otherwise indicated or unless it is clear to one of ordinary skill in the art that a contradiction or inconsistency would result. When elements are presented as a list (e.g., a Markush group or similar format), it should 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 referred to as "comprising" certain elements, features, etc., it should be understood that the particular embodiment or aspect "consists of" or "consists essentially of" such elements, features, etc. For the sake of brevity, these embodiments are not specifically described in any case in so many languages ​​herein. It should also be understood that any embodiment or aspect can be explicitly excluded from the claims, regardless of whether a specific exclusion is described in the specification.

[0012] 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, which represent adenosine, cytidine, guanosine, and thymidine, respectively), such polynucleotide or polypeptide will be presented in left to right, 5' to 3' or N-terminal to C-terminal order.

[0013] Administration: As used herein, the term "administration" typically refers to administration of a composition to a subject or system to achieve delivery of the agent to the subject or system. In some embodiments, the agent is a composition or is included in a composition. In some embodiments, the agent is produced via metabolism of the composition or one or more components thereof. Those skilled 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, injection through the round window membrane or into the scala tympani, injection into the scala tympani via the endolymph, delivery to the perilymph of the cochlea by the perilymph and / or endolymph after canalostomy. Local administration may also involve delivery to the eye, for example, by intraocular injection (e.g., intravitreal injection, intravitreal injection, subretinal injection, suprachoroidal injection (e.g., using Orbit™ Subretinal Delivery System (OrbitSDS) (Gyroscope Therapeutics)), or retinal injection). See, for example, Ochakovski et al., "Retinal Gene Therapy: Surgical Vector Delivery in the Translation to Clinical Trials," Front. Neurosci. April 3, 2017, or Ninel Z Gregori and Janet Louise David, "OCT-Assisted Delivery of Luxturna," https: / / www.aao.org / clinical-video / oct-assisted-delivery-of-luxturna (July 19, 2018), the disclosures of each of which are incorporated herein by reference in their entirety. 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 (eg, multiple doses separated by time) and / or periodic (eg, individual doses separated by common time intervals) dosing.In some embodiments, administration may involve continuous dosing (eg, perfusion) for at least a selected period of time.

[0014] Allele: As used herein, the term "allele" refers to one of two or more existing genetic variants at a particular polymorphic genomic locus.

[0015] Amelioration: As used herein, the term "amelioration" refers to the prevention, alleviation or alleviation of a condition in a subject, or the improvement of a condition. Amelioration may, but does not necessarily, include complete recovery or complete prevention of a disease, disorder or condition.

[0016] Amino Acid: As used herein, the term "amino acid" refers in its broadest sense to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure, e.g., H 2The amino acid has a NC(H)(R)-COOH. In some embodiments, the amino acid is a natural amino acid. In some embodiments, the amino acid is a non-natural amino acid. In some embodiments, the amino acid is a D-amino acid. In some embodiments, the 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 prepared synthetically 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 structure described above. For example, in some embodiments, the amino acid can be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) compared to the general structure. In some embodiments, such modifications can alter the circulating half-life of a polypeptide including the modified amino acid, for example, compared to a polypeptide including an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide that contains the modified amino acids, compared to a polypeptide that contains otherwise identical unmodified amino acids.

[0017] Approximately or about: As used herein, the term "approximately" or "about" may apply to one or more values ​​of interest, such as values ​​similar to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within ±10% (above or below) of a stated reference value, unless otherwise stated or otherwise clear from the context (except where such number may exceed 100% of possible values). For example, in some embodiments, the term "approximately" or "about" encompasses values ​​that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a reference value.

[0018] Associated: As used herein, the term "associated" refers to two events or entities as "associated" with each other when the presence, level and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, a gene signature, a metabolite, a microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition when its presence, level and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with each other when they directly or indirectly interact with each other, thereby being in and / or remaining in close physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other. In some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are non-covalently linked by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0019] Biological activity: As used herein, the term "biological activity" refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is determined by detection of a direct or indirect product produced by the biological pathway or event of interest.

[0020] 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 the 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 that is not found 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 one that comprises a contiguous stretch of amino acids, or a collection of contiguous stretches of amino acids, that together are characteristic of the protein or polypeptide. In some embodiments, each such contiguous stretch generally comprises 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 associated intact substance, in addition to the sequence and / or structural identity specified above. In some embodiments, a characteristic portion may be biologically active.

[0021] Characteristic sequences: As used herein, the term "characteristic sequences" are sequences that are found in all members of a family of polypeptides or nucleic acids and therefore can be used by those of skill in the art to define the members of the family.

[0022] Characteristic sequence element: As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., adjacently linked monomers). In some embodiments, a characteristic sequence element comprises at least first and second stretches of adjacent monomers separated by one or more spacer regions that may or may not vary in length between polymers sharing the sequence element.

[0023] 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 overlapping dosing regimens.

[0024] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, situations, sets of conditions, subjects, populations, etc., which may not be identical to one another, but are sufficiently similar to allow comparisons between them, so that one of skill in the art will understand that one may reasonably draw conclusions based on observed differences or similarities. In some embodiments, equivalent sets of agents, entities, situations, sets of conditions, subjects, populations, etc. are characterized by multiple substantially identical characteristics and one or a few different characteristics. One of skill in the art will understand what degree of identity is required in any given situation for two or more such agents, entities, situations, sets of conditions, subjects, populations, etc. to be considered equivalent in context. For example, one of skill in the art will understand that sets of agents, entities, situations, sets of conditions, subjects, populations, etc. are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to ensure a valid conclusion that differences in results obtained or phenomena observed under or by different sets of environments, stimuli, agents, entities, situations, sets of conditions, subjects, populations, etc. are caused by or indicate variations in the characteristics that vary.

[0025] 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, the construct may be a plasmid, a transposon, a cosmid, an artificial chromosome (e.g., a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC)) or a viral construct, and any Gateway® plasmid. The construct may, for example, contain sufficient cis-acting elements for expression, and other elements for expression may be provided by the host primate cell or in an in vitro expression system. The construct may contain any genetic element capable of replication when linked to appropriate control elements (e.g., a plasmid, a transposon, a cosmid, an artificial chromosome, or a viral construct, etc.). Thus, in some embodiments, a "construct" may include a cloning and / or expression construct and / or a viral construct (e.g., an adeno-associated virus (AAV) construct, an adenovirus construct, a lentivirus construct, or a retrovirus construct).

[0026] Conservative: As used herein, the term "conservative" refers to examples that represent conservative amino acid substitutions, such as 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). In general, conservative amino acid substitutions do not substantially change the desired functional property of a protein, such as, for example, the ability of a receptor to bind to a ligand. Examples of groups of amino acids with side chains that have 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 (G In, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains such as cysteine ​​(Cys, C) and methionine (Met, M). Conservative amino acid substitutions include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamic acid / aspartic acid (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, a conservative amino acid substitution can be the substitution of any naturally occurring residue in a protein with alanine, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions that have a positive value in the PAM250 log-likelihood matrix are made, as disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445, which is incorporated herein by reference in its entirety. In some embodiments, the substitutions are reasonably conservative substitutions that have non-negative values ​​in the PAM250 log-likelihood matrix.One of skill in the art will understand that amino acids that are not conserved between the same proteins from different species should be selected for mutation because changes (e.g., substitutions, additions, deletions, etc.) of these amino acids are unlikely to have an effect on the function of the protein. Amino acids that are conserved between the same proteins from different species should not be altered (e.g., deleted, added, substituted, etc.) because these mutations are likely to result in a change in the function of the protein. [Table 1]

[0027] Control: As used herein, the term "control" refers to its art-known meaning of a control, a standard against which results are compared. Typically, controls are used to enhance integrity in an experiment by isolating a variable in order to draw conclusions about such a variable. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparison. For example, in one experiment, the "test" (i.e., the variable being tested) is applied. In a second experiment, in the "control," the variable being tested is not applied. In some embodiments, a control is a pre-existing control (e.g., of a test or assay already performed, or of an amount or result already known). In some embodiments, a control is or includes a printed or otherwise stored record. In some embodiments, a control is a positive control. In some embodiments, a control is a negative control.

[0028] Determining, Measuring, Evaluating, Judging, Assaying, and Analyzing: As used herein, the terms "determining," "measuring," "evaluating," "determining," "assaying," and "analyzing" can 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. Assays can 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 absent.

[0029] Engineered: In general, the term "engineered" as used herein refers to an aspect of being manipulated by the hand of man. For example, a cell or organism is considered to be "engineered" if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example, by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been modified or removed, for example, by substitution or deletion mutations, or by mating protocols). As is common practice and will be understood by those skilled in the art, the progeny of an engineered polynucleotide or cell is typically still referred to as "engineered", even if the actual manipulation was performed on the previous entity.

[0030] Excipient: As used herein, the term "excipient" refers to an inert (e.g., non-therapeutic) agent that may be included in a pharmaceutical composition, for example, to 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, and the like.

[0031] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product (e.g., a transcript, e.g., mRNA, e.g., a polypeptide, etc.) from a nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, "expression" of a nucleic acid sequence refers to 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 a polypeptide or protein.

[0032] Functional: As used herein, the term "functional" refers to something that exists in a form that exhibits a characterized property and / or activity. For example, in some embodiments, a "functional" biomolecule is a biomolecule in a form that exhibits a characterized property and / or activity. In some such embodiments, a functional biomolecule is characterized relative to another biomolecule that is non-functional in that the "non-functional" form 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).

[0033] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that codes for a gene product (e.g., an RNA product, such as a polypeptide product). In some embodiments, a gene includes coding sequences (i.e., sequences that code for a particular product). In some embodiments, a gene includes non-coding sequences. In some particular embodiments, a gene may include both coding (e.g., exons) and non-coding (e.g., introns) sequences. In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences, which may, for example, control or affect one or more aspects of gene expression (e.g., cell type specific expression, inducible expression, etc.). As used herein, the term "gene" generally refers to a portion of a nucleic acid that codes for a polypeptide or a fragment thereof. The term can 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 is intended to clarify that in most cases, the term as used herein refers to a nucleic acid that codes for a polypeptide. In some embodiments, a gene may encode a polypeptide, but the polypeptide may not be functional, e.g., a gene variant may encode a polypeptide that does not function as well as, or at all, compared to a wild-type gene. 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.

[0034] Hearing Loss: As used herein, the term "hearing loss" may be used in reference to the partial or complete inability of an organism to hear. In some embodiments, the hearing loss may be acquired. In some embodiments, the hearing loss may be inherited. 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 agents that result in hearing loss, etc.). In some embodiments, the hearing loss may be due to one or more known genetic causes and / or syndromes. In some embodiments, the hearing loss may be of unknown etiology. In some embodiments, the hearing loss may or may not be alleviated by the use of hearing aids or other treatments.

[0035] 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 polypeptide domains that do not naturally occur together (e.g., in 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 are considered "heterologous" to each other because they do not naturally occur together.

[0036] 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., introducing gaps 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, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence, and 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., first and second) are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps and the length of each gap that need 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, the 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), which is 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.

[0037] Improve, increase, enhance, inhibit, or decrease: As used herein, the terms "improve," "increase," "enhance," "inhibit," "decrease," 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., in one individual) under otherwise comparable conditions in the absence of a particular agent or treatment (e.g., before and / or after) or in the presence of a suitable comparative reference substance. In some embodiments, a suitable reference measurement may be or include a measurement in a comparable system known or expected to respond in a particular way in the presence of a relevant agent or treatment. In some embodiments, a suitable reference is a negative reference. In some embodiments, a suitable reference is a positive reference.

[0038] 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, a "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, a "nucleic acid" refers to an oligonucleotide chain that includes individual nucleic acid residues. In some embodiments, a "nucleic acid" is or includes RNA. In some embodiments, a "nucleic acid" is or includes DNA. In some embodiments, a nucleic acid is, includes, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, includes, 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 in addition, 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, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, 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 those in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length. In some embodiments, the nucleic acid is partially or entirely single-stranded. 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, hi some embodiments, the nucleic acid has enzymatic activity.

[0039] Operably linked: As used herein, refers to a juxtaposition in which the components described are in a relationship that allows them to function in their intended manner. A control element "operably linked" to a functional element is joined such that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is adjacent (e.g., covalently linked) to a coding element of interest. In some embodiments, the control element acts in trans or otherwise on the functional element of interest. In some embodiments, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is "operably linked" to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. In some embodiments, for example, a 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 are contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0040] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in a unit 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, e.g., in solid or liquid form, e.g., an aqueous or non-aqueous solution or suspension, e.g., adapted for an injectable formulation, or as drops designed to be administered into the ear canal. In some embodiments, the pharmaceutical composition may be formulated for administration by injection into a particular organ or compartment, e.g., direct injection into the ear, or injection into the whole body, e.g., intravenous injection. In some embodiments, the formulation may be or include a drench (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste, capsule, powder, etc. In some embodiments, the active agent may be or include an isolated, purified, or pure compound.

[0041] Pharmaceutically acceptable: As used herein, the term "pharmaceutical acceptable," which may be used in reference to a carrier, diluent, or excipient used, for example, to formulate a pharmaceutical composition disclosed herein, means that the carrier, diluent, or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0042] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting the 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 may function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol, polyethylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and pH acid buffers, polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0043] Polyadenylation: As used herein, "polyadenylation" refers to the covalent linkage of a polyadenylyl moiety or modified variants thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. In some embodiments, the 3' poly(A) tail is a long sequence (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) of adenine nucleotides that are added to the pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, a poly(A) tail can be added to transcripts that contain a specific sequence, a polyadenylation signal or "poly(A) sequence." The poly(A) tail and its associated proteins help protect the mRNA from degradation by exonucleases. Polyadenylation can also affect transcription termination, nuclear export of mRNA, 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 by the action of an endonuclease complex associated with RNA polymerase. The cleavage site can be characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, adenosine residues can be added to the free 3' end of the cleavage site. As used herein, a "poly(A) sequence" is a sequence that triggers endonuclease cleavage of mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0044] 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 is naturally occurring. In some embodiments, a polypeptide has an amino acid sequence that is not naturally occurring. In some embodiments, a polypeptide has an amino acid sequence that has been engineered to be designed and / or generated by the action of the human hand. In some embodiments, a polypeptide may comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications, e.g., one or more amino acid side chain modifications or attachments, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be acetylation, amidation, lipidation, methylation, pegylation, and the like, e.g., combinations thereof. In some embodiments, a polypeptide may comprise L-amino acids, D-amino acids, or both, and may include any of a variety of amino acid modifications or analogs known in the art. In some embodiments, useful modifications may be or include, e.g., terminal acetylation, amidation, methylation, and the like. In some embodiments, a protein may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, a protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0045] 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, a polynucleotide analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively, or in addition, 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 contain one or more modified sugars compared to those in naturally occurring nucleic acids (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose).In some embodiments, the polynucleotide has a nucleotide sequence that encodes a functional gene product, such as an RNA or a protein. In some embodiments, the polynucleotide comprises one or more introns. In some embodiments, the polynucleotide is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, a polynucleotide is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length. In some embodiments, the polynucleotide is partially or entirely single-stranded. In some embodiments, the polynucleotide is partially or entirely double-stranded. In some embodiments, the polynucleotide has a nucleotide sequence that includes 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.

[0046] Protein: As used herein, "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan) and / or may be otherwise processed or modified. Those of skill in the art will understand that a "protein" may be a complete polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will further understand that a protein may include two or more polypeptide chains linked, for example, by one or more disulfide bonds or associated by other means.

[0047] Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide that is designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, including, for example, a polypeptide expressed using a recombinant expression construct transfected into a host cell, a polypeptide isolated from a recombinant combinatorial human polypeptide library, a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or 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, and / or a polypeptide prepared, expressed, created, or isolated by any other means, including splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or generating a nucleic acid that encodes and / or directs 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 of such selected sequence elements are the result of mutagenesis (e.g., in vivo or in vitro) of known sequence elements from natural or synthetic sources, such as in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0048] 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 past reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those of skill in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those being determined. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control. In some embodiments, the reference is a negative control reference. In some embodiments, the reference is a positive control reference.

[0049] Regulatory element: As used herein, the term "regulatory element" or "regulatory sequence" refers to a non-coding region of DNA that in some way regulates the expression of one or more specific genes. In some embodiments, such genes are close to or in the "neighborhood" of a given regulatory element. In some embodiments, such genes are located at a considerable distance from a given regulatory element. In some embodiments, a regulatory element impairs or enhances the transcription of one or more genes. In some embodiments, a regulatory element may be located in cis with respect to the gene being regulated. In some embodiments, a regulatory element may be located in trans with respect to the gene being regulated. For example, in some embodiments, a regulatory sequence refers to a nucleic acid sequence that regulates the expression of a gene product operably linked to the regulatory sequence. In some such embodiments, this sequence may be an enhancer sequence and other regulatory elements that regulate the expression of the gene product.

[0050] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism (e.g., a virus), a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some 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, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, flush or lavage (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other flush or lavage). In some embodiments, the biological sample is or includes 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 is clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). For example, filtration through a semipermeable membrane.Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting the primary sample to one or more techniques, such as amplification or reverse transcription of nucleic acids, isolation and / or purification of specific components.

[0051] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, in some embodiments, a prenatal human form, etc.). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a human having one or more characteristics characteristic of susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or who has been administered a diagnosis and / or therapy.

[0052] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the entire or nearly entire extent or degree of a desired characteristic or property. Those skilled in the art will understand that biological and chemical phenomena rarely, if ever, proceed to and / or completion 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.

[0053] Treatment: As used herein, the term "treatment" (also referred to as "treat" or "treating") refers to any administration of a therapy that partially or completely relieves, improves, eliminates, reverses, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of subjects who do not show signs of the associated disease, disorder, and / or condition and / or who show only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment of subjects who show established signs of one or more of the associated diseases, disorders, and / or conditions. In some embodiments, the treatment may be of subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, the treatment may be of subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing a given disease, disorder, and / or condition.

[0054] Variant: As used herein, the term "variant" refers to something that is different in some way from another version, e.g., a type of gene sequence. To determine if it is a variant, typically a reference type is selected and the variant is different from that reference type. In some embodiments, the variant can have the same or different (e.g., increased or decreased) level of activity or function as the wild-type sequence. For example, in some embodiments, the variant can have improved function compared to the wild-type sequence, e.g., if it is codon-optimized, e.g., to resist degradation by an inhibitory nucleic acid, e.g., miRNA. Such variants are referred to herein as gain-of-function variants. In some embodiments, the variant has a reduced or lost activity or functionality, or a change in activity that has a negative consequence (e.g., increased electrical activity that results in chronic depolarization leading to cell death). Such variants are referred to herein as loss-of-function variants. For example, in some embodiments, the CLRN1 gene sequence is a wild-type sequence that encodes a functional protein and is present in the majority of members of a species that has a genome that includes the CLRN1 gene. In some such embodiments, a gain-of-function variant can be a gene sequence of CLRN1 that contains one or more nucleotide differences compared to the wild-type CLRN1 gene sequence. In some embodiments, a gain-of-function variant is a codon-optimized sequence that encodes a transcript or polypeptide that can have improved properties (e.g., less susceptible to degradation, e.g., less susceptible to miRNA-mediated degradation) than its 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, a mutation in the CLRN1 sequence results in a non-functional or otherwise defective clarin1 protein. [Brief description of the drawings]

[0055] [Figure 1]A shows a simplified endogenous AAV genome. B shows a simplified recombinant AAV (rAAV) construct capable of expressing the CLRN1 gene. [Diagram 2] An exemplary rAAV construct containing the CLRN1 gene is shown. [Diagram 3] An exemplary rAAV construct containing the CLRN1 gene is shown. [Figure 4] An exemplary rAAV construct containing the CLRN1 gene is shown. [Diagram 5] An exemplary rAAV construct containing the CLRN1 gene is shown. [Figure 6] An exemplary rAAV construct containing the CLRN1 gene is shown. [Figure 7] An exemplary rAAV construct containing the CLRN1 gene is shown. [Figure 8] 1 shows an exemplary schematic diagram of the retinal structure of the eye. [Figure 9] 1 is a schematic diagram of representative anatomy of the human ear. [Figure 10] Schematic diagram of the inner ear showing the continuity of perilymphatic fluid between the vestibular system on the left side and the cochlea (scala tympani, scala vestibuli) on the right side. A is a schematic diagram of a coiled cochlea. The number of cochlear turns shown is representative of the mouse inner ear. B is a schematic diagram showing a cross section of the cochlea. In the schematic, the scala tympani and scala vestibuli are filled with perilymph, and the scala media is filled with endolymph (Talaei 2019, incorporated herein by reference in its entirety). [Figure 11] Schematic diagram of the administration method described herein. A includes an image of a delivery device described herein (see, e.g., WO2021 / 242926, incorporated herein by reference in its entirety). The illustrated delivery device includes a stopper to guide the insertion depth and is intended for intracochlear administration of fluid injected through the window window membrane. B includes an image showing the expected flow of fluid injected from the scala tympani to the scala vestibuli (via a communication at the cochlear foramen at the cochlear apex) and then exiting the cochlea through a stoma placed in the base of the stapes of the delivery device in the window vestibuli (Talei 2019, incorporated herein by reference in its entirety). [Figure 12] Representative fluorescent images showing in vivo cochlear transduction of naturally occurring AAV serotypes and AAV Anc80 variants in neonatal mice via round window membrane delivery are included. Mice (P1) were injected with various AAV capsids (AAV1, AAV2, AAV8, AAV6 [not shown], and AAV Anc80) containing a construct encoding enhanced GFP (eGFP). Actin was labeled using phalloidin. Quantification of eGFP-positive inner hair cells (IHCs) and outer hair cells (OHCs) showed approximately 90-100% transduction efficiency from base to apex following delivery of rAAV Anc80 containing a construct encoding enhanced GFP (rAAV Anc80-eGFP) (Landegger 2017, incorporated herein by reference in its entirety). [Figure 13] Representative fluorescent images showing in-vivo vestibular transduction of rAAV Anc80 particles in neonatal mice via round window membrane delivery are included. Mice (P1) were injected with AAV Anc80-eGFP. Phalloidin staining was used to label actin. Transduction was observed in both type I and type II hair cells of the utricle in A, and in cells of the semicircular canal ridge in B (Landegger 2017, incorporated herein by reference in its entirety). [Figure 14]Representative fluorescent images showing in-vivo cochlear transduction of rAAV Anc80 particles in adult mice via posterior semicircular canal delivery. Mice (7 weeks old) were injected with rAAV Anc80-eGFP particles. A includes low-magnification images of injected medial modiolar sections showing eGFP signal in IHCs (I), OHCs (O), the spiral limbus (SL), Reissner's membrane (RM), and the spiral ganglion (SG). B1 includes high-magnification views of the organ of Corti from the apical region of the cochlea. B2 includes high-magnification views of the organ of Corti from the central region of the cochlea. Quantification of eGFP-positive cells showed that approximately 100% of IHCs were transduced, whereas OHC transduction decreased from apex to base. C, Low magnification view showing that eGFP signal was detected in a subset of cells (neurons and satellite glial cells) within the spiral ganglion (Suzuki 2017, incorporated herein by reference in its entirety). [Figure 15] Representative fluorescent images showing in-vivo vestibular transduction of rAAV Anc80-eGFP in adult mice via posterior semicircular canal delivery. Mice (7 weeks old) were injected with rAAV Anc80-eGFP. A1 and A2 include low-magnification views of sections through the vestibule showing eGFP signal in both the utricle and saccule. B includes high-magnification views of sections through the vestibular end organ (utricle) showing eGFP expression in supporting cells and hair cells. C includes high-magnification views of sections through the vestibular end organ (crista ampullaris) showing eGFP expression in supporting cells and hair cells. Open arrows indicate examples of transduced supporting cells (hair cells not shown) (Suzuki 2017, incorporated herein by reference in its entirety). [Figure 16]Representative fluorescence images showing in-vivo cochlear and vestibular transduction of naturally occurring AAV2 serotypes via round window membrane delivery by canal fenestration compared to rAAV Anc80 variants in adult mice. Mice (4 weeks old) were injected with different AAV particles (AAV2 and rAAV Anc80 shown here, AAV1, AAV8, and AAV9 not shown) encoding eGFP. Compared to AAV2, rAAV Anc80-mediated transduction showed comparable rates of IHC and OHC transduction (A1 vs. A2), but more extensive transduction in spiral ganglion cells (B1 vs. B2) and hair cells of the saccule (C1 vs. C2: whole mount, D1 vs. D2: sections) (Omichi 2020, incorporated herein by reference in its entirety). [Figure 17]Figure 1 includes a Western blot showing HEK cell expression of CLRN1 protein with transduction of an exemplary rAAV-CLRN1 construct described herein. Lanes are shown along the top of the figure, and predicted protein sizes are shown on the left side of the figure. Lanes marked with "+" indicate PNGaseF treatment. Lane 1: prestained PageRuler™ protein ladder. Lane 2: non-transfected / negative control. Lane 3: non-transfected / negative control (+). Lane 4: transduction with rAAV-CLRN1 wild type (wt) particles at a multiplicity of infection (MOI) of 6.7x104. Lane 5: transduction with rAAV-CLRN1wt particles at a multiplicity of infection (MOI) of 6.7x104 (+). Lane 6: transduction with rAAV Anc80-CLRN1wt particles at a multiplicity of infection (MOI) of 1.3x105. Lane 7: transduction with rAAV Anc80-CLRN1wt particles (+) at a multiplicity of infection (MOI) of 1.3x105. Lane 8: transduction with rAAV Anc80-CLRN1wt particles (+) at a multiplicity of infection (MOI) of 2.3x105. Lane 9: transduction with rAAV Anc80-CLRN1wt particles (+) at a multiplicity of infection (MOI) of 2.3x105. Lane 10: transduction with rAAV Anc80-CLRN1-codon modified particles (+) at a multiplicity of infection (MOI) of 2.3x105. Lane 11: transduction with rAAV Anc80-CLRN1-codon modified particles (+) at a multiplicity of infection (MOI) of 2.3x105. Bands also indicate whether CLRN1 "iso1" or isoform A is glycosylated or deglycosylated. [Figure 18] A is a graphical representation of RNA expression in cochlear explants of wild-type (WT) newborn (P2) mice after transduction of the cochlear explants with rAAV Anc80 particles (rAAV Anc80-CLRN1) containing the clarin1 protein disclosed herein. RNA expression analysis is shown, demonstrating expression of mRNA encoding CLRN1 in cells of explants that received rAAV Anc80-CLRN1 particles (construct according to SEQ ID NO: 64). No expression was detected in explants that received vehicle ("mock"). Results show average expression. B shows a fluorescent image showing CLRN1 protein-expressing WT newborn mice. [Figure 19] 1 shows a perspective view of a device for delivering fluid to the inner ear according to an aspect of the present disclosure. [Figure 20] FIG. 1 illustrates a side view of a bent needle subassembly according to an aspect of the present disclosure. [Figure 21] 1 shows a perspective view of a device for delivering fluid to the inner ear according to an aspect of the present disclosure. [Figure 22] 1 illustrates a perspective view of a bent needle subassembly coupled to a distal end of a device according to an aspect of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Hearing loss In general, the ear can be described as including the outer ear, middle ear, inner ear, auditory nerve (auditory nerve), and auditory system (which processes sound as it travels from the ear to the brain). The ear not only senses 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.

[0057] Hearing loss may be the result of genetic factors, environmental factors, or a combination of genetic and environmental factors. Approximately half of people with tinnitus - auditory hallucinations in the auditory system (ringing, buzzing, whining, humming, or beating sounds) have hypersensitivity or reduced tolerance to certain sound frequencies and volume ranges. This is also known as hyperacusis (also spelled hyperacousis). A variety of non-syndromic and syndromic hearing losses are known to those skilled in the art (e.g., Usher syndrome, DFNB4, and Pendred syndrome, respectively). Environmental causes of hearing impairment or loss may include, for example, certain medications, certain prenatal or postnatal infections, and / or exposure to loud noise over time. In some embodiments, hearing loss may be due to noise, ototoxic agents, presbycusis, disease, infection, or cancer affecting certain parts of the ear. In some embodiments, ischemic damage may cause hearing loss via pathophysiological mechanisms. In some embodiments, intrinsic abnormalities, such as congenital mutations to genes that play important roles in the anatomy or physiology of the cochlea, or genetic or anatomical changes in supporting cells and / or hair cells, may cause or contribute to the hearing loss.

[0058] Hearing loss and / or profound hearing loss is one of the most common human sensory disorders and can occur for many reasons. In some embodiments, subjects may be born with or without hearing loss, while others may slowly lose hearing over time. Approximately 36 million American adults have some degree of hearing loss, with one in three people over the age of 60 and half of people over the age of 85 experiencing hearing loss. Approximately 1.5 out of every 1,000 children are born with extreme hearing loss, and another 2-3 out of every 1,000 children are born with some hearing loss (Smith et al., 2005, Lancet 365:879-890, incorporated herein by reference in its entirety). More than half of these cases have a genetic basis (Di Domenico, et al., 2011, J. Cell. Physiol. 226:2494-2499, incorporated herein by reference in its entirety).

[0059] Treatment of hearing loss currently consists of hearing amplification for mild to severe hearing loss and cochlear implants for severe to extreme hearing loss (Kral and O'Donoghue, 2010, N. Engl. J. Med. 363:1438-1450, incorporated herein by reference in its entirety). Recent research in this field has focused on regenerating cochlear hair cells, which can be applied to the most common forms of hearing loss, such as presbycusis, noise damage, infection, and ototoxicity. There remains a need for effective treatments, such as gene therapies, that can repair and / or alleviate the causes of hearing problems (see, e.g., WO2018 / 039375, WO2019 / 165292, and WO2020 / 097372, each of which is incorporated herein by reference in its entirety).

[0060] In some embodiments, non-syndromic hearing loss and / or severe hearing loss is not associated with other signs and symptoms. In some embodiments, syndromic hearing loss and / or severe hearing loss occurs in association with abnormalities in other parts of the body. Approximately 70%-80% of cases of hereditary hearing loss and / or severe hearing loss are non-syndromic, and the remaining cases are often caused by specific genetic syndromes. Non-syndromic severe hearing loss and / or deafness can have various inheritance patterns and can occur at any age. Types of non-syndromic severe hearing loss and / or deafness are generally named according to the inheritance pattern. For example, the autosomal dominant form is called DFNA, the autosomal recessive form is called DFNB, and the X-linked form is called DFN. Each type is also numbered in the order in which it was first described. For example, DFNA1 was the first autosomal dominant type of non-syndromic severe hearing loss described. 75%-80% of genetically caused deafness and / or profound hearing loss cases are inherited in an autosomal recessive pattern, meaning that both copies of the gene in each cell have a mutation (e.g., Usher syndrome). Usually, each parent of an individual with autosomal recessive deafness and / or profound hearing loss is a carrier of one copy of the mutated gene, but is not affected by this form of hearing loss. Another 20%-25% of nonsyndromic deafness and / or profound hearing loss cases are autosomal dominant, meaning that one copy of the altered gene in each cell is sufficient to result in profound deafness and / or hearing loss. Humans with autosomal dominant profound deafness and / or hearing loss most often inherit an altered copy of the gene from a parent with profound deafness and / or hearing loss. 1-2% of profound deafness and / or hearing loss cases show an X-linked inheritance pattern. This means that the mutated gene causing the condition is located on the X chromosome (one of the two sex chromosomes). Men with X-linked nonsyndromic and / or profound hearing loss tend to develop more severe hearing loss earlier in life than women who inherit a copy of the same gene mutation. A hallmark of X-linked inheritance is the inability of fathers to pass on the X-linked trait to their sons. Mitochondrial nonsyndromic profound hearing loss, caused by changes in mitochondrial DNA, occurs in less than 1% of cases in the United States.The altered mitochondrial DNA is passed from the mother to all sons and daughters. This type of profound hearing loss is not inherited from the father. The causes of syndromic and non-syndromic profound hearing loss and / or deafness are complex. Researchers have identified over 30 genes that, when altered, are associated with syndromic and / or non-syndromic profound hearing loss and / or deafness. However, some of these genes have not been fully characterized. Different mutations in the same gene may be associated with various types of profound hearing loss and / or deafness, and some genes are associated with both syndromic and non-syndromic profound hearing loss and / or deafness.

[0061] In some embodiments, the profound 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 profound hearing loss and / or hearing loss is associated with permanent hearing loss (profound sensorineural hearing loss) caused by damage to structures in the inner ear. In some embodiments, sensorineural hearing loss can be due to poor hair cell function. In some embodiments, sensorineural hearing impairment involves the eighth cranial nerve (vestibulocochlear nerve) or the auditory parts of the brain. In some such embodiments, only the auditory center of the brain is affected. In such situations, cortical profound hearing loss can occur, where sounds can be heard at normal thresholds, but the perceived quality of the sound is very poor and speech cannot be understood. Hearing loss due to changes in the middle ear is called conductive hearing loss. Some forms of non-syndromic profound hearing loss and / or hearing loss involve changes in both the inner and middle ear, called mixed hearing loss. Hearing loss and / or profound hearing loss that is present before a child acquires speech can be classified as prelingual or congenital. Hearing loss and / or profound hearing loss that occurs after speech development can be classified as postlingual. Most autosomal recessive loci associated with syndromic or nonsyndromic hearing loss cause prelingual severe-to-extreme hearing loss.

[0062] As known to those skilled in the art, hair cells are sensory receptors for both the auditory and vestibular systems of the vertebrate ear. Hair cells sense movement in the environment, and in mammals, hair cells are located in the organ of Corti within the cochlea of ​​the ear. It is known that there are two types of hair cells in the mammalian ear: 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 body. Inner hair cells convert vibrations of the cochlear fluid into electrical signals that the auditory nerve transmits to the brain. In some embodiments, hair cells can be abnormal at birth or damaged during an individual's life. In some embodiments, outer hair cells can regenerate. In some embodiments, inner hair cells cannot regenerate after disease or injury. In some embodiments, sensorineural hearing loss is due to abnormalities in hair cells.

[0063] As known to those skilled in the art, hair cells do not develop alone, but their functions are supported by a wide variety of cells that can be collectively referred to as supporting cells. Supporting cells perform numerous functions and include multiple cell types, including, but not limited to, Hensen's cells, Deiters' cells, pillar cells, Clausis cells, inner phalangeal cells, and border cells. In some embodiments, sensorineural hearing loss is due to abnormalities in supporting cells. In some embodiments, supporting cells may be abnormal at birth or damaged during an individual's life. In some embodiments, supporting cells can be regenerated. In some embodiments, certain supporting cells may not be able to regenerate.

[0064] decreased visual acuity The eye is an organ of the visual system that provides vision to animals, receives and processes visual details, and enables several light-responsive functions unrelated to vision. In general, the eye detects light and converts it into electrochemical impulses in neurons. In higher organisms, such as mammals, the eye is a complex optical system that collects light from the surrounding environment, regulates its intensity through a diaphragm, forms an image through an adjustable assembly of lenses, converts this image into a series of electrical signals, and transmits these signals to the brain through complex neural pathways that connect the eye to the visual cortex and other areas of the brain via the optic nerve. The anatomical structures of the eye generally include the cornea, pupil, iris, lens, anterior chamber, posterior chamber, tear, corneal limbus, ciliary muscle, suspensory ligament, vitreous chamber, sclera, choroid, retina, macula / fovea, optic nerve, and blind spot.

[0065] Vision loss can be the result of genetic factors, environmental factors, or a combination of genetic and environmental factors. Vision loss is any decrease in the ability to see, such as blurred vision, clouding, double vision, blind spots, poor night vision, and poor peripheral vision (tunnel vision). Vision loss can affect one or both eyes, can occur gradually or suddenly, and can be partial or complete. Environmental causes of vision impairment or loss can include, for example, exposure to certain medications, certain prenatal or postnatal infections, and / or physical contact of objects with the eye. In some embodiments, intrinsic abnormalities, such as congenital mutations to genes that play important roles in the anatomy or physiology of the eye, or genetic or anatomical changes in supporting cells and / or ocular cells, can cause or contribute to hearing loss.

[0066] Vision loss is one of the sensory disorders of humans, and can occur for many reasons.In some embodiments, subjects can be born with or without vision loss, while others can slowly lose vision over time.Approximately 26.9 million American adults are reported to have some degree of vision loss, and 7.8 million American adults over the age of 65 are reported to experience significant vision loss.

[0067] Retinitis pigmentosa is a genetic disorder that causes vision loss due to deterioration of retinal cells (e.g., photoreceptor cells). Usually, the rod cells of the retina are affected first, leading to early night blindness (nyctalopia) and a gradual decrease in peripheral vision. In other cases, early degeneration of the cone cells in the macula occurs, leading to a decrease in central vision. In some cases, foveal vision is impaired, leading to "doughnut vision." Central and peripheral vision are intact, but there is a halo around the central area where vision is impaired.

[0068] Symptoms of retinitis pigmentosa include difficulty seeing at night and reduced peripheral (side) vision. As peripheral vision deteriorates, a person may experience "tunnel vision." Complete blindness is possible, but uncommon. The onset of symptoms is generally slow, often beginning in childhood. Retinitis is generally inherited from a person's parents. Mutations in over 50 genes have been implicated. The underlying mechanism involves a progressive loss of rod photoreceptor cells at the back of the eye. This is typically followed by loss of cone photoreceptor cells. Diagnosis is made by examination of the retina, which looks for dark pigmentation. Other supporting tests may include electroretinograms, visual field tests, or genetic testing (see, e.g., "Facts About Retinitis Pigmentosa." National Eye Institute. May 2014, retrieved April 18, 2020, the contents of which are incorporated herein in their entirety).

[0069] Currently, there is no cure for retinitis pigmentosa. Efforts to address the problem may include the use of low vision aids, portable lighting, or orientation and mobility training. Vitamin A palmitate supplements may be useful in slowing deterioration. Artificial visual systems may be an option for certain people with severe disease. Retinitis pigmentosa is estimated to affect 1 in 4,000 people (see, e.g., "Facts About Retinitis Pigmentosa," National Eye Institute. May 2014, retrieved April 18, 2020, the contents of which are incorporated herein in their entirety, and Openshaw, Amanda (Feb 2008), "Understanding Retinitis Pigmentosa," University of Michigan Kellogg Eye Center. Archived from the original on August 29, 2017, the contents of which are incorporated herein by reference in their entirety).

[0070] As known to those skilled in the art, the eye is composed of various cell types. The cells in the optic nerve that connects the eye to the brain are the cells that transmit electrical signals to the brain, allowing the brain to interpret the signals as images. Retinal cells (e.g., photoreceptor cells) are located at the back of the eye and convert light into electrical signals. Other cells include rod cells and cone cells that allow humans to perceive color and shape. Expression of CLRN1 in the eye has been identified in the following exemplary layers and / or cells of the eye: [Table 2]

[0071] Usher Syndrome Usher syndrome is a condition that affects both hearing and vision. Usher syndrome can also affect balance. Some of the main symptoms of Usher syndrome are profound hearing loss or deafness, and eye disorders such as retinitis pigmentosa.

[0072] The severe hearing loss or deafness of Usher syndrome can be caused by abnormal development of hair cells (sound-receptor cells) in the inner ear. Children with Usher syndrome are typically born with moderate to severe hearing loss, depending on the type of Usher syndrome. Less commonly, hearing loss from Usher syndrome appears during or after puberty. Usher syndrome can cause severe balance problems due to abnormal development of vestibular hair cells, the sensory cells that detect gravity and head movement.

[0073] Retinitis pigmentosa initially causes night blindness and loss of peripheral vision due to progressive degeneration of cells in the retina, the light-sensitive tissue at the back of the eye that is important for vision. As retinitis pigmentosa progresses, the field of vision narrows until only central vision remains, a condition called tunnel vision. Cysts in the macula and cataracts (clouding of the lens) can cause early loss of central vision in people with Usher syndrome.

[0074] Usher syndrome affects approximately 4-17 per 100,000 people and accounts for approximately 50% of all inherited cases of profound deafness-blindness. The condition is believed to account for 3-6% of all profoundly hearing-impaired children and another 3-6% of deaf children (see, e.g., Boughman, JA, et al. (1983), "Usher syndrome: definition and estimate of prevalence from two high-risk populations", Journal of Chronic Diseases, 36(8), 595-603; Kimberling, W., et al. (2010), "Frequency of Usher syndrome in two pediatric populations: implications for genetic screening of deaf and hard of hearing children", Genetics in Medicine, 12(8), 512-516; Berson, EL (1998), "Treatment of retinitis pigmentosa with vitamin A", Digital Journal of Ophthalmology, 4(7), the disclosures of which are incorporated herein by reference in their entireties).

[0075] Usher syndrome is genetic, which means it is passed from parent to child through genes. Humans inherit two copies of the gene, one from each parent. Genes sometimes become altered or mutated. Mutated genes can cause cells to develop or act abnormally.

[0076] Usher syndrome is inherited as an autosomal recessive disorder. "Autosomal" means that men and women are equally likely to have the disorder and to pass it on to children of either sex. "Recessive" means that the condition can only occur if a child inherits two copies of the same defective gene, one from each parent. People with one abnormal Usher gene do not have the disorder, but have a 50% chance of being carriers, passing the abnormal gene to each of their children.

[0077] Diagnosis of Usher syndrome includes appropriate questions about the patient's medical history, as well as tests of hearing, balance, and vision. Early diagnosis is important to improve treatment success. An eye specialist can use a dilating medication to examine the retina for signs of retinitis pigmentosa. A visual field test measures peripheral vision and may aid in the diagnosis of Usher syndrome. An electroretinogram measures the electrical response of the eye's light-sensitive cells in the retina. Optical coherence tomography may be useful in determining macular cystic changes. A videonystagmus test measures involuntary eye movements that may indicate balance problems. A hearing test measures hearing sensitivity in a range of frequencies.

[0078] There are three types of Usher syndrome: Type I, Type II, and Type III. People with Usher syndrome Type III are not born profoundly deaf, but experience progressive hearing loss, and nearly half have balance problems. For example, children with Usher syndrome Type III have normal hearing and normal to near-normal balance at birth, which may decline with age. Hearing and vision loss vary. Children with Usher syndrome Type III often develop hearing loss by adolescence and require hearing aids by mid-to-late adulthood. Night blindness also usually begins during adolescence. Blind spots appear in the late teens to early twenties. Legal blindness also occurs by middle age.

[0079] Genetic testing can aid in the diagnosis of Usher syndrome. For example, mutations in the CLRN1 gene have been associated with Usher syndrome type III. CLRN1 encodes clarin1, a protein important in the development and maintenance of the inner ear and retina. However, the function of this protein and how mutations in clarin1 cause hearing loss and vision loss are poorly understood.

[0080] Currently, there is no cure for Usher Syndrome. Treatment includes addressing hearing, vision, and balance problems. Early diagnosis helps tailor educational programs to account for the severity of hearing and vision loss, as well as the child's age and abilities. Therapeutic and communication services may include hearing aids, assistive listening devices, cochlear implants, auditory (hearing) training, and / or learning American Sign Language. Independent living training may include gait training for balance span problems, Braille instruction, and low vision services.

[0081] Vitamin A may slow the progression of retinitis pigmentosa, according to results from a long-term clinical trial supported by the National Eye Institute and the Foundation Fighting Blindness. Based on this study, adults with a common form of retinitis pigmentosa may benefit from a daily supplement of 15,000 IU (international units) of vitamin A in the palmitic acid form.

[0082] CLRN1 The CLRN1 gene encodes "clarin 1" (CLRN1), a protein expressed in hair cells of the inner ear (eg, inner ear hair cells, outer ear hair cells) and in the retina.

[0083] For example, the CLRN1 gene encodes a protein that contains a cytosolic N-terminus, a multihelical transmembrane domain, and the endoplasmic reticulum membrane retention signal TKGH at the C-terminus. CLRN1 is thought to be necessary for hair cell and / or eye cell function and associated with neural activation (see, e.g., Geng et al., "Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation," Hum Mol Genet. 2009 Aug 1;18(15):2748-60. doi:10.1093 / hmg / ddp210. Epub 2009 May 3, the contents of which are incorporated herein by reference in their entirety; see also, e.g., Dinculescu et al., "AAV-mediated Clarin-1 expression in the mouse retina: implications for USH3A gene therapy," PLoS One. 2016;11(2):e0148874, published online February 16, 2016, the contents of which are incorporated herein by reference in their entirety).

[0084] The human CLRN1 gene is located on chromosome 3q25.1. It contains at least eight exons (referred to as exons 0, 0b, 1, 1b, 2, 2b, 3a, and 3b) and comprises approximately 47 kilobases (kb) (see, e.g., Vastinsalo et al. (2011) Eur J Hum Genet 19(1):30-35, incorporated herein by reference in its entirety; NCBI accession number NG_009168.1, incorporated herein by reference in its entirety). Multiple transcript variants encoding different isoforms have been identified for this gene (see, e.g., NCBI Gene ID:7401, incorporated herein by reference in its entirety).

[0085] Various mutations in the CLRN1 gene have been associated with Usher syndrome type III (e.g., Usher syndrome type IIIA (MIM number 606397; see Fields et al. (2002) Am J Hum Genet 71:607-617, which is incorporated by reference in its entirety; and Joensuu et al. (2001) Am J Hum Genet 69:673-684, which is incorporated by reference in its entirety)) and retinitis pigmentosa (see, e.g., Khan et al. (2011) Ophthalmology 118:1444-1448, which is incorporated by reference in its entirety). Mutations causing Usher syndrome type III are found primarily within exon 3 of CLRN1. Usher syndrome type III severe hearing loss can be modeled by generating CLRN1-deficient mice (see, e.g., Geng et al. (2017) Sci Rep 7(1):13480, which is incorporated by reference in its entirety. Exemplary CLRN1 mutations associated with Usher syndrome type III include T528G, M120K, M44K, N48K, and C40G.

[0086] Exemplary CLRN1 mutations associated with retinitis pigmentosa include L154W and P31L (see, e.g., Khan et al. (2011) Ophthalmology 118:1444-1448, which is incorporated by reference in its entirety).

[0087] Further exemplary mutations in the CLRN1 gene detected in subjects with hearing loss and methods for sequencing the nucleic acid encoding CLRN1 are described, for example, in Fields et al. (2002) Am J Hum Genet 71:607-617, Joensuu et al. (2001) Am J Hum Genet 69:673-684, Adato et al. (2002) Europ J Hum Genet 10:339-350, Aller et al. (2004), Clin Genet 66:525-529, 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, sequencing, Southern blotting, and Northern blotting.

[0088] Clarin1 is found in several areas of the body, including sensory cells in the inner ear called hair cells. These cells help transmit sound and movement signals to the brain. The protein is also active in the retina, the light-sensing tissue that lines the back of the eye. The function of Clarin1 is unclear, but studies suggest that Clarin1 is involved in communication between nerve cells (neurons) in the inner ear and retina. Clarin1 may be important in the development and function of synapses, the junctions between neurons where cell-to-cell communication occurs. Other names for the CLRN1 gene include USH3, USH3A, USH3A_Human, Usher Syndrome 3A, and Usher Syndrome Type 3 protein.

[0089] Mutations in CLRN1 have been associated with hearing loss and profound hearing loss (see, e.g., 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 CLRN1 gene alter the structure or function of clarin1, disrupting its intrinsic function. Several mutations in CLRN1 associated with hearing loss have been reported. For example, point mutations A123D, N48K, Y176X, and L54P have been reported in USH3 patients (see, e.g., Isosomppi et al., "Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane," Molecular vision 15(191-121):1806-18, published September 2009, 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.

[0090] Mutations in the CLRN1 gene and the encoded clarin 1 protein are associated with Usher syndrome type III. Usher syndrome type III is an autosomal recessive disorder characterized by progressive sensorineural hearing loss, vestibular dysfunction, and retinitis pigmentosa. Usher syndrome type III (USH3 [MIM#276902]) is unique among clinical subtypes of Usher syndrome, showing progressive hearing loss after language acquisition, and late onset of retinitis pigmentosa (RP), and progressive loss of vestibular function (see, e.g., Kimberling WJ, Orten D, Pieke-Dahl S (2000) Genetic heterogeneity of Usher syndrome. Adv Otorhinolaryngol 56:11-18, the contents of which are incorporated herein by reference in their entirety). The disease locus was originally mapped to chromosome 3q25, a region of approximately 700 kb between markers WI-17533 and 486D12SP6 (see, e.g., Joensuu et al. 1996, incorporated herein by reference in its entirety). In a recent publication (see, e.g., Joensuu T, Blanco G, Pakarinen L, Sistonen P, Kaariainen H, Brown S, Chapelle A, Sankila EM (1996) Refined mapping of the Usher syndrome type III locus on chromosome 3, exclusion of candidate genes, and identification of the putative mouse homologous region. Genomics 38:255-263, the contents of which are incorporated herein by reference in their entirety), the USH3 locus was assigned to a 250 kb region between 107G19CA7 and D3S3625 by haplotype and linkage disequilibrium analysis in Finnish carriers of a putative founder mutation.

[0091] Wild-type CLRN1 is a glycoprotein that is localized to the plasma membrane of transfected BHK-21 cells. Mutant CLRN1 proteins are mislocalized. It has been suggested that part of the pathogenesis of USH3 may be related to defects in intracellular trafficking and reduced stability of mutant CLRN1 proteins (see, e.g., Isosomppi et al., "Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane," Molecular vision 15(191-121):1806-18, published September 2009, which is incorporated herein by reference in its entirety).

[0092] CLRN1 polynucleotides Among other things, the disclosure provides polynucleotides, such as polynucleotides that include the CLRN1 gene or characteristic portions thereof, as well as compositions that include such polynucleotides, and methods that utilize such polynucleotides and / or compositions.

[0093] In some embodiments, the polynucleotide comprising the CLRN1 gene or a characteristic portion thereof may be DNA or RNA. In some embodiments, the DNA may be genomic DNA or cDNA. In some embodiments, the RNA may be mRNA. In some embodiments, the polynucleotide comprises exons and / or introns of the CLRN1 gene.

[0094] In some embodiments, a gene product is expressed from a polynucleotide comprising the CLRN1 gene or a characteristic portion thereof. In some embodiments, expression of such a polynucleotide can utilize one or more control 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 control elements.

[0095] In some embodiments, the CLRN1 gene is a mammalian CLRN1 gene. In some embodiments, the CLRN1 gene is a mouse CLRN1 gene. In some embodiments, the CLRN1 gene is a primate CLRN1 gene. In some embodiments, the CLRN1 gene is a human CLRN1 gene. In some embodiments, the CLRN1 gene is a human CLRN1 isoform. Human CLRN1 isoform variants are described, for example, in Vastinsalo et al. (2011) Eur J Hum Genet 19(1):30-35, which is incorporated herein by reference in its entirety, and have NCBI accession number NG_009168.1, which is incorporated herein by reference in its entirety. Exemplary human CLRN1 cDNA sequences are or include the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. Exemplary human CLRN1 genomic DNA sequences are found within SEQ ID NO:5. Exemplary human CLRN1 cDNA sequences, including untranslated regions, are or include SEQ ID NO:6, 7, 8, or 9.

[0096] Exemplary human CLRN1 cDNA coding sequence (isoform A) (SEQ ID NO:1) ATGCCAAGCCAACAGAAGAAAATCATTTTTTGCATGGCCGGAGTGTTCAGTTTTGCATGTGCCCTCGGAGTTGTGACAGCCTTGGGGACACCGTTGTGGATCAAAGCCACTGTCCTCTGCAAAACGGGAGCTCTGCTCGTCAATGCCTCAGGGCAGGAGCTGGACAAGTTTATGGGTGAAATGCAGTACGGGCTTTTCCACGGAGAGGGTGTGAGGCAGTGTGGGTTGGGAGCAAGGCCCTTTCGGTTCTCATTTTTTCCAGATTTGCTCAAAGCAATCCCAGTGAGCATCCACGTCAATGTCATTCTCTTCTCTGCCATCCTTATTGTGTTAACCATGGTGGGGACAGCCTTCTTCATGTACAATGCTTTTGGAAAACCTTTTGAAACTCTGCATGGTCCCCTAGGGCTGTACCTTTTGAGCTTCATTTCAGGCTCCTGTGGCTGTCTTGTCATGATATTGTTTGCCTCTGAAGTGAAAATCCATCACCTCTCAGAAAAAATTGCAAATTATAAAGAAGGGACTTATGTCTACAAAACGCAAAGTGAAAAATATACCACCTCATTCTGGGTCATTTTCTTTTGCTTTTTTGTTCATTTTCTGAATGGGCTCCTAATACGACTTGCTGGATTTCAGTTCCCTTTTGCAAAATCTAAAGACGCAGAAACAACTAATGTAGCTGCAGATCTAATGTAC

[0097] Exemplary human CLRN1 cDNA coding sequence (isoform C) (SEQ ID NO: 2) ATGCAGGCCCTGCAGCAGCAACCAGTTTTTCCAGATTTGCTCAAAGCAATCCCAGTGCATCCACGTCAATGTCATTCTCTTCTCTGCCATCCTTATTGTGTTAACCATGGTGGGGACAGCCTTCTTCATGTACAATGCTTTTGGAAAACCTTTTGAAACTCTGCATGGTCCCCTAGGGC TGTACCTTTTGAGCTTCATTTCAGGCTCCTGTGGCTGTCTTGTCATGATATTGTTTGCCTCTGAAGTGAAAATCCATCACCTCTCAGAAAAAATTGCAAATTATAAAGAAGGGACTTATGTCTACAAAACGCAAAGTGAAAAATATACCACCTCATTCTGGCTGACTAAAGGCCACAGCTGA

[0098] Exemplary human CLRN1 cDNA coding sequence (isoform D) (SEQ ID NO:3) ATGCCAAGCCAACAGAAGAAAATCATTTTTTGCATGGCCGGAGTGTTCAGTTTTGCATGTGCCCTCGGAGTTGTGACAGCCTTGGGGACACCGTTGTGGATCAAAGCCACTGTCCTCTGCAAAACGGGAGCTCTGCTCGTCAATGCCTCAGGGCAGGAGCTGGACAAGTTTATGGGTGAAATGCAGTACGGGCTTTTCCACGGAGAGGGTGTGAGGCAGTGTGGGTTGGGAGCAAGGCCCTTTCGGTTCTCATTTTTTCCAGATTTGCTCAAAGCAATCCCAGTGAGCATCCACGTCAATGTCATTCTCTTCTCTGCCATCCTTATTGTGTTAACCATGGTGGGGACAGCCTTCTTCATGTACAATGCTTTTGGAAAACCTTTTGAAACTCTGCATGGTCCCCTAGGGCTGTACCTTTTGAGCTTCATTTCAGTTGCCCTTTGGCTGCCAGCTACCAGGCACCAGGCTCAAGGCTCCTGTGGCTGTCTTGTCATGATATTGTTTGCCTCTGAAGTGAAAATCCATCACCTCTCAGAAAAAATTGCAAATTATAAAGAAGGGACTTATGTCTACAAAACGCAAAGTGAAAAATATACCACCTCATTCTGGGTCATTTTCTTTTGCTTTTTTGTTCATTTTCTGAATGGGCTCCTAATACGACTTGCTGGATTTCAGTTCCCTTTTGCAAAATCTAAAGACGCAGAAACAACTAATGTAGCTGCAGATCTAATGTACTGA

[0099] Exemplary human CLRN1 cDNA coding sequence (isoform E) (SEQ ID NO: 4) ATGCCAAGCCAACAGAAGAAAATCATTTTTTGCATGGCCGGAGTGTTCAGTTTTGCATGTGCCCTCGGAGTTGTGACAGCCTTGGGGACACCGTTGTGGATCAAAGCCACTGTCCTCTGCAAAACGGGAGCTCTGCTCGTCAATGCCTCAGGGCAGGAGCTGGACAAGTTTATGGGTGAAATGCAGTACGGGCTTTTCCACGGAGAGGGTGTGAGGCAGTGTGGGTTGGGAGCAAGGCCCTTTCGGTTCTCATGCTATTTTCTTGACCCCTTCATGGGACTCCCAACAGGGGTACCCCATTTACTCAGCCTGCCCTGCTCAACCTCTTGCAGGAGGGAGCACACGAGTGAACGAGTGCAGGAACCAGCTGGCTGCTTTAGTGCTGTGAGGAGTAAACTCCATGCAGGCCCTGCAGCAGCAACCAGTTTTTCCAGATTTGCTCAAAGCAATCCCAGTGAGCATCCACGTCAATGTCATTCTCTTCTCTGCCATCCTTATTGTGTTAACCATGGTGGGGACAGCCTTCTTCATGTACAATGCTTTTGGAAAACCTTTTGA

[0100] Exemplary human CLRN1 cDNA coding sequence (codon-optimized) (SEQ ID NO: 19) ATGCCTAGCCAGCAGAAGAAAATCATCTTCTGCATGGCCGGCGTGTTCAGCTTCGCCTGTGCTCTGGGAGTTGTGACAGCCCTGGGAACCCCTCTGTGGATCAAAGCCACAGTGCTGTGCAAGACAGGCGCCCTGCTGGTTAATGCCTCTGGCCAAGAGCTGGACAAGTTCATGGGCGAGATGCAGTACGGCCTGTTCCATGGCGAAGGCGTCAGACAGTGTGGCCTGGGAGCCAGACCTTTCAGATTCAGCTTCTTCCCAGACCTGCTGAAGGCTATCCCCGTGTCCATCCACGTGAACGTGATCCTGTTCAGCGCCATCCTGATCGTGCTGACAATGGTCGGAACCGCCTTCTTCATGTACAACGCCTTCGGCAAGCCCTTCGAGACACTGCATGGACCTCTGGGCCTGTACCTGCTGAGCTTTATCAGCGGCAGCTGTGGCTGCCTGGTCATGATTCTGTTCGCCAGCGAAGTGAAGATCCACCACCTGAGCGAGAAGATCGCCAACTACAAAGAGGGCACCTACGTCTACAAGACCCAGTCCGAGAAGTACACCACCAGCTTTTGGGTTATCTTCTTCTGTTTCTTCGTGCACTTCCTGAACGGCCTGCTGATCAGACTGGCCGGCTTCCAGTTTCCATTCGCCAAGAGCAAGGACGCCGAAACCACAAACGTGGCCGCCGATCTGATGTAC

[0101] Exemplary human CLRN1 genomic DNA sequence (SEQ ID NO: 5) TTTTTTTCCCCTGAATTACCCATCAAATTGATCTGCAGCTGACTAAAGGCCACAGCTGAGCCTGGAACTGACCCTTCCTTCATCCTCAACCTGCTGTCCTCCAGAAAGCACCAAGGAAAAAAGCAGAGAATGACAGCAAACAGATCACTAGGCCTCTGACCACAGGTGCTGAGTACTCAGCAGCCCTC ATATAATAGGTTTGAAAGTACTCCTTAAAATAAAACACTGTTTCCTTTGGAACTATTTACAAGGATGAAAACAACCGTATACCTGAGAAATAACTTGCTCTGGTGTCAATTCGCTATTCGCCAGCAGACATCAGAACACACCGAGTTTCCAGATGCTGGTTTTTCCCCTTAAATCAGGAAATACACC TGGACAATTTCTAGAAGACTACAATTCAGTCTAGCCACAAAGGGGATTTTTTTTTTTTGGTAACAGGCTAGAGCCCGGTTCTGTAAGTCTTTAGCTGAAATGGTCCAGTACAAAAGCACTGGAAATGAGTGGGCTAGGAGGACAAGGACCGTCTCCTGCGTGAGGAGTTGGTTGGAGGTCCCCAAGG CCAGGTACCCCCTGCACTCTTATTGGATTCCTCTCTGTCTTCTTGGAGTTTTGAAAAACTCCTTCGAACACCAGGCTTTTTTCTTTAGAAAACAAGTCTCCAATCGTTCTCTGTTCCGTAGAAAGAGAAAGAAAACCTGGAGCAGCTGCTGAAAAATCTAATGAGGAACTAAGAGGCAAACCCACCA

[0102] Exemplary human CLRN1 cDNA sequence (isoform A) including untranslated regions (SEQ ID NO:6)

[0103] Exemplary human CLRN1 cDNA sequence (isoform C) including untranslated regions (SEQ ID NO:7)

[0104] Exemplary human CLRN1 cDNA sequence (isoform D) including untranslated regions (SEQ ID NO:8)

[0105] An exemplary human CLRN1 cDNA sequence including untranslated regions (isoform E (SEQ ID NO: 9)

[0106] It is recognized in the present disclosure that certain changes to the polynucleotide sequence do not affect its expression or the protein encoded by the polynucleotide. In some embodiments, the polynucleotide comprises a CLRN1 gene with one or more silent mutations. In some embodiments, the present disclosure provides a polynucleotide comprising a CLRN1 gene with one or more silent mutations, e.g., a CLRN1 gene with a sequence different from SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, but encoding the same amino acid sequence as a functional CLRN1 gene. In some embodiments, the present disclosure provides a polynucleotide comprising a CLRN1 gene with a sequence different from SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 that encodes an amino acid sequence comprising one or more mutations (e.g., a different amino acid sequence when compared to that produced from a functional CLRN1 gene), where the one or more mutations are conservative amino acid substitutions. In some embodiments, the present disclosure provides a polynucleotide comprising a CLRN1 gene having a sequence different from SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 that encodes an amino acid sequence that includes one or more mutations (e.g., a different amino acid sequence when compared to that produced from a functional CLRN1 gene), where the one or more mutations are not within a characteristic portion of the CLRN1 gene or the encoded clarin1 protein. In some embodiments, a polynucleotide according to the present disclosure comprises a CLRN1 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, 5, 6, or 7. As can be understood in the art, SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 can be optimized (e.g., codon optimized) to achieve increased or optimized expression in an animal, e.g., a mammal, e.g., a human.

[0107] Polypeptides encoded by the CLRN1 gene In particular, the present disclosure provides a polypeptide encoded by the CLRN1 gene or a characteristic portion thereof. In some embodiments, the CLRN1 gene is a mammalian CLRN1 gene. In some embodiments, the CLRN1 gene is a mouse CLRN1 gene. In some embodiments, the CLRN1 gene is a primate CLRN1 gene. In some embodiments, the CLRN1 gene is a human CLRN1 gene.

[0108] In some embodiments, the polypeptide comprises a clarin 1 protein or a characteristic portion thereof. In some embodiments, the clarin 1 protein or a characteristic portion thereof is a mammalian clarin 1 protein or a characteristic portion thereof, such as a primate clarin 1 protein or a characteristic portion thereof. In some embodiments, the clarin 1 protein or a characteristic portion thereof is a human clarin 1 protein or a characteristic portion thereof.

[0109] In some embodiments, the polypeptides provided herein comprise post-translational modifications. In some embodiments, the clarin 1 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.

[0110] Exemplary human clarin 1 protein sequences are or include the sequences of SEQ ID NO: 10, 11, 12, or 13. Exemplary human clarin 1 protein sequences with a c-terminal flag tag are or include the sequences of SEQ ID NO: 14.

[0111] Exemplary human clarin 1 protein sequence (isoform A) (SEQ ID NO: 10) MPSQQKKIIIFCMAGVFSFACALGVVTALGTPLWIKATVLCKTGALLVNASGQELDKFMGEMQYGLFHGEGVRQCGLGARPFRFSFFPDLLKAIPVSIHVNVILFSAILIVLTMVGT AFFMYNAFGKPFETLHGPLGLYLLSFISGSCGCLVMILFASEVKIHHLSEKIANYKEGTYVYKTQSEKYTTSFWVIFFCFFVHFLNGLLIRLAGFQFPFAKSKDAETTNVAADLMY

[0112] Exemplary human clarin 1 protein sequence (isoform C) (SEQ ID NO:11) MQALQQQPVFPDLLKAIPVSIHVNVILFSAILIVLTMVGTAFFMYNAFGKPFETLHGPLGLYLLSFISGSCGCLVMILFASEVKIHHLSEKIANYKEGTYVYKTQSEKYTTSFWLTKGHS

[0113] Exemplary human clarin 1 protein sequence (isoform D) (SEQ ID NO: 12) MPSQQKKIIFCMAGVFSFACALGVVTALGTPLWIKATVLCKTGALLVNASGQELDKFMGEMQYGLFHGEGVRQCGLGARPFRFSFFPDLLKAIPVSIHVNVILFSAILIVLTMVGTAFFMYN AFGKPFETLHGPLGLYLLSFISVALWLPATRHQAQGSCGCLVMILFASEVKIHHLSEKIANYKEGTYVYKTQSEKYTTSFWVIFFCFFVHFLNGLLIRLAGFQFPFAKSKDAETTNVAADLMY

[0114] Exemplary human clarin 1 protein sequence (isoform E) (SEQ ID NO: 13) MPSQQKKIIFCMAGVFSFACALGVVTALGTPLWIKATVLCKTGALLVNASGQELDKFMGEMQYGLFHGEGVRQCGLGARPFRFSCYFLDPFMGLPTGVPHLLSLPCSTSCRREHTSERVQEPAGCFSAVRSKLHAGPAAATSFSRFAQSNPSEHPRQCHSLLCHPYCVNHGGDSLLHVQCFWKTF

[0115] Exemplary human clarin 1 protein sequence with C-terminal flag tag (isoform A) (SEQ ID NO: 14) MPSQQKKIIFCMAGVFSFACALGVVTALGTPLWIKATVLCKTGALLVNASGQELDKFMGEMQYGLFHGEGVRQCGLGARPFRFSFFPDLLKAIPVSIHVNVILFSAILIVLTMVGTAFFMYNAFGKPFE TLHGPLGLYLLSFISGSCGCLVMILFASEVKIHHLSEKIANYKEGTYVYKTQSEKYTTSFWVIFFCFFVHFLNGLLIRLAGFQFPFAKSKDAETTNVAADLMYGSRADYKDHDGDYKDHDIDYKDDDDK

[0116] The present disclosure recognizes that certain mutations in the amino acid sequence of a polypeptide (e.g., including clarin1 or a characteristic portion thereof) described herein do not affect the expression, folding, or activity of the polypeptide. In some embodiments, a polypeptide (e.g., including clarin1 or a characteristic portion thereof) comprises one or more mutations, wherein the one or more mutations are conservative amino acid substitutions. In some embodiments, a polypeptide according to the present disclosure comprises a clarin1 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:8, 9, 10, or 11. In some embodiments, a polypeptide according to the present disclosure comprises a clarin1 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:14. In some embodiments, a polypeptide according to the present disclosure comprises a clarin 1 protein that is identical to the sequence of SEQ ID NO:14, or a characteristic portion thereof.

[0117] Constructs 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, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus constructs) incorporating polynucleotides containing CLRN1 gene or characteristic portions thereof. Those skilled in the art can select suitable constructs and cells to generate any of the polynucleotides described herein. In some embodiments, the construct is a plasmid (i.e., a circular DNA molecule that can replicate autonomously inside a cell). In some embodiments, the construct can be a cosmid (e.g., pWE or sCos series).

[0118] In some embodiments, the construct is a viral construct. In some embodiments, the viral construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus construct. In some embodiments, the construct is an adeno-associated virus (AAV) construct (see, e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, incorporated herein by reference in its entirety). In some embodiments, the viral construct is an adenovirus construct. In some embodiments, the viral construct may also be based on or derived from an alphavirus. Alphaviruses include Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreatic disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Wataroa virus. Generally, the genomes of such viruses encode nonstructural proteins (e.g., replicons) and structural proteins (e.g., capsid and envelope) that can be translated in the cytoplasm of the host cell. Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to generate viral constructs for delivery of coding sequences. Pseudotyped viruses can be formed by combining the envelope glycoproteins of an alphavirus with the capsid of a retrovirus.Examples of alphavirus constructs can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819, the constructs and methods for making them being incorporated herein by reference in their entirety.

[0119] The constructs provided herein may be of different sizes. In some embodiments, the constructs are plasmids and may comprise 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 may 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.

[0120] In some embodiments, the construct is a viral construct and can have a total number of nucleotides of up to 10 kb. In some embodiments, the viral construct may be from about 1 kb to about 2 kb, 1 kb to about 3 kb, from about 1 kb to about 4 kb, from about 1 kb to about 5 kb, from about 1 kb to about 6 kb, from about 1 kb to about 7 kb, from about 1 kb to about 8 kb, from about 1 kb to about 9 kb, from about 1 kb to about 10 kb, from about 2 kb to about 3 kb, from about 2 kb to about 4 kb, from about 2 kb to about 5 kb, from about 2 kb to about 6 kb, from about 2 kb to about 7 kb, from about 2 kb to about 8 kb, from about 2 kb to about 9 kb, from about 2 kb to about 10 kb, from about 3 kb to about 4 kb, from about 3 kb to about 5 kb, from about 3 kb to about 6 kb, from about 3 kb to about 7 kb, from about 3 kb to about 8 kb, from about 3 kb to about 9 kb. kb, 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.

[0121] In some embodiments, the construct is a lentiviral construct and can have a total number of nucleotides of up to 8 kb. In some examples, the lentiviral construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb. b, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb.

[0122] In some embodiments, the construct is an adenoviral construct and can have a total number of nucleotides of up to 8 kb. In some embodiments, the adenoviral construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb. , 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.

[0123] Any of the constructs described herein may further comprise a control sequence selected from the group of, for example, transcription initiation sequences, transcription termination sequences, promoter sequences, enhancer sequences, RNA splicing sequences, polyadenylation (poly(A)) sequences, Kozak consensus sequences, and / or additional untranslated regions that may house pre- or post-transcriptional regulatory and / or control elements. In some embodiments, the promoter may be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of these control sequences are described herein.

[0124] 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, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990), which is incorporated herein by reference in its entirety). Generally, the ITRs can form hairpins. The ability to form hairpins contributes to the self-priming ability of the ITRs, allowing synthesis of a second DNA strand independent of primase. The ITRs may also assist in efficient encapsidation of the AAV construct within the AAV particle.

[0125] The rAAV particles (e.g., AAV2 / Anc80 particles) of the present disclosure may include rAAV constructs that include coding sequences (e.g., CLRN1 gene) and associated elements flanking the 5' and 3' AAV ITR sequences. In some embodiments, the ITRs are or include about 145 nucleic acids. In some embodiments, all or substantially all of the ITR coding sequences are used. The AAV ITR sequences can be obtained from any known AAV, such as currently identified mammalian AAV types. In some embodiments, the ITRs are AAV2 ITRs.

[0126] An example of a construct molecule used in this disclosure is a "cis-acting" construct that contains a transgene, where the selected transgene sequence and associated regulatory elements are flanked by 5' or "left" and 3' or "right" AAV ITR sequences. The 5' and left designations refer to the location of the ITR sequence relative to the entire construct, reading from left to right in the sense orientation. For example, in some embodiments, the 5' or left ITR is the ITR that is closest to the promoter (as opposed to the polyadenylation sequence) of a given construct when the construct is linearly depicted in the sense orientation. At the same time, the 3' and right designations refer to the location of the ITR sequence relative to the entire construct, reading from left to right in the sense orientation. For example, in some embodiments, the 3' or right ITR is the ITR that is closest to the polyadenylation sequence (as opposed to the promoter sequence) of a given construct when the construct is linearly depicted in the sense orientation. The ITRs provided herein are shown in the order 5' to 3' according to the sense strand. Thus, one skilled in the art will understand that when converting from sense to antisense orientation, a 5' or "left" oriented ITR can be depicted as a 3' or "right" ITR. Moreover, it is well within the capabilities of one skilled in the art to convert a given sense ITR sequence (e.g., 5' / left AAV ITR) to an antisense sequence (e.g., 3' / right ITR sequence). One skilled in the art will understand how to modify a given ITR sequence for use as either a 5' / left or 3' / right ITR, or an antisense version thereof.

[0127] For example, an ITR (e.g., a 5'ITR) can have a sequence according to SEQ ID NO: 15, 17, 20, or 21. In some embodiments, an ITR (e.g., a 3'ITR) can have a sequence according to SEQ ID NO: 16, 18, 20, or 22. In some embodiments, an ITR comprises one or more modifications, such as truncations, deletions, substitutions, or insertions, as known in the art. In some embodiments, an ITR comprises fewer than 145 nucleotides, such as 127, 130, 134, or 141 nucleotides. For example, in some embodiments, the ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 nucleotides. In some embodiments, the ITR (e.g., the 5'ITR) can have a sequence according to SEQ ID NO: 15. In some embodiments, the ITR (e.g., the 5'ITR) can have a sequence according to SEQ ID NO: 17. In some embodiments, the ITR (e.g., the 5'ITR) can have a sequence according to SEQ ID NO: 20. In some embodiments, an ITR (e.g., the 5'ITR) can have a sequence according to SEQ ID NO: 21. In some embodiments, an ITR (e.g., the 3'ITR) can have a sequence according to SEQ ID NO: 16. In some embodiments, an ITR (e.g., the 3'ITR) can have a sequence according to SEQ ID NO: 18. In some embodiments, an ITR (e.g., the 3'ITR) can have a sequence according to SEQ ID NO: 20. In some embodiments, an ITR (e.g., the 3'ITR) can have a sequence according to SEQ ID NO: 22.

[0128] Non-limiting examples of 5'AAV ITR sequences are SEQ ID NO: 15, 17, 20, or 21. Non-limiting examples of 3'AAV ITR sequences are SEQ ID NO: 16, 18, 20, or 22. In some embodiments, the rAAV constructs of the disclosure comprise a 5'AAV ITR and / or a 3'AAV ITR. In some embodiments, the ITR (e.g., the 5'ITR) can have a sequence according to SEQ ID NO: 17. In some embodiments, the 5'AAV ITR sequence can have a sequence according to SEQ ID NO: 20. In some embodiments, the 5'AAV ITR sequence can have a sequence according to SEQ ID NO: 21. In some embodiments, the 3'AAV ITR sequence is SEQ ID NO: 16. In some embodiments, the 3'AAV ITR sequence is SEQ ID NO: 18. In some embodiments, the 3'AAV ITR sequence is SEQ ID NO: 20. In some embodiments, the 3'AAV ITR sequence is SEQ ID NO: 22. In some embodiments, the 5' and 3' AAV ITRs (e.g., SEQ ID NO: 15, 17, or 21, or 16, 18, 20, or 22) flank a portion of the coding sequence, e.g., all or part of the CLRN1 gene (e.g., SEQ ID NO: 1, 2, 3, or 4). The ability to modify these ITR sequences is within the skill of one of ordinary skill 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, the 5' ITR sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the 5' ITR sequence represented by SEQ ID NO: 15, 17, 20, or 21. In some embodiments, the 3'ITR sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the 3'ITR sequence represented by SEQ ID NO:16, 18, 20, or 22.

[0129] Exemplary 5’ AAV ITR (SEQ ID NO: 15) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0130] Exemplary 3’ AAV ITR (SEQ ID NO: 16) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA

[0131] Exemplary 5’ AAV ITR (SEQ ID NO: 17) CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0132] Exemplary 3’ AAV ITR (SEQ ID NO: 18) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0133] Exemplary 5’ AAV ITR (SEQ ID NO: 20) CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0134] Exemplary 5' AAV ITR (SEQ ID NO:21) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0135] Exemplary 3' AAV ITR (SEQ ID NO:22) AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGGGAGTGGCCAA

[0136] promoter In some embodiments, the construct (e.g., 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., CLRN1 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, the construct (e.g., rAAV construct) comprises a promoter operably linked to one of the non-limiting examples of promoters described herein.

[0137] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art. In some embodiments, the promoter is an RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some embodiments, the promoter is an RNA polymerase III promoter, including but not limited to an HI promoter, a human U6 promoter, a mouse U6 promoter, or a porcine U6 promoter. Generally, the promoter is a promoter that can promote transcription in inner ear cells or ocular cells. In some embodiments, the promoter is a cochlea-specific promoter or a cochlea-directed promoter. In some embodiments, the promoter is a hair cell-specific promoter or a supporting cell-specific promoter.

[0138] 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 entirety of which is 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, retrovirus 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 Kb, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone alpha gene, immunoglobulin light chain, T cell receptor, HLA DQa and DQ, interleukin-2 receptor, MHC class II, MHC class II HLA-DRa, 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) promoter. Further examples of promoters are known in the art. See, for example, 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 in SEQ ID NO: 16. In certain embodiments, the promoter comprises a CMV / CBA enhancer / promoter construct exemplified in SEQ ID NO: 17. In certain embodiments, the promoter comprises a CAG promoter or a CMV / CBA / SV-40 enhancer / promoter construct exemplified in SEQ ID NO: 43. In certain embodiments, the promoter comprises a CAG promoter or a CMV / CBA / SV-40 enhancer / promoter construct exemplified in 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 represented by SEQ ID NO:16, 17, 43, or 44.

[0139] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a nucleic acid encoding a protein (e.g., a clarin 1 protein), causes transcription of RNA from the nucleic acid in a cell under most or all physiological conditions.

[0140] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al, Cell 41:521-530, 1985, which is incorporated herein by reference in its entirety), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1-alpha promoter (Invitrogen).

[0141] Inducible promoters allow for the regulation of gene expression and can be controlled by exogenously supplied compounds, environmental factors such as temperature, or the presence of certain physiological conditions (e.g., only in acute phase, in certain differentiation states of cells, or in replicating cells). Inducible promoters and induction 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.

[0142] Examples of inducible promoters regulated by an exogenously supplied compound include the zinc-inducible sheep metallothionein (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 ecdysone insect promoter (see, e.g., No et al, Proc. Natl. Acad. Sci. USA 93:3346-3351, 1996, incorporated herein by reference in its entirety), the tetracycline repression system (see, e.g., Gossen et al, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992, incorporated herein by reference in its entirety), the tetracycline inducible system (see, e.g., Gossen et al, Science 268:1766-1769, 1995; and Harvey et al. al, Curr. Opin. Chem. Biol. 2:512-518, 1998, each of which is incorporated herein by reference in its entirety), the RU486-inducible system (see, e.g., 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 (see, e.g., Magari et al. J Clin. Invest. 100:2865-2872, 1997, each of which is incorporated herein by reference in its entirety).

[0143] 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 will only occur in cells that express a transcriptional regulatory and / or control protein that binds to the tissue-specific promoter).

[0144] In some embodiments, the regulatory and / or control sequences confer tissue-specific gene expression. In some cases, the tissue-specific regulatory and / or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.

[0145] 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, ATOH1 promoter, POU4F3 promoter, LHX3 promoter, MYO7A promoter, MYO6 promoter, α9ACHR promoter, and α10ACHR promoter. In some embodiments, the promoter is a cochlear hair cell-specific promoter, such as a PRESTIN promoter or an ONCOMOD promoter. (See, e.g., Zheng et al., Nature 405:149-155, 2000; Tian et al. Dev. Dyn. 23 l:199-203, 2004; and Ryan et al., Adv. Otorhinolaryngol. 66:99-115, 2009, each of which is incorporated herein by reference in its entirety.)

[0146] 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.

[0147] In some embodiments, the tissue-specific promoter is an ocular cell-specific promoter.Non-limiting examples of ocular cell-specific promoters include, but are not limited to, RPE65, RLBP1, VMD2, IRBP, GNAT2, PR1.7, PR2.1, HB569, CAR, GRK1, RK, B-PDE, GRM6, Nefh, Tyh1, SYN, GFAP, or other opsin or rhodopsin promoters.Non-limiting examples of ocular cell-specific promoters and illustrative content describing such promoters are as follows: [Table 3-1] [Table 3-2]

[0148] In some embodiments, the AAV construct provided 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 only AAV construct further comprises at least one promoter sequence selected from a cochlear and / or inner ear specific promoter.

[0149] In certain embodiments, the promoter is a CBA promoter set forth in SEQ ID NO: 23. 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: 23.

[0150] Exemplary CBA Promoter (SEQ ID NO:23) GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCCAGGCGGGG CGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0151] Exemplary CBA Promoter (SEQ ID NO:24) GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGCGCGCCAGGCGGGGC GGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0152] In certain embodiments, the promoter is a CMV / CBA enhancer / promoter represented by SEQ ID NO: 25. 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:25.

[0153] Exemplary CMV / CBA enhancer / promoter (SEQ ID NO:25) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0154] Exemplary CMV / CBA enhancer / promoter (SEQ ID NO: 26) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0155] Exemplary CAG enhancer / promoter (SEQ ID NO: 27)

[0156] Exemplary CAG enhancer / promoter (SEQ ID NO:28)

[0157] In certain embodiments, the promoter is the endogenous human ATOH1 enhancer-promoter represented by SEQ ID NO: 29. 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: 29.

[0158] Exemplary Human ATOH1 Enhancer-Promoter (SEQ ID NO:29)

[0159] In certain embodiments, the promoter is the endogenous human SLC26A4 immediate-initiation promoter represented by SEQ ID NO: 30 or 31. In certain embodiments, the promoter is the endogenous human SLC26A4 enhancer-promoter represented by SEQ ID NO: 32, 33 or 34. 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: 30, 31, 32, 33, or 34. In certain embodiments, the promoter is the human SLC26A4 endogenous enhancer-promoter sequence contained within SEQ ID NO: 32, 33, or 34.

[0160] Exemplary human SLC26A4 immediate early promoter (SEQ ID NO:30) CTGCCTTCTGAGAGCGCTATAAAAGGCAGCGGAAGGGTAGTCCGCGGGGCATTCCGGGCGG

[0161] Exemplary human SLC26A4 immediate early promoter (SEQ ID NO:31) CTCTAGGCGGGCTCTGCTCTTCTTTAAGGAGTCCCACAGGGCCTGGCCCGCCCCTGACCT

[0162] Exemplary Human SLC26A4 Enhancer-Promoter (SEQ ID NO:32)

[0163] Exemplary human SLC26A4 enhancer-promoter (SEQ ID NO:33)

[0164] Exemplary human SLC26A4 enhancer-promoter (SEQ ID NO:34) CGGAAGGTTGATGTACAGAGGTCTGTATTTTGGAGCCTCTTCTGTATTTACTTCAGAACACTAACAATCAGGCGAGAATGTTCTGGTTTATCAAACCCTTCCTGCCTTTCATCTTAACCATGCATTAGTTTTAACAAAGTTCATCCCAACAGAAGACAAAACACTGATGAGGTAGGATAGCTCCAGCTCCTCCTCCCTCTCTTCTAGTCTTGATTTCCATGT AGTCCAGTTTATTCCTTCCCTGATTGTCCAGGAGAATGAGAAAAAGAAAAAACAGAGTCTAGTGGGTAAGAAAGGGCCACCTGGACGGCTTGATTTGGATTGTGAAATAAAACACACACACATGCACACGTAGAATAAGTGGCTAAAATCTGAGTAAATCGTGAACTCTCTGTATCCTCCACCCATTGAATACTCCTAAAAGACTTTCTAGAAATTCAAGGACTT ATTAATATAGAAACCTGGCCATTGTTCCTCTTCTCCTCCCCATGTGGTATGAGAGCACCTGTGGCAGGCTCCCAGAGACCACGGACCTCTTCCTCTAGGCGGGCTCTGCTCTTTTAAGGAGTCCCACAGGGCCTGGCCCGCCCCTGACCTCGCAACCCTTGAGATTAGTAACGGGATGAGTGAGGATCCGGGTGGCCCCTGCGTGGCAGCCAGTAAGAGTCTC AGCCTTCCCGGTTCGGGAAAGGGGAAGAATGCAGGAGGGGTAGGATTTCTTTCCTGATAGGATCGGTTGGGAAAGACCGCAGCCTGTGTGTGTCTTTCCCTTCGACCAAGGTGTCTGTTGCTCCGTAAATAAAACGTCCCACTGCCTTCTGAGAGCGCTATAAAGGCAGCGGAAGGGTAGTCCGCGGGGCATTCCGGGCGGGGCGCGAGCAGAGACAGGTGAGTT

[0165] In certain embodiments, the promoter is the human LGR5 enhancer-promoter represented by SEQ ID NO: 35. 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: 35. In some embodiments, the promoter is the human LGR5 endogenous enhancer-promoter sequence contained within SEQ ID NO: 35.

[0166] Exemplary human LGR5 enhancer-promoter (SEQ ID NO:35)

[0167] In certain embodiments, the promoter is the human SYN1 enhancer-promoter represented by SEQ ID NO: 36. 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: 36. In some embodiments, the promoter is the human SYN1 endogenous enhancer-promoter sequence contained within SEQ ID NO: 36.

[0168] Exemplary human SYN1 enhancer-promoter (SEQ ID NO:36) TGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCCCACCGCCGC CTCAGCACTGAAGGCGCCGCTGACGTCACTCGCCGGTCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGCAGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGTCGAGAA

[0169] In certain embodiments, the promoter is the human GFAP enhancer-promoter represented by SEQ ID NO: 37. 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: 37. In some embodiments, the promoter is the human GFAP endogenous enhancer-promoter sequence contained within SEQ ID NO: 37.

[0170] Exemplary human GFAP enhancer-promoter (SEQ ID NO:37)

[0171] Enhancer In some examples, the construct may include an enhancer sequence. The term "enhancer" refers to a nucleotide sequence that can increase the transcription level of a nucleic acid encoding a protein of interest (e.g., a clarin 1 protein). Enhancer sequences (generally 50-1500 bp in length) generally increase the transcription level by providing additional binding sites for transcription-associated proteins (e.g., transcription factors). In some embodiments, enhancer sequences are found within intron sequences. Unlike promoter sequences, enhancer sequences can act much further away from the transcription start site (e.g., compared to promoters). Non-limiting examples of enhancers include RSV enhancers, CMV enhancers, and / or SV40 enhancers. In some embodiments, the construct includes a CMV enhancer exemplified by SEQ ID NO:38. 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:38. In some embodiments, the enhancer from SV-40 is the SV-40 T intron sequence exemplified by SEQ ID NO: 39. 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:39.

[0172] Exemplary CMV enhancer (SEQ ID NO:38) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG

[0173] An exemplary SV-40 synthetic intron (SEQ ID NO:39)

[0174] Flanking untranslated regions, 5'UTR and 3'UTR In some embodiments, any of the constructs described herein can include an untranslated region (UTR), such as a 5'UTR or a 3'UTR. The UTR of a gene is transcribed but not translated. The 5'UTR starts at the transcription start site and continues up to but not including the start codon. The 3'UTR starts just after the stop codon and continues to the transcription termination signal. The regulatory and / or control functions of the UTR can be incorporated into any of the constructs, compositions, kits, or methods described herein to enhance or otherwise regulate the expression of the clarin 1 protein.

[0175] Natural 5'UTRs contain sequences involved in translation initiation. In some embodiments, 5'UTRs can contain sequences such as the Kozak sequence, which is commonly known to be involved in the process by which ribosomes initiate the translation of many genes. The Kozak sequence has the consensus sequence CCR(A / G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), which is followed by another "G". 5'UTRs are also known to form secondary structures involved in the binding of elongation factors.

[0176] 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 a nucleic acid molecule (e.g., mRNA), including those from the following genes: albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, and factor VIII.

[0177] In some embodiments, a 5'UTR from an mRNA transcribed by cells in the cochlea may be included in any of the constructs, compositions, kits, and methods described herein. In some embodiments, the 5'UTR is derived from the endogenous CLRN1 locus and may include all or a portion of the endogenous sequence exemplified by SEQ ID NO: 40. 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: 40.

[0178] 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 may be included in any of the constructs, compositions, kits, and methods described herein. In some embodiments, the 3'UTR is derived from the endogenous CLRN1 locus and may include all or a portion of the endogenous sequence exemplified by SEQ ID NO:41. 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:41.

[0179] 3'UTRs are known to have stretches of adenosines and uridines (in RNA form) or thymidines (in DNA form) embedded within them. These AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on these sequence features and functional properties, AU-rich elements (AREs) can be separated into three classes (see, e.g., 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 copies of the AUUUA motif dispersed within the U-rich region. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs. Class III AREs are less well defined. Although their U-rich regions do not contain AUUUA motifs, two well-studied examples of this class are c-Jun and myogenin mRNAs.

[0180] Most proteins that bind to AREs are known to destabilize messengers, whereas members of the ELAV family, particularly HuR, have been demonstrated to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding and thus stabilization of the message in vivo.

[0181] In some embodiments, the introduction, removal, or modification of the 3'UTR ARE can be used to modulate the stability of the mRNA encoding the clarin1 protein, in other embodiments, the ARE can be removed or mutated to increase the intracellular stability of the clarin1 protein and thus its translation and production.

[0182] In other embodiments, non-ARE sequences may be incorporated into the 5' or 3' UTR. In some embodiments, introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the constructs, compositions, kits, and methods provided herein. Incorporation of intron sequences may increase protein production and mRNA levels.

[0183] Exemplary 5'UTR Sequence (SEQ ID NO:40) AGGAGATACTTGAAGGCAGTTTGAAAGACTTGTTTTACAGATTCTTAGTCCAAAGATTTCCAATTAGGGAGAAGAAGCAGCAGAAAAGGAGAAAAGCCAAGTATGAGTGATGATGAGGCCTTCATCTACTGACATTTAACCTGGC GAGAACCGTCGATGGTGAAGTTGCCTTTTCAGCTGGGAGCTGTCCGTTCAGCTTCCGTAATAAATGCAGTCAAAGAGGCAGTCCCTTCCCATTGCTCACAAAGGTCTTGTTTTTGAACCTCGCCCTCACAGAAGCCGTTTCTCATC

[0184] Exemplary 3'UTR sequence (SEQ ID NO:41):

[0185] Internal ribosome entry site (IRES) In some embodiments, the construct encoding the clarin 1 protein may contain an internal ribosome entry site (IRES), which forms a complex secondary structure that allows translation initiation to occur from any position with the mRNA immediately downstream from the IRES position (see, e.g., Pelletier and Sonenberg, Mal. Cell. Biol. 8(3):1103-1112, 1988).

[0186] There are several IRES sequences known to those of skill in the art, including, for example, IRES sequences 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).

[0187] In some embodiments, the IRES sequence incorporated into the construct encoding the clarin 1 protein or the C-terminal portion of the clarin 1 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 been previously demonstrated in artificial systems, including plasmids and gene therapy constructs (AAV and retroviruses) (e.g., 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).

[0188] IRES can be utilized in AAV constructs. In some embodiments, the construct encoding the C-terminal portion of the clarin 1 protein can include a polynucleotide internal ribosome entry site (IRES). In some embodiments, the IRES can be part of a composition that includes multiple constructs. In some embodiments, the IRES is used to produce more than one polypeptide from a single gene transcript.

[0189] Splice Site In some embodiments, any of the constructs provided herein can include splice donor and / or splice acceptor sequences that function during RNA processing that occurs during transcription, hi some embodiments, the splice sites are involved in trans-splicing.

[0190] Exemplary splice donor intron (SEQ ID NO: 42) GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTCT

[0191] Exemplary splice acceptor intron (SEQ ID NO: 43) GATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG

[0192] Polyadenylation sequence In some embodiments, the constructs provided herein may include a polyadenylation (poly(A)) signal sequence. Most nascent mRNAs in eukaryotes have a poly(A) tail at their 3' end, which is added in a complex process involving cleavage of the primary transcript and coupled polyadenylation driven by the poly(A) signal sequence (see, e.g., Proudfoot et al., Cell 108:501-512, 2002, which is incorporated herein by reference in its entirety). The poly(A) tail confers mRNA stability and translocation (see, e.g., Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994, which is incorporated herein by reference in its entirety). In some embodiments, the poly(A) signal sequence is located 3' to the coding sequence.

[0193] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety, or modified variants thereof, to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) of adenine nucleotides that are added to pre-mRNA by the action of the enzyme polyadenylate polymerase. In some embodiments, poly(A) tails are added to transcripts that contain specific sequences, e.g., poly(A) signals. Poly(A) tails and associated proteins help protect mRNA from exonuclease degradation. Polyadenylation is also involved in transcription termination, export of mRNA from the nucleus, and translation. Polyadenylation signals typically occur in the nucleus immediately following 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.

[0194] As used herein, a "poly(A) signal sequence" or "polyadenylation signal sequence" is a sequence that triggers endonucleolytic cleavage of mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0195] There are several poly(A) signal sequences that may be used, including those from bovine growth hormone (bGH) (see 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 (see, e.g., Orkin et al., EMBO J 4(2):453-456, 1985; Thein et al., Blood 71(2):313-319, 1988, each of which is incorporated herein by reference in its entirety), human collagen, polyoma virus (see, e.g., Batt et al., Mal. Cell 2001, 1997, 1999, 1990, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 19 ... Biol. 15(9):4783-4790, 1995, incorporated herein by reference in its entirety), herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy chain gene polyadenylation signal (US2006 / 0040354, incorporated herein by reference in its entirety), human growth hormone (hGH) (see, e.g., Szymanski et al., Mal. Therapy 15(7):1340-1347, 2007, incorporated herein by reference in its entirety), the group of SV40 poly(A) sites, such as those derived from the SV40 late and early poly(A) sites (see, e.g., Schek et al., Mal. Cell Biol. 12(12):5386-5393, 1992, incorporated herein by reference in its entirety).

[0196] The poly(A) signal sequence can be AATAAA. The AATAAA sequence may be replaced with other hexanucleotide sequences having homology to AATAAA and capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e.g., WO06 / 12414, which is incorporated herein by reference in its entirety).

[0197] In some embodiments, the poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., Promega's pCl-neo expression construct based on Levitt el al, Genes Dev. 3(7):1019-1025, 1989, which is incorporated herein by reference in its entirety). In some embodiments, the poly(A) signal sequence is the polyadenylation signal of soluble neuropilin-1 (sNRP) (AAATAAAATACGAAATG) (see, e.g., WO05 / 073384, which is 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 sequence comprises or consists of SEQ ID NO:45. 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:44. Additional 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 represented by SEQ ID NO: 44 or 45.

[0198] Exemplary bGH poly(A) signal sequence (SEQ ID NO:44) CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGTGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0199] Exemplary SV40 poly(A) signal sequence (SEQ ID NO:45) AACTTGTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

[0200] 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 appreciated by those skilled in the art, cloning sites may vary significantly in primary sequence while retaining their desired function. In some embodiments, constructs may include any combination of cloning sites, with exemplary cloning sites being represented by SEQ ID NOs: 46-56.

[0201] Exemplary Cloning Site A (SEQ ID NO:46) TTGTCGACGCGGCCGCACGCGT

[0202] Exemplary Cloning Site B (SEQ ID NO:47) CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTCGCTAGCCACC

[0203] Exemplary Cloning Site C (SEQ ID NO:48) TAAGAGCTCGCTGATCAGCCTCGA

[0204] Exemplary cloning site D (SEQ ID NO:49) AAGCTTGAATTCAGCTGACGTGCCTCGGACCGTCCTAGG

[0205] Exemplary cloning site E (SEQ ID NO:50) GCGGCCGCACGCGT

[0206] Exemplary cloning site F (SEQ ID NO:51) CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC

[0207] Exemplary cloning site G (SEQ ID NO:52) TAAGAGCTCGCTGATCAGCCTCGA

[0208] Exemplary cloning site H (SEQ ID NO:53) AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT

[0209] Exemplary Cloning Site I (SEQ ID NO:54) CTCCTGGGCAACGTGCTGGTTATTGTGACCGGT

[0210] Exemplary cloning site J (SEQ ID NO:55) GCCACC

[0211] Exemplary cloning site K (SEQ ID NO:56) TAAGAGCTC

[0212] Destabilizing Domains In some embodiments, any of the constructs provided herein may optionally include a sequence encoding a destabilization domain ("destabilization sequence") for temporally regulating protein expression. Non-limiting examples of destabilization sequences include sequences encoding FK506 sequences, dihydrofolate reductase (DHFR) sequences, or other exemplary destabilization sequences.

[0213] 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, protein degradation is inhibited, thereby allowing the protein sequence operably linked to the destabilizing sequence to be actively expressed. As a positive control for the stabilization of protein expression, protein expression can be detected by conventional means, including enzyme, radiographic, colorimetric, fluorescent, or other spectrophotometric assays; fluorescence-activated cell sorting (FACS) assays; immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0214] Further examples of destabilizing sequences are known in the art. In some embodiments, the destabilizing sequence is an FK506- and rapamycin-binding protein (FKBP12) sequence and the stabilizing ligand is Shield-1 (Shld1) (see, e.g., Banaszynski et al. (2012) Cell 126(5):995-1004, which is incorporated herein by reference in its entirety). In some embodiments, the destabilizing sequence is a DHFR sequence and the stabilizing ligand is trimethoprim (TMP) (see, e.g., Iwamoto et al. (2010) Chem Biol 17:981-988, which is incorporated herein by reference in its entirety).

[0215] 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 outer cochlear hair cell) is detected by Western blotting. In some embodiments, the destabilization sequence can be used to verify the time-specific activity of any of the AAV constructs described herein.

[0216] Exemplary DHFR destabilizing amino acid sequence (SEQ ID NO:57) MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR

[0217] Exemplary DHFR destabilizing nucleotide sequence (SEQ ID NO:58) GGTACCATCAGTTCTGATTGCGGCGTTAGCGGTAGATTACGTTATCGGCATGGAAAACGCCATGCCGTGGAACCTGCCTGCCGATCTCGCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGGCCGCCATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGCAGTCAACCGAGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGATG AAGCCATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTGATTGGCGGCGGTCGCGTTATTGAACAGTTCTTGCCAAAAGCGCAAAAACTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGATTACGAGCCGGATGACTGGGAATCGGTATTCAGCGAATTCCACGATGCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGATAA

[0218] Exemplary Destabilization Domains (SEQ ID NO:59) ATCAGTCTGATTGCGGCGTTAGCGGTAGATTACGTTATCGGCATGGAAAACGCCATGCCGTGGAACCTGCCTGCCGATCTCGCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGCCGCCATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGCAGTCAACCGAGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGATGAA GCCATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTGATTGGCGGCGGTCGCGTTATTGAACAGTTCTTGCCAAAAGCGCAAAAACTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGATTACGAGCCGGATGACTGGGAATCGGTATTCAGCGAATTCCACGATGCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGA

[0219] Exemplary FKBP12 destabilizing peptide amino acid sequence (SEQ ID NO:60) MGVEKQVIRPGNGPKPAPGQTVTVHCTGFGKDGDLSQKFWSTKDEGQKPFSFQIGKGAVIKGWDEGVIGMQIGEVARLRCSSDYAYGAGGFPAWGIQPNSVLDFEIEVLSVQ

[0220] Reporter sequence or element In some embodiments, the constructs provided herein may optionally include a sequence encoding a reporter polypeptide and / or protein ("reporter sequence"). Non-limiting examples of reporter sequences include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein, mCherry fluorescent protein, yellow fluorescent protein, chloramphenicol acetyltransferase (CAT), and luciferase. Additional examples of reporter sequences are known in the art. When associated with a control element that drives expression of the reporter sequence, the reporter sequence can provide a signal that is detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescent, or other spectrophotometric assays; fluorescence-activated cell sorting (FACS) assays; immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0221] In some embodiments, the reporter sequence is the LacZ gene, and the presence of the construct carrying the LacZ gene in mammalian cells (e.g., cochlear hair cells) is detected by assaying for beta-galactosidase activity. If the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of the construct carrying the fluorescent protein or luciferase in mammalian cells (e.g., cochlear hair cells) may be measured by fluorescent techniques (e.g., fluorescent microscopy or FACS) or by light production in a luminometer (e.g., a spectrophotometer or IVIS imager). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulation and / or control activity of any of the constructs described herein.

[0222] In some embodiments, the reporter sequence is a FLAG tag (e.g., a 3xFLAG tag), and the presence of the construct bearing the FLAG tag in mammalian cells (e.g., inner ear cells, such as cochlear hair or supporting cells, such as ocular cells) is detected by protein binding or detection assays (e.g., Western blot, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry). An exemplary 3xFLAG tag sequence is provided as SEQ ID NO:61.

[0223] Exemplary 3xFLAG tag sequence (SEQ ID NO:61) GGATCCCGGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG

[0224] AAV particles In particular, the present disclosure provides AAV particles comprising a construct encoding the CLRN1 gene or a characteristic portion thereof as described herein, and a capsid as described herein. In some embodiments, the AAV particles can be described as having a serotype that indicates the construct strain and the capsid strain. For example, in some embodiments, the AAV particles can be described as AAV2, which has an AAV2 capsid and a construct that includes a characteristic AAV2 inverted terminal repeat (ITR). In some embodiments, the AAV particles can be described as pseudotypes in which the capsid and construct are derived from different AAV strains, for example, AAV2 / 9 refers to an AAV particle that includes a construct that utilizes AAV2 ITRs and AAV9 capsid.

[0225] AAV constructs The present disclosure provides a polynucleotide construct comprising the CLRN1 gene or a characteristic portion thereof. In some embodiments described herein, the polynucleotide comprising the CLRN1 gene or a characteristic portion thereof can be included in an AAV particle.

[0226] In some embodiments, polynucleotide construct comprises one or more components derived from or modified from naturally occurring AAV genome construct.In some embodiments, the sequence derived from AAV construct is AAV1 construct, AAV2 construct, AAV3 construct, AAV4 construct, AAV5 construct, AAV6 construct, AAV7 construct, AAV8 construct, AAV9 construct, AAV2.7m8 construct, AAV8BP2 construct, AAV293 construct or AAV Anc80 construct.In some embodiments, rAAV Anc80 capsid is rAAV Anc80L65 capsid. Additional exemplary AAV constructs that may be used herein are known in the art (see, e.g., Kanaan et al., Mol. Ther. Nucleic Acids 8:184-197, 2017; Li et al., Mol. Ther. 16(7):1252-1260, 2008; Adachi et al., Nat. Commun. 5:3075, 2014; Isgrig et al., Nat. Commun. 10(1):427, 2019; and Gao et al., J. Virol. 78(12):6381-6388, 2004; each of which is incorporated herein by reference in its entirety).

[0227] In some embodiments, the provided constructs include a coding sequence, such as a CLRN1 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 constructs may be referred to as recombinant AAV (rAAV) constructs. In some embodiments, the provided rAAV constructs are packaged into AAV capsids to form AAV particles.

[0228] In some embodiments, the AAV-derived sequences (included in the polynucleotide construct) typically include cis-acting 5' and 3' ITR sequences (see, e.g., BJ Carter, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168, 1990, which is incorporated herein by reference in its entirety). A typical AAV2-derived ITR sequence is about 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 of ordinary skill 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 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. The AAV ITR sequences can be obtained from any known AAV, including the AAV types identified herein.

[0229] In some embodiments, polynucleotide constructs described in accordance with the present disclosure in a pattern known in the art (see, e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, which are incorporated herein by reference in their entirety) typically consist of a coding sequence or a portion thereof, at least one and / or control sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). In some embodiments, the provided constructs can be packaged into capsids to create AAV particles. The AAV particles can be delivered to selected target cells. In some embodiments, the provided constructs include additional optional coding sequences that are heterologous nucleic acid sequences (e.g., inhibitory nucleic acid sequences) to the construct sequence that encode 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 the components in a manner that allows for transcription, translation, and / or expression of the coding sequence in cells of the target tissue.

[0230] As shown in FIG. 1A, the unmodified AAV endogenous genome includes two open reading frames, "cap" and "rep", flanked by ITRs. As shown in FIG. 1B, an exemplary rAAV construct also includes a coding region, e.g., ITRs flanking a coding sequence (e.g., CLRN1 gene). In some embodiments, the rAAV construct also includes conventional control elements operably linked to the coding sequence in a manner that allows for its transcription, translation and / or expression in cells transfected with a plasmid construct or infected with a virus produced by the present disclosure. In some embodiments, the rAAV construct optionally includes a promoter (as shown in FIG. 1B), an enhancer, a non-translated 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 (as shown in FIG. 1B), or any combination thereof. Such additional elements are further described herein.

[0231] In some embodiments, the construct is a rAAV construct.In some embodiments, the rAAV construct can comprise at least 500bp, at least 1kb, at least 1.5kb, at least 2kb, at least 2.5kb, at least 3kb, at least 3.5kb, at least 4kb, or at least 4.5kb.In some embodiments, the AAV construct can comprise up to 7.5kb, up to 7kb, up to 6.5kb, up to 6kb, up to 5.5kb, up to 5kb, up to 4.5kb, up to 4kb, up to 3.5kb, up to 3kb, or up to 2.5kb. In some embodiments, the AAV construct may comprise about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

[0232] Any of the constructs described herein can further comprise regulatory and / or control sequences, such as control sequences selected from the group of transcription initiation sequences, transcription termination sequences, promoter sequences, enhancer sequences, RNA splicing sequences, polyadenylation (poly(A)) sequences, Kozak consensus sequences, and / or any combination thereof. In some embodiments, the promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of these control sequences are described herein.

[0233] 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 AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rh10, rh39, rh43, or Anc80 serotype AAV capsid or one or more hybrids thereof. In some embodiments, the AAV capsid is from an AAV ancestral serotype. In some embodiments, the AAV capsid is an ancestral (Anc) AAV capsid. Anc capsids are generated from constructed construct sequences using evolutionary probability and evolutionary modeling to determine putative ancestral sequences. Therefore, it is unknown whether the Anc capsid / construct sequence existed in nature. For example, in some embodiments, the AAV capsid is an Anc80 capsid (e.g., Anc80L65 capsid).

[0234] In some embodiments, the AAV capsid is generated using a template nucleotide coding sequence comprising SEQ ID NO: 62. In some embodiments, the capsid comprises a polypeptide represented by SEQ ID NO: 63. 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: 63.

[0235] As provided herein, any combination of AAV capsids and AAV constructs (e.g., including AAV ITRs) can be used in the recombinant AAV (rAAV) particles of the present disclosure. For example, wild-type or variant AAV2 ITRs and Anc80 capsid, wild-type or variant AAV2 ITRs and AAV6 capsid, etc. In some embodiments of the present disclosure, the AAV particles are entirely composed of AAV2 components (e.g., the capsid and ITRs are of the AAV2 serotype). In some embodiments, the AAV particles are AAV2 / 6, AAV2 / 8, or AAV2 / 9 particles (e.g., AAV6, AAV8, or AAV9 capsids with AAV constructs with AAV2 ITRs). In some embodiments of the present disclosure, the AAV particle is an AAV2 / Anc80 particle that includes an Anc80 capsid (e.g., a polypeptide of SEQ ID NO: 63) that encapsidates an AAV construct (e.g., SEQ ID NOs: 15-22) having AAV2 ITRs flanking a portion of a coding sequence, such as the CLRN1 gene or a characteristic portion thereof (e.g., SEQ ID NOs: 1, 2, 3, or 4). Other AAV particles are known in the art and are described, for example, in Sharma et al., Brain Res Bull. 2010 Feb 15;81(2-3):273, which is incorporated herein by reference in its entirety. In some embodiments, the capsid sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the capsid nucleotide or amino acid sequence represented by SEQ ID NOs: 62 or 63, respectively.

[0236] In some embodiments, the platform delivery approach disclosed herein combines a library of synthetic AAV capsids known as ancestral AAV (AAVAnc) capsids that recapitulate the evolutionary lineage of the current natural virus. In some embodiments, these AAV capsids are combined with a novel, minimally invasive administration procedure to deliver product candidates directly to the cochlea. In some embodiments, the delivery approach utilizes an AAV Anc80 capsid variant (e.g., Anc80L65) from this library. In some embodiments, such capsids are utilized to generate rAAV particles, which are generated by adding a construct described herein, such as a construct containing a CLRN1 cDNA encoding the CLRN1 protein described herein, to generate rAAV Anc80-CLRN1.

[0237] In some embodiments, the compositions disclosed herein include AAV Anc80 capsid, a rationally designed synthetic AAV capsid whose sequence was inferred by ancestral sequence reconstruction. Ancestral sequence reconstruction uses sequence information available from natural adeno-associated viruses to identify ancestral states of sequences at various intermediate evolutionary nodes as a result of phylogenetic and statistical predictions. During the creation of AAV Anc80, nine nodes were reconstructed and in silico-derived sequences of AAV lineages were de novo synthesized and characterized. This led to the identification of the Anc80 library node (Anc80Lib), which is the putative ancestor of the widely studied AAV serotypes 1, 2, 8, and 9. The Anc80Lib protein sequence was then reverse translated and generated by gene synthesis, and individual clones were individually evaluated for packaging, infectivity, and biological properties. In some embodiments, based on these results, AAV Anc80, the 65th Anc80Lib clone (Anc80L65), was selected for further characterization. AAV Anc80 capsid variants have unique compositions. Although the sequences of AAV8 and AAV2 differ from AAVAnc80 by only approximately 9% and 12%, respectively, structural modeling of AAV Anc80 shows that approximately 20% of the outer surface of the particle is different from known circulating AAVs, distributed across the entire capsid surface (see, e.g., Zinn 2015, which is incorporated herein by reference in its entirety).

[0238] In some embodiments, the performance of AAV Anc80 as a gene therapy particle in vivo has been characterized, and rAAV particles containing AAV Anc80 have been demonstrated to have the potential to act as broadly applicable gene therapy particles. In some embodiments, studies performed in mice and non-human primates (NHPs) have shown that AAV Anc80 has similar transduction efficiency to AAV8 when targeting the liver after intravenous injection, without obvious signs of systemic toxicity (see, e.g., Zinn 2015; Murillo 2019, each of which is incorporated herein by reference in its entirety). Additionally, in some embodiments, AAV Anc80 has demonstrated tropism and efficient transduction of the mouse anterior eye (Wang 2017, incorporated herein by reference in its entirety), mouse and NHP retina (see, e.g., Zinn 2015; Carvalho 2018, each of which is incorporated herein by reference in its entirety), mouse skeletal muscle (see, e.g., Zinn 2015, incorporated herein by reference in its entirety), mouse central nervous system (CNS) by systemic and intraparenchymal delivery (Hudry 2018, incorporated herein by reference in its entirety), and mouse kidney (see, e.g., Ikeda 2018, incorporated herein by reference in its entirety).

[0239] In some embodiments, a composition described herein (e.g., comprising rAAV-CLRN1) comprises an AAV Anc80 capsid. In some embodiments, the rAAV Anc80 capsid is an rAAV Anc80L65 capsid. In some embodiments, the AAV Anc80 capsid exhibits high transduction efficiency for cochlear and vestibular cells. In some embodiments, the AAV Anc80 capsid exhibits high transduction efficiency for ocular cells.

[0240] AAV Anc80 is a rationally designed AAV capsid inferred by ancestral sequence reconstruction (see, e.g., Zinn 2015, which is incorporated herein by reference in its entirety). Ancestral sequence reconstruction uses available sequence information from natural AAVs to identify ancestral states of sequences at various intermediate evolutionary nodes as a result of phylogenetic and statistical predictions. As described in the literature, de novo synthesis and characterization of in silico derived sequences across the AAV lineage identified the Anc80 Library (Anc80Lib) node, which is the putative ancestor of the widely tested AAV serotypes 1, 2, 8, and 9. Subsequent evaluation of Anc80Lib sequences led to further characterization of the 65th Anc80Lib clone (Anc80L65), AAV Anc80. These studies showed that AAV Anc80 capsid variants have a distinctive composition with a different outer surface particle distribution resulting in stable and functional AAV variants with transduction efficiency similar to AAV8 (see, e.g., Zinn 2015, incorporated herein by reference in its entirety). The first reported use of AAV Anc80 in the mammalian inner ear revealed high transduction efficiency in cochlea and vestibular hair cells (see, e.g., Landegger 2017, incorporated herein by reference in its entirety). Subsequent independent studies confirmed the increased transduction efficiency of cochlea and vestibular cells for AAV Anc80 compared to other AAV serotypes. In mice of various ages (see, e.g., Landegger 2017; Tao 2018; Yoshimura 2018; and Omichi 2020, each of which is incorporated by reference in its entirety) and non-human primates (see, e.g., Andres-Mateos 2019, which is incorporated by reference in its entirety), AAV Anc80 has higher transduction efficiency and broader tropism compared to multiple other AAV capsids.

[0241] Gene therapy using AAV particles is a promising treatment modality for inner ear disorders for several reasons: (1) the inner ear, including the auditory and vestibular sensory epithelia, has altered immune surveillance similar to that within the central nervous system (Fujioka 2014, incorporated herein by reference in its entirety); (2) the sensory and supporting cells of the cochlea are postmitotic, allowing for long-term expression potential following a single dose of AAV; and (3) the combined clinical experience with rAAV delivery in both adults and children by multiple routes of administration suggests a strong safety profile of AAV as a delivery vehicle, especially for local delivery and / or at low-to-moderate doses.

[0242] Beginning with the first clinical trials more than 20 years ago, rAAV particles have been administered to hundreds of participants in dozens of clinical trials at doses up to approximately 1E15vg or more for systemic administration (see, e.g., Flotte 1996; Flotte 2013; Parente 2018; and Wang 2019, each of which is incorporated herein by reference in its entirety). The number of clinical trials in which AAV particles have been used for in vivo gene transfer has steadily increased. The safety profile, along with the highly efficient transduction of a wide range of target tissues, has established rAAV particles as the platform of choice for in vivo gene therapy (see, e.g., Wang 2019, each of which is incorporated herein by reference in its entirety). rAAV technology has been successfully applied in the clinic in a variety of conditions, such as coagulation disorders, inherited blindness, and neurodegenerative diseases (see, e.g., Colella 2018; and Wang 2019, each of which is incorporated herein by reference in its entirety).

[0243] An rAAV particle product (alipogenet iparvovec; Glybera®) was first approved by the European Medicines Agency (EMA) for the treatment of lipoprotein lipase deficiency in 2012. Two other rAAV products were subsequently approved by the US Food and Drug Administration (FDA) in 2017 and 2019, respectively: the voretigene neparvovec-rzyl (Luxturna®) for the treatment of confirmed biallelic RPE65 mutation-associated retinal dystrophy, and the onasemnogene abeparvovec-xioi (Zolgensma®) for the treatment of spinal muscular atrophy (SMA) with biallelic mutations in the SMN1 gene. The voretigene neparvovec-rzyl (Luxturna®) has also been approved by the EMA for treating vision loss due to inherited retinal dystrophy, when the disease is caused by mutations in the gene RPE65.

[0244] In some embodiments, drugs and biologics, including rAAV particles, can reach many target cells in the inner ear by delivering them to the perilymph. The perilymph is a fluid that is very similar in composition to and diffusely connected to cerebrospinal fluid (CSF) (see, e.g., Lysaght 2011, incorporated herein by reference in its entirety). The perilymph bathes most of the sensory, neuronal, and supporting cells of the cochlea and vestibular systems and is contained in the bony labyrinth of the inner ear (FIGS. 11 and 12). In some embodiments, the perilymphatic space of the cochlea, into which the compositions disclosed herein, e.g., rAAV-CLRN1, are delivered, includes two scala or passages, the scala tympani and the scala vestibuli, which are connected to each other at the apex of the cochlear spiral via the cochlear foramen. Many cells of the inner ear are in fluid communication with the perilymph through gaps in the tissue.

[0245] In some embodiments, disclosed herein is a single-use sterile delivery device for intracochlear administration to deliver a composition disclosed herein, e.g., rAAV-CLRN1, to the perilymphatic fluid of the inner ear through a cochlear window membrane with a pore located in the base of the stapes. In some embodiments, in this intracochlear administration approach, a composition disclosed herein, e.g., rAAV-CLRN1, is administered to the scala tympani through a cochlear window membrane with a pore at the base of the stapes in the vestibular window, whereby the composition is perfused through the scala tympani and then through the scala vestibuli via a connection at the cochlear foramen, following a fluid path to a pore in the base of the stapes (FIG. 11). In some embodiments, the presence of a pore distinct from the injection port allows for a more uniform distribution of the drug along the length of the cochlea and prevents the harmful buildup of additional fluid pressure in the inner ear. In some embodiments, this delivery approach also allows for diffusion of the compositions disclosed herein, e.g., injectates, into the vestibular system, as evidenced by the transduction of vestibular cells using this double window, injection and venting technique. In some embodiments, the entire process can be accomplished in the subject using a relatively atraumatic approach via the ear canal. See FIG. 9. Also see the Examples for additional information regarding the surgical administration procedure.

[0246] Several studies on AAV Anc80 transduction in mice have been published. Different types of viral vectors (e.g., adenoviral vectors, herpes simplex viral vectors) have been considered for gene delivery to the inner ear in animal models (Chen 2001; Wenzel 2007; Husseman 2009, each of which is incorporated herein by reference in its entirety). However, given the acceptable safety profile and long-term transgene expression, including restoration of hearing, cochlear, and vestibular function in knockout and knockin mouse models, rAAV particles are considered to be a promising tool for delivering genes directly to the cochlea (see, e.g., Akil 2012; Kim 2016; Pan 2017; Akil 2019; Al-Moyed 2019; Gyorgy 2019, each of which is incorporated herein by reference in its entirety). Several AAV serotypes have been delivered to the inner ear in both neonatal and adult mice using different surgical approaches and doses (see, e.g., Akil 2012; Askew 2015; Chien 2016; Landegger 2017; Suzuki 2017; Tao 2018; Yoshimura 2018; Akil 2019; Al-Moyed 2019; Gyorgy 2019; Kim 2019; Omichi 2020, each of which is incorporated herein by reference in its entirety). Transduction efficiency, as determined by GFP expression in different cell types in the cochlea and vestibular apparatus, varies depending on the postnatal age of the mouse, the method used to deliver the particles, and the serotype or capsid variant evaluated.

[0247] In some embodiments, AAV Anc80 variants have shown highly efficient targeting of cochlear and vestibular sensory cells (hair cells) and accessory cells of the cochlea and vestibular apparatus in neonatal and adult mice compared to other AAV capsids (see, e.g., Landegger 2017; Suzuki 2017; Omichi 2020, each of which is incorporated herein by reference in its entirety). Anc80 neonatal tropism and gene transfer efficiency were evaluated in vivo in C57BL / 6 mice injected via window window administration on postnatal day 1 (P1) (see, e.g., Landegger 2017, which is incorporated herein by reference in its entirety). Consistent with previous studies, AAV2, AAV6, and AAV8 targeted a low percentage of IHCs, and AAV1 was able to transduce IHCs with higher efficiency, but OHC transduction was minimal. In contrast, AAV Anc80 (1.7E9vg / cochlea) was able to transduce nearly 100% of IHCs and about 90% of OHCs without any adverse effects on cochlea or hearing function (Figure 12) (see, e.g., Landegger 2017, incorporated herein by reference in its entirety). In some embodiments, in the vestibular system, AAV Anc80 transduced type I and type II hair cells of the utricle and cells of the semicircular canal ridge without affecting vestibular function (Figure 13) (see, e.g., Landegger 2017, incorporated herein by reference in its entirety).

[0248] AAV Anc80 tropism and gene transfer efficiency was evaluated in adult (7-week-old) CBA / CaJ mice using different administration routes via the posterior semicircular canal (see, e.g., Suzuki 2017, incorporated herein by reference in its entirety). AAV Anc80 (9.6E8vg / cochlea) targeted cochlear sensory and accessory cells, such as approximately 100% IHCs throughout the length of the cochlea, as well as a significant proportion of OHCs, cells of the spiral limbus and Reissner's membrane, and cells of the modiolus (e.g., spiral ganglion neurons and satellite glial cells) (FIG. 14), while maintaining normal cochlear and hearing function (see, e.g., Suzuki 2017, incorporated herein by reference in its entirety). Multiple cell types in the vestibular system, such as a subset of hair cells and virtually 100% of supporting cells in the utricle and semicircular canal ridge, and saccule (Figure 15), were also transduced, all without adverse effects on vestibular function (see, e.g., Suzuki 2017, incorporated herein by reference in its entirety).

[0249] Recently, AAV Anc80 in vivo transduction in adult (4 weeks) C3H / FeJ mice was evaluated using the delivery route utilized herein (via round window membrane delivery with posterior semicircular canal fenestration) and directly compared to transduction with naturally occurring serotypes AAV1, AAV2, AAV8, and AAV9 (see, e.g., Omichi 2020, incorporated herein by reference in its entirety). All particles resulted in some degree of transduction without adversely affecting hearing function, as demonstrated by control-like (uninjected) ABR thresholds. AAV Anc80 (5.5E9vg / cochlea) transduced virtually 100% of IHCs along the cochlear length and approximately 27-66% of OHCs depending on location in the cochlea (Figure 16). Despite slightly higher transduction efficiency of OHCs by AAV2 (3.68E9vg / cochlea) compared to AAV Anc80, AAV Anc80 maintained a significantly broader tropism than AAV2, as evidenced by eGFP-positive hair cells of the sacculus and spiral ganglion neurons in the cochlea, where AAV2 produced little or no transduction in these same cell types (Figure 16) (see, e.g., Omichi 2020, incorporated herein by reference in its entirety).

[0250] In some embodiments, the ability of AAV Anc80 to target a wide range of inner ear cell types, such as cochlear IHCs and OHCs, supporting cells, cells of the cochlear spiral ganglion, vestibular hair cells of the utricle, saccule, and crista ampullaris, and cochlear and vestibular supporting / accessory cells, in neonatal to adult mice suggests, for example, that AAV Anc80 may advance the development of gene therapy approaches for inner ear disorders.

[0251] Exemplary AAV Anc80 capsid DNA sequence (SEQ ID NO:62)

[0252] Exemplary AAV Anc80L65 capsid amino acid sequence (SEQ ID NO:63) *

[0253] Exemplary AAV Anc80 scaffold amino acid sequence (SEQ ID NO:XX) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAX 1 KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMX 2 AGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGX 3 STNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKX 4 LNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX 5 FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRX 6 LQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTX 7 NQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITX 8 EEEIKTTNPVATEX 9 YGTVATNLQSX 10 NTAPATGTVNSQGALPGMVWQX 11 RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL

Table 4

[0254] composition Among other things, the present disclosure provides compositions. In some embodiments, the compositions comprise a construct as described herein. In some embodiments, the compositions comprise one or more constructs as described herein. In some embodiments, the compositions comprise a plurality of constructs as described herein. In some embodiments, when a composition comprises a plurality of constructs, each of the constructs is different.

[0255] In some embodiments, the composition comprises an AAV particle as described herein. In some embodiments, the composition comprises one or more AAV particles as described herein. In some embodiments, the composition comprises a plurality of AAV particles as described herein. In some embodiments, when a plurality of AAV particles is included in the composition, each of the AAV particles is different.

[0256] In some embodiments, the composition comprises a clarin 1 protein.In some embodiments, the composition comprises a cell.

[0257] In some embodiments, the composition is or comprises a pharmaceutical composition.

[0258] One AAV construct composition In some embodiments, the present disclosure provides compositions or systems that include AAV particles that are composed of a single construct. In some such embodiments, a single construct can deliver a polynucleotide that encodes a functional (e.g., wild-type or other functional, e.g., codon-optimized) copy of the CLRN1 gene. In some embodiments, the construct is or includes an rAAV construct. In some embodiments described herein, a single rAAV construct can express full-length CLRN1 messenger RNA or a characteristic protein thereof in a target cell (e.g., an inner ear cell, e.g., an ocular cell). In some embodiments, a single construct (e.g., any of the constructs described herein) can include a sequence that encodes a functional clarin1 protein (e.g., any construct that generates a functional clarin1 protein). In some embodiments, a single construct (e.g., any of the constructs described herein) can include a sequence that encodes a functional clarin1 protein (e.g., any construct that generates a functional clarin1 protein) and optionally additional polypeptide sequences (e.g., regulatory sequences and / or reporter sequences).

[0259] In some embodiments, a construct composition or system may include any or all of the exemplary construct components described herein. In some embodiments, an exemplary construct is represented by SEQ ID NO: 64. In some embodiments, an exemplary construct is at least 85%, 90%, 95%, 98%, or 99% identical to the sequence represented by SEQ ID NO: 64. One of skill in the art will recognize that the construct may be subject to additional modifications, such as codon optimization, introduction of novel but functionally equivalent (e.g., silent mutations), addition of reporter sequences, and / or other routine modifications.

[0260] In some embodiments, a construct composition or system may include any or all of the exemplary construct components described herein. In some embodiments, an exemplary construct is represented by SEQ ID NO: 68. In some embodiments, an exemplary construct is at least 85%, 90%, 95%, 98%, or 99% identical to the sequence represented by SEQ ID NO: 68. One of skill in the art will recognize that the construct may be subject to additional modifications, such as codon optimization, introduction of novel but functionally equivalent (e.g., silent mutations), addition of reporter sequences, and / or other routine modifications.

[0261] In some embodiments, an exemplary construct comprises a 5'ITR exemplified by SEQ ID NO:21, optionally a cloning site exemplified by SEQ ID NO:46, a CMV enhancer exemplified by SEQ ID NO:38, a CBA promoter exemplified by SEQ ID NO:23, a chimeric intron exemplified by SEQ ID NO:39, optionally a cloning site exemplified by SEQ ID NO:54, a 5'UTR exemplified by SEQ ID NO:40, a CLRN1 coding region exemplified by SEQ ID NO:1, optionally a 3'UTR exemplified by SEQ ID NO:41, a poly(A) site exemplified by SEQ ID NO:44, optionally a cloning site exemplified by SEQ ID NO:49, and a 3'ITR exemplified by SEQ ID NO:22 (see Table 1 below). [Table 5]

[0262] In some embodiments, an exemplary construct comprises a 5'ITR exemplified by SEQ ID NO:21, optionally a cloning site exemplified by SEQ ID NO:46, a CMV enhancer exemplified by SEQ ID NO:38, a CBA promoter exemplified by SEQ ID NO:23, a chimeric intron exemplified by SEQ ID NO:39, optionally a cloning site exemplified by SEQ ID NO:54, a 5'UTR exemplified by SEQ ID NO:40, a CLRN1 coding region exemplified by SEQ ID NO:19, optionally a 3'UTR exemplified by SEQ ID NO:41, a poly(A) site exemplified by SEQ ID NO:44, optionally a cloning site exemplified by SEQ ID NO:49, and a 3'ITR exemplified by SEQ ID NO:22 (see Table 2 below). [Table 6]

[0263] Exemplary construct sequence 1 (SEQ ID NO:64)

[0264] Exemplary construct sequence 2 (SEQ ID NO:68)

[0265] Multiple AAV construct compositions The present disclosure recognizes that some coding sequences encoding proteins (e.g., clarin 1 proteins) can be delivered by splitting the coding sequence into multiple portions, each of which is contained in a different construct. In some embodiments, compositions or systems are provided herein that include at least two different constructs (e.g., 2, 3, 4, 5, or 6). In some embodiments, each of the at least two different constructs comprises a coding sequence encoding a different portion of the coding region (e.g., encoding a target protein, e.g., an inner ear target protein, e.g., an eye target protein, e.g., a clarin 1 protein), wherein each of the encoded 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, 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 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), where the amino acid sequence of each of the encoded portions can optionally overlap with the amino acid sequence of a different one of the encoded portions. One of the at least two different constructs does not code for an active target protein. When introduced into a subject cell (e.g., an animal cell, e.g., a primate cell, e.g., a human cell), the at least two different constructs undergo homologous recombination with each other, where the recombinant nucleic acid codes for an active target protein (e.g., a gene product encoded by the CLRN1 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 clarin 1 protein), where the encoded portion is up to about 820 amino acids (e.g., up to about 10 amino acids, up to about 20 amino acids, up to about 30 amino acids, up to about 60 amino acids, up to about 70 amino acids, up to about 80 amino acids, up to about 90 amino acids, up to about 100 amino acids, up to about 110 amino acids, up to about 120 amino acids,up to about 130 amino acids, up to about 140 amino acids, up to about 150 amino acids, up to about 160 amino acids, up to about 170 amino acids, up to about 180 amino acids, up to about 190 amino acids, up to about 200 amino acids, up to about 210 amino acids, up to about 220 amino acids, up to about 230 amino acids, up to about 240 amino acids, up to about 250 amino acids, up to about 260 amino acids, up to about 270 amino acids, up to about 280 amino acids, up to about 290 amino acids, up to about 300 amino acids, amino acids, up to about 310 amino acids, up to about 320 amino acids, up to about 330 amino acids, up to about 340 amino acids, up to about 350 amino acids, up to about 360 amino acids, up to about 370 amino acids, up to about 380 amino acids, up to about 390 amino acids, up to about 400 amino acids, up to about 410 amino acids, up to about 420 amino acids, up to about 430 amino acids, up to about 440 amino acids, up to about 450 amino acids, up to about 460 amino acids, up to about 470 amino acids, up to about 480 amino acids, up to about 490 amino acids, up to about 500 amino acids, up to about 510 amino acids, up to about 520 amino acids, up to about 530 amino acids, up to about 540 amino acids, up to about 550 amino acids, up to about 560 amino acids, up to about 570 amino acids, up to about 580 amino acids, up to about 590 amino acids, up to about 600 amino acids, up to about 610 amino acids, up to about 620 amino acids, up to about 630 amino acids, up to about 640 amino acids, up to about 650 amino acids , up to about 660 amino acids, up to about 670 amino acids, up to about 680 amino acids, up to about 690 amino acids, up to about 700 amino acids, up to about 710 amino acids, up to about 720 amino acids, up to about 730 amino acids, up to about 740 amino acids, up to about 750 amino acids, up to about 760 amino acids, up to about 770 amino acids, up to about 780 amino acids, up to about 790 amino acids, up to about 800 amino acids, up to about 810 amino acids, or up to about 820 amino acids.

[0266] In some embodiments, at least one of the constructs comprises a nucleotide sequence that spans two adjacent exons of a target genomic DNA (e.g., an inner ear target genomic DNA, e.g., a CLRN1 genomic DNA) and lacks any naturally occurring intron sequence between the two adjacent exons.

[0267] In some embodiments, the amino acid sequence of each encoded portion of the construct does not even partially overlap with the amino acid sequence of a different one of the encoded portions. In some embodiments, the amino acid sequence of the encoded portion of the construct partially overlaps with the amino acid sequence of the encoded portion of a different construct. In some embodiments, the amino acid sequence of the encoded portion of each construct partially overlaps with the amino acid sequence of the encoded portion of at least one different construct.In some embodiments, the overlapping amino acid sequences are between about 10 amino acid residues and about 300 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, about 190 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 600 amino acids, about 610 amino acids, about 62 Amino acids, about 180 amino acids, 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 610 600 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.

[0268] 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 the nucleotide sequence of an intron (e.g., any of the exemplary introns of SEQ ID NO:5 described herein) present in the target genomic DNA (e.g., inner ear cell target genomic DNA or ocular cell target genomic DNA (e.g., CLRN1 genomic DNA)). In some embodiments, the different intron segments overlap. In some embodiments, the different intron segments are up to about 12,000 nucleotides in length (e.g., up to about 100 nucleotides, up to about 200 nucleotides, up to about 300 nucleotides, up to about 600 nucleotides, up to about 700 nucleotides, up to about 800 nucleotides, up to about 900 nucleotides, up to about 1,000 nucleotides, up to about 1,100 nucleotides, up to about 1,200 nucleotides, up to about 1,300 nucleotides, up to about 1,400 nucleotides, up to about 1,500 nucleotides, up to about 1,600 nucleotides, up to about 1,700 nucleotides, up to about 1,800 nucleotides, up to about 1,900 nucleotides, up to about 2,000 nucleotides, up to about 2,100 nucleotides, up to about 2,200 nucleotides, up to about 2,300 nucleotides, up to about 2,400 nucleotides, up to about 2,500 nucleotides, up to about 2,600 nucleotides, up to about 2,700 nucleotides, up to about 2,800 nucleotides, up to about 2,900 nucleotides, up to about 3,000 nucleotides, up to about 3,100 nucleotides, up to about 3,200 nucleotides, up to about 3,300 nucleotides, up to about 3,400 nucleotides, up to about 3,500 nucleotides, up to about 3,600 nucleotides, up to about 3,700 nucleotides, up to about 3,800 nucleotides, up to about 3,900 nucleotides, up to about 4,000 nucleotides, up to about 4,100 nucleotides, up to about 4,200 nucleotides, up to about 4,300 nucleotides, up to about 4,400 nucleotides, up to about 4,500 nucleotides, up to about 4,600 nucleotides, up to about 4,700 nucleotides, up to about 4,800 nucleotides, up to about 4,900 nucleotides, up to about 5,000 nucleotides, up to about 5,100 nucleotides, up to about 5,200 nucleotides, up to about 5,300 nucleotides, up to about 5,400 nucleotides, up to about 5,500 nucleotides, up to about 5,600 nucleotides, up to about 5,700 nucleotides, up to about 5,800 nucleotides, up to about 5,900 nucleotides, up to about 6,000 nucleotides, up to about 6,100 nucleotides, up to about 6,200 nucleotides, up to about 6,300 nucleotides, up to about 6,400 nucleotides, up to about 6,500 nucleotides up to about 6,600 nucleotides, up to about 6,700 nucleotides, up to about 6,800 nucleotides, up to about 6,900 nucleotides, up to about 7,000 nucleotides, up to about 7,100 nucleotides, up to about 7,200 nucleotides, up to about 7,300 nucleotides, up to about 7,400 nucleotides, up to about 7,500 nucleotides, up to about 7,600 nucleotides, up to about 7,700 nucleotides, up to about 7,800 nucleotides, up to about 7,900 nucleotides, up to about 8,000 nucleotides, up to about 8,100 nucleotides, up to about 8, 200 nucleotides, up to about 8,300 nucleotides, up to about 8,400 nucleotides, up to about 8,500 nucleotides, up to about 8,600 nucleotides, up to about 8,700 nucleotides, up to about 8,800 nucleotides, up to about 8,900 nucleotides, up to about 9,000 nucleotides, up to about 9,100 nucleotides, up to about 9,200 nucleotides, up to about 9,300 nucleotides, up to about 9,400 nucleotides, up to about 9,500 nucleotides, up to about 9,600 nucleotides, up to about 9,700 nucleotides, up to about 9,800 nucleotides up to about 9,900 nucleotides, up to about 10,000 nucleotides, up to about 10,100 nucleotides, up to about 10,200 nucleotides, up to about 10,300 nucleotides, up to about 10,400 nucleotides, up to about 10,500 nucleotides, up to about 10,600 nucleotides, up to about 10,700 nucleotides, up to about 10,800 nucleotides, up to about 10,900 nucleotides, up to about 11,000 nucleotides, up to about 11,100 nucleotides, up to about 11,200 nucleotides, up to about 11,300 nucleotides, up to about 11,400 nucleotides, up to about 11,500 nucleotides, up to about 11,600 nucleotides, up to about 11,700 nucleotides, up to about 11,800 nucleotides, up to about 11,900 nucleotides, or up to about 12,000 nucleotides), the sequences overlap. In some embodiments, the nucleotide sequence that overlaps in any two of the different constructs can include part or all of one or more exons (e.g., any one or more of the exemplary exons of SEQ ID NO:5 described herein) of a target gene (e.g., an inner ear cell target gene or an eye cell target gene (e.g., the CLRN1 gene)).

[0269] 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 can comprise a coding sequence encoding the N-terminal portion of a protein (e.g., a clarin 1 protein), which can be referred to as the lead portion, the first construct, or the 5' portion (e.g., the N-terminal portion of an inner ear cell protein, e.g., the N-terminal portion of an eye cell protein, e.g., the N-terminal portion of a clarin 1 protein). In some examples, the N-terminal portion of the target gene is at least about 10 amino acids in length (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, no 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). In some examples, the first construct includes one or both of a promoter (e.g., any promoter described herein or known in the art) and a Kozak sequence (e.g., any exemplary Kozak sequence 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 examples, the second of the two distinct 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 an ocular cell targeted protein, e.g., a C-terminal portion of a clarin 1 protein). In some examples, the C-terminal portion of the target protein is at least about 10 amino acids in length (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, The second construct may be 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. In some examples, the second construct further comprises a poly(A) sequence. ,

[0270] 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 an N-terminal portion of the inner ear cell targeting protein or the eye cell targeting protein (e.g., SEQ ID NO: 10, 11, 12, 13, or 14) that is encoded by a portion including amino acid 1 to about amino acid 820, 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 820, or any subrange within this range (e.g., amino acid 1 to at least about amino acid 9, In all cases, the amino acid sequence is about amino acid position 70, amino acid position 1 to at least about amino acid position 80, amino acid position 1 to at least about amino acid position 90, amino acid position 1 to at least about amino acid position 100, amino acid position 1 to at least about amino acid position 110, amino acid position 1 to at least about amino acid position 120, amino acid position 1 to at least about amino acid position 130, amino acid position 1 to at least about amino acid position 140, amino acid position 1 to at least about amino acid position 150, amino acid position 1 to at least about amino acid position 160, amino acid position 1 to at least about amino acid position 170, amino acid position 1 to at least about amino acid position 180, amino acid position 180, amino acid position 1 to at least about amino acid position 190, amino acid position 1 to at least about amino acid position 200, amino acid position 1 to at least about amino acid position 210, amino acid position 1 to at least about amino acid position 220, amino acid position 1 to at least about amino acid position 230, amino acid position 1 to at least about amino acid position 240, amino acid position 1 to at least about amino acid position 250, amino acid position 1 to at least about amino acid position 260, amino acid position 1 to at least about amino acid position 270, amino acid position 1 to at least about amino acid position 280, amino acid position 1 to at least about amino acid position 29 0, amino acid position 1 to at least about amino acid position 300, amino acid position 1 to at least about amino acid position 310, amino acid position 1 to at least about amino acid position 320, amino acid position 1 to at least about amino acid position 330, amino acid position 1 to at least about amino acid position 340, amino acid position 1 to at least about amino acid position 350, amino acid position 1 to at least about amino acid position 360, amino acid position 1 to at least about amino acid position 370, amino acid position 1 to at least about amino acid position 380, amino acid position 1 to at least about amino acid position 390, amino acid position 1 to at least about amino acid position 400,Amino acid position 1 to at least about amino acid position 410, amino acid position 1 to at least about amino acid position 420, amino acid position 1 to at least about amino acid position 430, amino acid position 1 to at least about amino acid position 440, amino acid position 1 to at least about amino acid position 450, amino acid position 1 to at least about amino acid position 460, amino acid position 1 to at least about amino acid position 470, amino acid position 1 to at least about amino acid position 480, amino acid position 1 to at least about amino acid position 490, amino acid position 1 to at least about amino acid position 500, amino acid position 1 to at least about amino acid position 510, amino acid position 510, amino acid position 1 to at least about amino acid position 520, amino acid position 1 to at least about amino acid position 530, amino acid position 1 to at least about amino acid position 540, amino acid position 1 to at least about amino acid position 550, amino acid position 1 to at least about amino acid position 560, amino acid position 1 to at least about amino acid position 570, amino acid position 1 to at least about amino acid position 580, amino acid position 1 to at least about amino acid position 590, amino acid position 1 to at least about amino acid position 600, amino acid position 1 to at least about amino acid position 610, amino acid position 1 to at least about amino acid position 620, at least about amino acid position 620, amino acid position 1 to at least about amino acid position 630, amino acid position 1 to at least about amino acid position 640, amino acid position 1 to at least about amino acid position 650, amino acid position 1 to at least about amino acid position 660, amino acid position 1 to at least about amino acid position 670, amino acid position 1 to at least about amino acid position 680, amino acid position 1 to at least about amino acid position 690, amino acid position 1 to at least about amino acid position 700, amino acid position 1 to at least about amino acid position 710, amino acid position 1 to at least about amino acid position 720, amino acid 1 to at least about amino acid 730, amino acid 1 to at least about amino acid 740, amino acid 1 to at least about amino acid 750, amino acid 1 to at least about amino acid 760, amino acid 1 to at least about amino acid 770, amino acid 1 to at least about amino acid 780, amino acid 1 to at least about amino acid 790, amino acid 1 to at least about amino acid 800, amino acid 1 to at least about amino acid 810, or amino acid 1 to at least about amino acid 820). In some examples where the number of different constructs in the composition is two,The N-terminal portion of the precursor inner ear cell targeting protein or eye cell targeting protein can be from at most amino acid 1 to amino acid 820 of the inner ear cell targeting protein or eye cell targeting protein (e.g., SEQ ID NO: 10, 11, 12, 13, or 14), or any subrange within this range (e.g., amino acid 1 up to about amino acid 10, amino acid 1 up to about amino acid 20, amino acid 1 up to about amino acid 30, amino acid 1 up to about amino acid 60, amino acid 1 up to about amino acid 70, amino acid 1 up to about amino acid 80, amino acid 1 up to about amino acid 90, amino acid 1 up to about amino acid 100, amino acid 1 up to about amino acid 110, amino acid 1 up to about amino acid 120, amino acid 1 up to about amino acid 130, amino acid 1 up to about amino acid 140, amino acid 1 up to about amino acid 150, amino acid 1 up to about amino acid 160, amino acid 1 up to about amino acid 170, amino acid 1 up to about amino acid 180, amino acid 1 up to about amino acid 190, amino acid 1 up to about amino acid 200, amino acid 1 up to about amino acid 210, amino acid 1 up to about amino acid 220, amino acid 1 up to about amino acid 230, amino acid 1 up to about amino acid 240, amino acid 1 up to about amino acid 250, amino acid 1 up to about amino acid 260, amino acid 1 up to about amino acid 270, amino acid 1 up to about amino acid 280, amino acid 1 up to about amino acid 290, amino acid 1 up to about amino acid 290, amino acid 1 up to about amino acid 290, amino acid 1 up to about amino acid 290, amino acid Amino acid position 90, amino acid position 1 to approximately amino acid position 100, amino acid position 1 to approximately amino acid position 110, amino acid position 1 to approximately amino acid position 120, amino acid position 1 to approximately amino acid position 130, amino acid position 1 to approximately amino acid position 140, amino acid position 1 to approximately amino acid position 150, amino acid position 1 to approximately amino acid position 160, amino acid position 1 to approximately amino acid position 170, amino acid position 1 to approximately amino acid position 180, amino acid position 1 to approximately amino acid position 190, amino acid position 1 to approximately amino acid position 200, amino acid position 1 to approximately amino acid position 210, amino acid position 1 to approximately amino acid position 220, amino acid 1 to approximately 230, amino acid 1 to approximately 240, amino acid 1 to approximately 250, amino acid 1 to approximately 260, amino acid 1 to approximately 270, amino acid 1 to approximately 280, amino acid 1 to approximately 290, amino acid 1 to approximately 300, amino acid 1 to approximately 310, amino acid 1 to approximately 320, amino acid 1 to approximately 330, amino acid 1 to approximately 340, amino acid 1 to approximately 35 0, amino acid position 1 to a maximum of about amino acid position 360, amino acid position 1 to a maximum of about amino acid position 370, amino acid position 1 to a maximum of about amino acid position 380, amino acid position 1 to a maximum of about amino acid position 390, amino acid position 1 to a maximum of about amino acid position 400, amino acid position 1 to a maximum of about amino acid position 410, amino acid position 1 to a maximum of about amino acid position 420, amino acid position 1 to a maximum of about amino acid position 430, amino acid position 1 to a maximum of about amino acid position 440, amino acid position 1 to a maximum of about amino acid position 450, amino acid position 1 to a maximum of about amino acid position 460, amino acid position 1 to a maximum of about amino acid position 470, amino acid position 1 to a maximum of about amino acid position 480,Amino acid position 1 to approximately amino acid position 490, amino acid position 1 to approximately amino acid position 500, amino acid position 1 to approximately amino acid position 510, amino acid position 1 to approximately amino acid position 520, amino acid position 1 to approximately amino acid position 530, amino acid position 1 to approximately amino acid position 540, amino acid position 1 to approximately amino acid position 550, amino acid position 1 to approximately amino acid position 560, amino acid position 1 to approximately amino acid position 570, amino acid position 1 to approximately amino acid position 580, amino acid position 1 to approximately amino acid position 590, amino acid position 1 to approximately amino acid position 600, amino acid position 1 to approximately amino acid position 610, amino acid position 1 to approximately amino acid position 620, amino acid position 1 to approximately amino acid position 630, amino acid position 1 to approximately amino acid position 640, amino acid position 1 to approximately amino acid position 650, amino acid position 1 amino acid position 1 to about amino acid position 660 at most, amino acid position 1 to about amino acid position 670 at most, amino acid position 1 to about amino acid position 680 at most, amino acid position 1 to about amino acid position 690 at most, amino acid position 1 to about amino acid position 700 at most, amino acid position 1 to about amino acid position 710 at most, amino acid position 1 to about amino acid position 720 at most, amino acid position 1 to about amino acid position 730 at most, amino acid position 1 to about amino acid position 740 at most, amino acid position 1 to about amino acid position 750 at most, amino acid position 1 to about amino acid position 760 at most, amino acid position 1 to about amino acid position 770 at most, amino acid position 1 to about amino acid position 780 at most, amino acid position 1 to about amino acid position 790 at most, amino acid position 1 to about amino acid position 800 at most, amino acid position 1 to about amino acid position 810 at most, or amino acid position 1 to about amino acid position 820 at most).

[0271] 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 final amino acid (e.g., about amino acid position 820) to about amino acid position 1 of the inner ear cell targeting protein or the eye cell targeting protein (e.g., SEQ ID NO: 10, 11, 12, 13, or 14), or any subrange within this range (e.g., from amino acid position 820 to at least about amino acid position 10, from amino acid position 820 to at least about amino acid position 20, from amino acid position 820 to at least about amino acid position 30, from amino acid position 820 to at least about amino acid position 40, from amino acid position 820 to at least about amino acid position 50, from amino acid position 820 to at least about amino acid position 60, from amino acid position 820 to at least about amino acid position 70, from amino acid position 820 to at least about amino acid position 81, amino acid position 60, amino acid position 820 to at least about amino acid position 70, amino acid position 820 to at least about amino acid position 80, amino acid position 820 to at least about amino acid position 90, amino acid position 820 to at least about amino acid position 100, amino acid position 820 to at least about amino acid position 110, amino acid position 820 to at least about amino acid position 120, amino acid position 820 to at least about amino acid position 130, amino acid position 820 to at least about amino acid position 140, amino acid position 820 to at least about amino acid position 150, amino acid position 820 to at least about amino acid position 160, amino acid position 20 to at least about amino acid position 170, amino acid position 820 to at least about amino acid position 180, amino acid position 820 to at least about amino acid position 190, amino acid position 820 to at least about amino acid position 200, amino acid position 820 to at least about amino acid position 210, amino acid position 820 to at least about amino acid position 220, amino acid position 820 to at least about amino acid position 230, amino acid position 820 to at least about amino acid position 240, amino acid position 820 to at least about amino acid position 250, amino acid position 820 to at least about amino acid position 260, amino acid position 820 to at least about amino acid position Positions 820 to at least about amino acid 270, 820 to at least about amino acid 280, 820 to at least about amino acid 290, 820 to at least about amino acid 300, 820 to at least about amino acid 310, 820 to at least about amino acid 320, 820 to at least about amino acid 330, 820 to at least about amino acid 340, 820 to at least about amino acid 350, 820 to at least about amino acid 360, 820 to at least about amino acid 370,amino acid position 820 to at least about amino acid position 380, amino acid position 820 to at least about amino acid position 390, amino acid position 820 to at least about amino acid position 400, amino acid position 820 to at least about amino acid position 410, amino acid position 820 to at least about amino acid position 420, amino acid position 820 to at least about amino acid position 430, amino acid position 820 to at least about amino acid position 440, amino acid position 820 to at least about amino acid position 450, amino acid position 820 to at least about amino acid position 460, amino acid position 820 to at least about amino acid position 470, amino acid position 820 to at least about amino acid position 480, 0 to at least about amino acid position 480, 820 to at least about amino acid position 490, 820 to at least about amino acid position 500, 820 to at least about amino acid position 510, 820 to at least about amino acid position 520, 820 to at least about amino acid position 530, 820 to at least about amino acid position 540, 820 to at least about amino acid position 550, 820 to at least about amino acid position 560, 820 to at least about amino acid position 570, 820 to at least about amino acid position 580 In each case, the amino acid sequence is from about amino acid position 580, from amino acid position 820 to at least about amino acid position 590, from amino acid position 820 to at least about amino acid position 600, from amino acid position 820 to at least about amino acid position 610, from amino acid position 820 to at least about amino acid position 620, from amino acid position 820 to at least about amino acid position 630, from amino acid position 820 to at least about amino acid position 640, from amino acid position 820 to at least about amino acid position 650, from amino acid position 820 to at least about amino acid position 660, from amino acid position 820 to at least about amino acid position 670, from amino acid position 820 to at least about amino acid position 680, amino acid position 680, amino acid position 820 to at least about amino acid position 690, amino acid position 820 to at least about amino acid position 700, amino acid position 820 to at least about amino acid position 710, amino acid position 820 to at least about amino acid position 720, amino acid position 820 to at least about amino acid position 730, amino acid position 820 to at least about amino acid position 740, amino acid position 820 to at least about amino acid position 750, amino acid position 820 to at least about amino acid position 760, amino acid position 820 to at least about amino acid position 770, amino acid position 820 to at least about amino acid position 780,In some examples where the number of distinct constructs in the composition is two, the C-terminal portion of the precursor inner ear cell targeted protein or ocular cell targeted protein can include the final amino acid (e.g., about amino acid 820) to about amino acid 1 of the inner ear cell targeted protein or ocular cell targeted protein (e.g., SEQ ID NOs: 10, 11, 12, 13, or 14), or any subrange within this range (e.g., amino acid 820 up to about amino acid 10, amino acid 820 up to about amino acid 20, amino acid 820 up to about amino acid 21, amino acid 820 up to about amino acid 22, amino acid 820 up to about amino acid 23, amino acid 820 up to about amino acid 24, amino acid 820 up to about amino acid 25, amino acid 820 up to about amino acid 26, amino acid 820 up to about amino acid 27, amino acid 820 up to about amino acid 28, amino acid 820 up to about amino acid 29, amino acid 820 up to about amino acid 30, amino acid 820 up to about amino acid 31, amino acid 820 up to about amino acid 32, amino acid 820 up to about amino acid 33, amino acid 820 up to about amino acid 34, amino acid 820 up to about amino acid 35, amino acid 820 up to about amino acid 36, amino acid 820 up to about amino acid 37, amino acid 820 up to about amino acid 38, amino acid 820 up to about amino acid 39 ... amino acid position 30 at most, amino acid position 820 to approximately amino acid position 60 at most, amino acid position 820 to approximately amino acid position 70 at most, amino acid position 820 to approximately amino acid position 80 at most, amino acid position 820 to approximately amino acid position 90 at most, amino acid position 820 to approximately amino acid position 100 at most, amino acid position 820 to approximately amino acid position 110 at most, amino acid position 820 to approximately amino acid position 120 at most, amino acid position 820 to approximately amino acid position 130 at most, amino acid position 820 to approximately amino acid position 140 at most, amino acid position 820 to approximately amino acid position 150 at most, amino acid position 820 to approximately amino acid position 160, amino acid position 820 to approximately amino acid position 170, amino acid position 820 to approximately amino acid position 180, amino acid position 820 to approximately amino acid position 190, amino acid position 820 to approximately amino acid position 200, amino acid position 820 to approximately amino acid position 210, amino acid position 820 to approximately amino acid position 220, amino acid position 820 to approximately amino acid position 230, amino acid position 820 to approximately amino acid position 240, amino acid position 820 to approximately amino acid position 250, amino acid position 820 to approximately amino acid position 260 amino acid position 260, amino acid position 820 to a maximum of approximately amino acid position 270, amino acid position 820 to a maximum of approximately amino acid position 280, amino acid position 820 to a maximum of approximately amino acid position 290, amino acid position 820 to a maximum of approximately amino acid position 300, amino acid position 820 to a maximum of approximately amino acid position 310, amino acid position 820 to a maximum of approximately amino acid position 320, amino acid position 820 to a maximum of approximately amino acid position 330, amino acid position 820 to a maximum of approximately amino acid position 340, amino acid position 820 to a maximum of approximately amino acid position 350, amino acid position 820 to a maximum of approximately amino acid position 360,820 to approximately 370 amino acids, 820 to approximately 380 amino acids, 820 to approximately 390 amino acids, 820 to approximately 400 amino acids, 820 to approximately 410 amino acids, 820 to approximately 420 amino acids, 820 to approximately 430 amino acids, 820 to approximately 440 amino acids, 820 to approximately 450 amino acids, 820 to approximately 460 amino acids, 820 to approximately 470 amino acids, 820 to approximately 480 amino acids, 820 to approximately 490 amino acids, 820 to approximately 500 amino acids, 820 to approximately 510 amino acids, 820 to approximately 520 amino acids, 820 to approximately 530 amino acids, 820 to approximately 540 amino acids, 820 to approximately 550 amino acids, 820 to approximately 560 amino acids, 820 to approximately 570 amino acids, 820 to approximately 580 amino acids, 820 to approximately 590 amino acids, 820 to approximately 600 amino acids, 820 to approximately 610 amino acids, 820 to approximately 620 amino acids, 820 to approximately 630 amino acids, 820 to approximately 640 amino acids, 820 to approximately 650 amino acids, 820 to approximately 660 amino acids, 820 to approximately 670 amino acids, 820 to approximately 680 amino acids, 820 to approximately 690 amino acids, 820 to approximately 700 amino acids, 820 to approximately 710 amino acids, 820 to approximately 720 amino acids, 820 to approximately 730 amino acids, 820 to approximately 740 amino acids, 820 to approximately 750 amino acids, 820 80, 820 to approximately 490, 820 to approximately 500, 820 to approximately 510, 820 to approximately 520, 820 to approximately 530, 820 to approximately 540, 820 to approximately 550, 820 to approximately 560, 820 to approximately 570, 820 to approximately 580, 820 to approximately 590, 820 to approximately 590 amino acid position 600, amino acid position 820 to a maximum of about amino acid position 610, amino acid position 820 to a maximum of about amino acid position 620, amino acid position 820 to a maximum of about amino acid position 630, amino acid position 820 to a maximum of about amino acid position 640, amino acid position 820 to a maximum of about amino acid position 650, amino acid position 820 to a maximum of about amino acid position 660, amino acid position 820 to a maximum of about amino acid position 670, amino acid position 820 to a maximum of about amino acid position 680, amino acid position 820 to a maximum of about amino acid position 690, amino acid position 820 to a maximum of about amino acid position 700, amino acid position 820 to a maximum of about amino acid position 710, amino acid position 820 to a maximum of about amino acid position 700 amino acid position 720, amino acid position 820 to a maximum of about amino acid position 730, amino acid position 820 to a maximum of about amino acid position 740, amino acid position 820 to a maximum of about amino acid position 750, amino acid position 820 to a maximum of about amino acid position 760, amino acid position 820 to a maximum of about amino acid position 770, amino acid position 820 to a maximum of about amino acid position 780, amino acid position 820 to a maximum of about amino acid position 790, amino acid position 820 to a maximum of about amino acid position 800, amino acid position 820 to a maximum of about amino acid position 810, or amino acid position 820 to a maximum of about amino acid position 820, or a sequence of any length therebetween.

[0272] In some embodiments, the splice site is involved in trans-splicing. In some embodiments, a splice donor site (see, for example, Trapani et al. EMBO Mol. Med. 6(2):194-211, 2014, incorporated herein by reference in its entirety) follows the coding sequence in the N-terminal construct. In the C-terminal construct, a splice acceptor site can be subcloned immediately before the coding sequence of CLRN1. In some embodiments, a silent mutation can be introduced into the coding sequence, which generates an additional site for restriction digestion.

[0273] In some embodiments, any of the constructs provided herein can be included in a composition suitable for administration to an animal to ameliorate symptoms associated with symptomatic and / or non-syndromic hearing loss.

[0274] In some embodiments, any of the constructs provided herein can be included in a composition suitable for administration to an animal to ameliorate symptoms associated with vision loss.

[0275] Pharmaceutical Compositions Among other things, the present disclosure provides pharmaceutical compositions. In some embodiments, the compositions provided herein are suitable for administration to animals to improve symptoms associated with symptomatic and / or non-syndromic hearing loss. In some embodiments, the compositions provided herein are suitable for administration to animals to improve symptoms associated with vision loss.

[0276] In some embodiments, a pharmaceutical composition of the present disclosure can include, for example, a polynucleotide, such as one or more constructs, as described herein. In some embodiments, a pharmaceutical composition can include one or more AAV particles, such as one or more rAAV constructs encapsulated by one or more AAV serotype capsids, as described herein.

[0277] In some embodiments, the pharmaceutical composition may include one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients. As used herein, the term "pharma-ceutically acceptable carriers" includes solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like, that are compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into any of the compositions described herein. Such compositions may include one or more buffers (e.g., neutral buffered saline, phosphate buffered saline, and the like); 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, for example, an injectable solution, an injectable gel, a drug-releasing capsule, and the like.

[0278] 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, polymer nanoparticles, minicircle DNA, and / or CELiD DNA.

[0279] In some embodiments, the therapeutic composition is formulated to include a synthetic perilymph solution. For example, in some embodiments, the synthetic perilymph solution includes 20-200 mM NaCl; 1-5 mM KCl; 0.1-10 mM CaCl2; 1-10 mM glucose; and 2-50 mM HEPES at a pH of about 6 to about 9. In some embodiments, the therapeutic composition is formulated to include a physiologically suitable solution. For example, in some embodiments, the physiologically suitable solution includes commercially available 1x PBS containing pluronic acid F68, prepared to a final concentration of 8.10 mM disodium phosphate, 1.5 mM monopotassium phosphate, 2.7 mM potassium chloride, 172 mM sodium chloride, and 0.001% pluronic acid F68). In some embodiments, an alternative pluronic acid is utilized. In some embodiments, an alternative ion concentration is utilized.

[0280] In some embodiments, any of the pharmaceutical compositions described herein may further comprise one or more agents that facilitate the entry of any of the nucleic acids or 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, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.), formulations from Mirus Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PhaseRX polymer formulations, including, but not limited to, SMARTT POLYMER TECHNOLOGY® (PhaseRX, Seattle, Wash.), DMRI / DOPE, poloxamers, VAXFECTIN® adjuvants from Vical (San Diego, Calif.), chitosan, cyclodextrin, 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 other polymers from other companies. 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 delivering oligonucleotides to mammalian cells in vivo (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 herein).

[0281] In some embodiments, the composition comprises a pharma- ceutically acceptable carrier (e.g., phosphate buffered saline, saline, or bacteriostatic water). Once formulated, the solution is administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulation is easily administered in a variety of dosage forms, such as, for example, injectable solutions, injectable gels, drug release capsules, and the like.

[0282] In some embodiments, the compositions provided herein can be formulated to be compatible with, for example, its intended route of administration, non-limiting examples of which include local administration (e.g., intracochlear administration).

[0283] In some embodiments, the delivery approaches disclosed herein include synthetic AAV capsids (e.g., AAV Anc80) for transduction of inner ear cells, and / or devices for targeted delivery directly to the cochlea. In certain embodiments, the present disclosure provides methods and compositions suitable for transduction of inner ear cells. In some embodiments, transduction of inner ear cells can allow long-term expression of CLRN1 protein in the cochlea with minimal systemic exposure.

[0284] In some embodiments, the delivery approaches disclosed herein include synthetic AAV capsids (e.g., AAV Anc80) for transduction of ocular cells, and / or devices for targeted delivery directly to the eye. In certain embodiments, the present disclosure provides methods and compositions suitable for transduction of ocular cells. In some embodiments, transduction of ocular cells can allow long-term expression of CLRN1 protein in the eye with minimal systemic exposure.

[0285] In some embodiments, the compositions provided include one nucleic acid construct. In some embodiments, the compositions provided include two or more different constructs. In some embodiments, the compositions include one nucleic acid construct comprising a coding sequence encoding a clarin 1 protein and / or a functional characteristic portion thereof. In some embodiments, the compositions include one nucleic acid construct comprising a coding sequence encoding a clarin 1 protein and / or a functional characteristic portion thereof, which, when introduced into a mammalian cell, integrates the coding sequence into the genome of the mammalian cell. In some embodiments, the compositions include at least two different constructs, e.g., constructs comprising coding sequences encoding different portions of the clarin 1 protein, which can be combined to generate a sequence encoding an active clarin 1 protein (e.g., a full-length clarin 1 protein) in a mammalian cell, thereby treating associated symptomatic or non-syndromic sensorineural hearing loss in a subject in need thereof.

[0286] Also provided is a kit that includes any of the compositions described herein.In some embodiments, the kit can include a solid composition (e.g., a lyophilized composition that includes at least two different constructs described herein) and a liquid for solubilizing the lyophilized composition.In some embodiments, the kit can include a pre-filled syringe that includes any of the compositions described herein.

[0287] In some embodiments, the kit includes a vial containing any of the compositions described herein (eg, formulated as an aqueous composition, eg, an aqueous pharmaceutical composition).

[0288] In some embodiments, the kits may include instructions for practicing any of the methods described herein.

[0289] Genetically modified cells The 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., monkey cells, feline cells, canine cells, etc.). One of skill in the art will appreciate 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.

[0290] In some embodiments, the animal cell can be any cell of the inner ear, such as a hair and / or supporting cell. Non-limiting examples of such cells include Hensen's cells, Deiters' cells, cells of the endolymphatic sac and duct, transitional cells of the saccule, utricle, and ampulla, inner and outer hair cells, spiral ligament cells, spiral ganglion cells, spiral eminence cells, outer globular cells, marginal cells, intermediate cells, basal cells, inner pillar cells, outer pillar cells, Clausis cells, inner border cells, inner phalangeal cells, or cells of the stria vascularis.

[0291] In some embodiments, the animal cell is a specific 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.

[0292] In some embodiments, the animal cell can be any cell of the eye, such as a supporting cell, non-limiting examples of which include a rod cell, a cone cell, a pigment cell, a horizontal cell, a bipolar cell, an amacrine cell, a ganglion cell, a photoreceptor cell, a Muller cell, or a retinal cell.

[0293] In some embodiments, the animal cell is an eye-specific cell. In some embodiments, the animal cell is an ocular cell. In some embodiments, the animal cell is a retinal cell.

[0294] In some embodiments, the animal cells are in vitro. In some embodiments, the animal cells are of a cell type that is endogenously present in an animal, such as a primate and / or human. In some embodiments, the animal cells are autologous cells obtained from an animal and cultured ex vivo.

[0295] method Hearing loss Among other things, the 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, provided herein, in some embodiments, is a method that includes 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 and / or auditory cell target gene having a mutation that results in reduced expression and / or activity of a supporting 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 may further include detecting a mutation in the inner ear cell target gene in the subject. Some embodiments of any of these methods may further include identifying or diagnosing the subject as having nonsyndromic or syndromic sensorineural hearing loss.

[0296] In some embodiments, provided herein are methods of correcting an inner ear cell target gene defect (e.g., a defect in CLRN1) 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 a therapeutically effective amount of any of the compositions described herein to the inner ear of the subject, wherein administering repairs and / or ameliorates the inner ear cell target gene defect in any cell subset of the inner ear of the subject. In some embodiments, the inner ear target cells can be sensory cells, such as hair cells, and / or non-sensory cells, such as supporting cells, and / or all or any subset of inner ear cells.

[0297] Also provided herein is a method of 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), the method comprising administering a therapeutically effective amount of any of the compositions described herein to the inner ear of the subject, where the administration results in an increase in the expression level of an inner ear cell target protein (e.g., clarin 1 protein) in any subset of cells of the inner ear of the subject. In some embodiments, the inner ear target cells can be sensory cells, such as hair cells, and / or non-sensory cells, such as supporting cells, and / or all or any subset of inner ear cells.

[0298] Also provided herein are methods of treating hearing loss, e.g., nonsyndromic 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, comprising administering a therapeutically effective amount of any of the compositions described herein to the inner ear of the subject. In some embodiments of any of the methods provided herein, the subject has Usher syndrome type III.

[0299] Also provided herein are methods of restoring synapses and / or preserving spiral ganglion nerves in a subject identified or diagnosed as having an inner ear disorder, comprising administering a therapeutically effective amount of any of the compositions described herein to the subject's inner ear.

[0300] Also provided herein are methods comprising administering a composition disclosed herein, e.g., rAAV-CLRN1, to a subject, e.g., a mammal, e.g., a human, e.g., a patient, having Usher syndrome type III. In some embodiments, the composition disclosed herein is delivered by surgical delivery, e.g., to the cochlea.

[0301] Also provided herein are methods of reducing the size of the vestibular aqueduct and / or restoring the vestibular aqueduct to an appropriate size.Also provided herein are methods of restoring endolymphatic pH to an appropriate and / or acceptable level in a subject identified or diagnosed with an inner ear disorder, comprising administering a therapeutically effective amount of any of the compositions described herein to the subject's inner ear.

[0302] Also provided herein are methods comprising administering a therapeutically effective amount of any of the compositions described herein to the inner ear of a subject.

[0303] Also provided herein is a surgical method 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 administering intracochlearly 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 into or through the first incision.

[0304] 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 oval window membrane of the cochlea. 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 oval window membrane of the cochlea. In some embodiments of any of the methods described herein, the composition is administered with a medical device capable of creating multiple incision points in the oval 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 substantially circular first surface, 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 capable of transporting the composition. In some embodiments, the medical device comprises a means for generating at least a partial vacuum.

[0305] 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 of CLRN1.Those skilled in the art will understand that many different mutations in CLRN1 can result in pathogenic variants.In some such embodiments, the pathogenic variant causes or is at risk of causing hearing loss.

[0306] 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 CLRN1 gene product or function is evaluated (e.g., via 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 young individual, a juvenile individual, an infant individual, an infant individual, or a neonatal individual. In some embodiments of any of the methods described herein, the age of the animal, subject or mammal is 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 2-5, 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90 , 10-100, 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.

[0307] In some embodiments of any of the methods described herein, the method results in improved hearing (e.g., any of the metrics for determining improved hearing described herein) 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 in a subject in need thereof.

[0308] 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 CLRN1 gene. In some embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) has any of the mutations in the CLRN1 gene described herein or known in the art to be associated with syndromic or non-syndromic sensorineural hearing loss.

[0309] In some embodiments, the subject (e.g., an animal, e.g., a mammal, e.g., a human) has been identified (e.g., via genetic testing) as a carrier of a mutation in the CLRN1 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 CLRN1 gene and has been diagnosed with syndromic or nonsyndromic 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 nonsyndromic sensorineural hearing loss.

[0310] 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., a CLRN1 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 risk for hearing loss (e.g., a known parent carrier, an affected sibling, 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 (e.g., via genetic testing) as being a carrier of a mutation in the CLRN1 gene that has not been previously identified (i.e., a variant of CLRN1 that has not been published or is unknown). In some such embodiments, the identified mutation may be novel (i.e., not previously described in the literature), and treatment methods for subjects suffering from or susceptible to hearing loss are individualized to the specific patient's mutation(s).

[0311] In some embodiments, the therapeutic success 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.Non-limiting examples of functional hearing tests are various types of audiometric assays (e.g., pure tone test, speech test, middle ear test, auditory brainstem response, and otoacoustic emission).

[0312] 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 may 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 ​​the subject that is found not to have normal range hearing function (e.g., as determined using any hearing test known in the art).

[0313] In some embodiments of any method provided herein, the composition can be formulated for intracochlear administration.In some embodiments of any method described herein, the composition described herein can be administered via intracochlear administration or local administration.In some embodiments of any method described herein, the composition is administered by using a medical device (e.g., any exemplary medical device described herein).

[0314] In some embodiments, intracochlear administration can be performed using any method 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 cleaned and a round knife is used to sharply delineate the outline of the ear canal tympanic membrane approximately 5 mm. The ear canal tympanic membrane is then elevated and the middle ear is entered from behind. The chorda tympani nerve is identified and divided, and a curette is used to remove the scutal bone of the superior tympanic cavity to expose the window window membrane. To enhance the distribution of the administered or introduced composition to the apex, a small 2 mm fenestration can be made in the window oval using a surgical laser to allow perilymphatic displacement during the window window membrane injection of the composition. The microinjection device is then primed and brought to the surgical field. The device is steered to the fenestra cochlea and the tip is placed within the bony prominence of the fenestra cochlea to allow penetration of the membrane by the microneedle(s). The foot pedal is engaged to allow the composition to be injected in a measured, constant amount. The device is then retracted and the fenestra cochlea and base of the stapes are sealed with a Gelfoam patch.

[0315] 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, the gene being a gene that can be expressed in supporting cells and / or hair cells.

[0316] In some embodiments of any of the methods provided herein, the subject is identified (e.g., via 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 is identified as having a mutation in an inner ear cell target gene and is 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 is identified as having hearing loss (e.g., nonsyndromic sensorineural hearing loss or syndromic sensorineural hearing loss). In some embodiments, the treatment success of hearing loss (e.g., nonsyndromic sensorineural hearing loss or syndromic sensorineural hearing loss) can be determined in the subject using any conventional functional hearing test known in the art. Non-limiting examples of functional hearing tests include various types of audiometric assays (e.g., pure tone test, speech test, middle ear test, auditory brainstem response, and otoacoustic emissions).

[0317] 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 been previously determined to be defective in an inner ear cell target gene. In some embodiments, the subject cells have been previously determined to be defective in a hair cell target gene. In some embodiments, the subject cells have been previously determined to be defective in a supporting cell target gene.

[0318] In some embodiments of these methods, expression of an active inner ear cell target protein (e.g., clarin 1 protein) is increased following treatment, e.g., one or more administrations of a composition described herein. In some embodiments, the increase in expression of an active inner ear target protein (e.g., clarin 1 protein) described herein is relative to a control level, e.g., compared to the expression level of the inner ear cell target protein prior to introduction of a composition comprising any of the construct(s) described herein.

[0319] Methods for detecting the expression and / or activity of a target protein (e.g., clarin 1 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., detection of an 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., CLRN1 gene product and / or clarin 1 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 (e.g., by functional hearing testing).

[0320] In some embodiments, the safety and tolerability of the compositions disclosed herein can be assessed in subjects with hearing loss. In some embodiments, the safety and tolerability of the compositions disclosed herein, such as rAAV-CLRN1, can be assessed in subjects with hearing loss.

[0321] Administration 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.

[0322] Also provided herein is a surgical method 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 administering intracochlearly 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 into or through the first incision.

[0323] 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 oval window membrane of the cochlea. 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 oval window membrane of the cochlea. In some embodiments of any of the methods provided herein, the composition is administered using a medical device capable of creating multiple incisions in the oval 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 substantially circular first surface, 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 capable of transporting the composition. In some embodiments, the medical device comprises a means for generating at least a partial vacuum.

[0324] In some embodiments, any of the methods disclosed herein includes a dose escalation study to determine safety and tolerability in a subject, e.g., a mammal, e.g., a human, e.g., a patient, with hearing loss. In some embodiments, a composition disclosed herein, e.g., rAAV-CLRN1, is administered in a dosing regimen disclosed herein. In some embodiments, the dosing regimen includes unilateral or bilateral intracochlear administration of a composition disclosed herein, e.g., rAAV-CLRN1, e.g., at a dose described herein. In some embodiments, the dosing regimen comprises delivery of a volume of at least 0.01 mL, at least 0.02 mL, at least 0.03 mL, at least 0.04 mL, at least 0.05 mL, at least 0.06 mL, at least 0.07 mL, at least 0.08 mL, at least 0.09 mL, at least 0.10 mL, at least 0.11 mL, at least 0.12 mL, at least 0.13 mL, at least 0.14 mL, at least 0.15 mL, at least 0.16 mL, at least 0.17 mL, at least 0.18 mL, at least 0.19 mL, or at least 0.20 mL per cochlea. In some embodiments, the dosing regimen includes delivery of a volume of up to 0.30 mL, up to 0.25 mL, up to 0.20 mL, up to 0.15 mL, up to 0.14 mL, up to 0.13 mL, up to 0.12 mL, up to 0.11 mL, up to 0.10 mL, up to 0.09 mL, up to 0.08 mL, up to 0.07 mL, up to 0.06 mL, or up to 0.05 mL per cochlea. In some embodiments, the dosing regimen includes delivery of a volume of about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 0.10 mL, about 0.11 mL, about 0.12 mL, about 0.13 mL, about 0.14 mL, or about 0.15 mL per cochlea depending on the population.In some embodiments, the dosing regimen comprises delivery of a volume of at least 0.01 mL, at least 0.02 mL, at least 0.03 mL, at least 0.04 mL, at least 0.05 mL, at least 0.06 mL, at least 0.07 mL, at least 0.08 mL, at least 0.09 mL, at least 0.10 mL, at least 0.11 mL, at least 0.12 mL, at least 0.13 mL, at least 0.14 mL, at least 0.15 mL, at least 0.16 mL, at least 0.17 mL, at least 0.18 mL, at least 0.19 mL, or at least 0.20 mL per cochlea. In some embodiments, the dosing regimen includes delivery of a volume of up to 0.30 mL, up to 0.25 mL, up to 0.20 mL, up to 0.15 mL, up to 0.14 mL, up to 0.13 mL, up to 0.12 mL, up to 0.11 mL, up to 0.10 mL, up to 0.09 mL, up to 0.08 mL, up to 0.07 mL, up to 0.06 mL, or up to 0.05 mL per cochlea. In some embodiments, the dosing regimen includes delivery of a volume of about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 0.10 mL, about 0.11 mL, about 0.12 mL, about 0.13 mL, about 0.14 mL, or about 0.15 mL per cochlea depending on the population.

[0325] In some embodiments, the methods disclosed herein evaluate the safety and tolerability of increasing doses of a composition disclosed herein, e.g., rAAV-CLRN1, administered via intracochlear injection to a subject with hearing loss, e.g., a subject between the ages of 18 and 80.

[0326] In some embodiments, the methods disclosed herein evaluate the safety and tolerability of escalating doses of a composition disclosed herein, e.g., rAAV-CLRN1, administered via intraocular injection to a subject with hearing loss, e.g., a subject between the ages of 18 and 80.

[0327] In some embodiments, any of the methods disclosed herein include evaluating the safety and tolerability of a composition disclosed herein, such as rAAV-CLRN1. In some embodiments, evaluation of the efficacy of a composition disclosed herein, such as rAAV-CLRN1, for treating hearing loss is performed in a randomized, controlled setting (using a concurrent non-interventional observational arm).

[0328] In some embodiments, any of the methods disclosed herein include evaluating the safety and tolerability of a composition disclosed herein, e.g., rAAV-CLRN1. In some embodiments, evaluation of the efficacy of a composition disclosed herein, e.g., rAAV-CLRN1, for treating vision loss is performed in a randomized, controlled setting (using a concurrent non-interventional observational arm).

[0329] Devices and Surgical Methods The present disclosure provides, among other things, in some embodiments, techniques (e.g., systems, methods, devices, etc.) that can be used to treat profound hearing loss and other hearing-related diseases, disorders, and conditions. Examples of such techniques are also included, for example, in WO2017223193 and WO2019084145, each of which is incorporated herein by reference in its entirety. In some embodiments, for example, the present disclosure provides a therapeutic delivery system for treating hearing loss (e.g., non-syndromic sensorineural hearing loss). In some such embodiments, the therapeutic delivery system can include (i) a medical device capable of creating one or more incisions in the round window membrane of the subject's inner ear, and (ii) an effective dose of a composition (e.g., any composition described herein). In some embodiments, the medical device includes a plurality of microneedles.

[0330] The AAV construct can construct full-length auditory polypeptide messenger RNA in target cells of the inner ear. In some embodiments, the present disclosure provides a means for performing a surgical method, the method comprising administering an effective dose of the therapeutic composition of the present disclosure intracochlearly to a human subject in need thereof. The therapeutic composition can be administered by using a medical device comprising: a) a means for making one or more incisions in the round window membrane; and b) an effective dose of the therapeutic composition.

[0331] The present disclosure provides, inter alia, a surgical method for the treatment (e.g., prevention, restoration, mitigation, attenuation) of hearing loss. In one aspect, the method includes the steps of: introducing a first incision into the cochlea of ​​a human subject at a first incision point, and intracochlearly administering an effective dose of a therapeutic composition provided herein (any composition described herein). In one embodiment, the therapeutic composition (e.g., any composition described herein) is administered to the subject at the first incision point. In some embodiments, the therapeutic composition is administered to the subject into or through the first incision. In one embodiment, the therapeutic composition is administered to the subject in or through the oval window membrane of the cochlea. In one embodiment, the therapeutic composition is administered to the subject in or through the oval window membrane of the cochlea.

[0332] For example, in some embodiments, the therapeutic composition is administered by using a medical device capable of creating 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 surface, each microneedle comprising a diameter of at least about 10 microns. In some embodiments, the medical device comprises a base and / or reservoir capable of holding the therapeutic composition. In some embodiments, the medical device comprises a plurality of hollow microneedles each comprising a lumen capable of transporting the therapeutic composition. In some embodiments, the medical device comprises a means for generating at least a partial vacuum.

[0333] Cochlear Introduction Method The present disclosure also provides, inter alia, a method of introducing a therapeutically effective amount of any of the compositions or systems described herein into the cochlea of ​​a mammal (e.g., human). Also provided is a method of increasing the expression of functional CLRN1 protein in cells (e.g., hair cells, e.g., outer hair cells, e.g., inner ear cells, e.g., spinal ganglion neurons (SGNs)) in the cochlea of ​​a mammal (e.g., human), comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​a subject.

[0334] Also provided are methods of treating hearing loss in a subject (e.g., a human) identified as having a defective (i.e., non-functional) CLRN1 gene product. In some such embodiments, the method comprises administering a therapeutically effective amount of any of the compositions described herein to the cochlea of ​​the subject. In some embodiments, the method of treatment may further comprise administering to the subject a cochlear implant (e.g., substantially simultaneously with any of the compositions described herein being administered to the subject).

[0335] In some embodiments, the method of treatment comprises administering two or more doses of any of the compositions described herein. In some such embodiments, the composition is introduced or administered to the cochlea of ​​a mammal or subject. In some embodiments, the method of treatment comprises introducing or administering an initial dose of the composition to the cochlea of ​​the subject, determining the hearing function of the subject after the introduction or administration of the initial dose, and if the subject is found not to have hearing function within normal range (e.g., as determined using any hearing test known in the art), administering at least one additional dose of the composition to the cochlea of ​​the subject.

[0336] In some embodiments, the method of treatment includes intracochlear administration. In some such embodiments of any of the methods described herein, the composition is administered via the use of a medical device (e.g., any of the exemplary medical devices described herein). In some embodiments, intracochlear administration can be performed as 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 cleaned and a round knife is used to sharply delineate the outline of the approximately 5 mm ear canal tympanic flap. The ear canal tympanic flap is then elevated and the middle ear is entered from behind. The chorda tympani nerve is identified and divided, and a curette is used to remove the scutal bone of the superior tympanic lateral wall to expose the round window membrane. To enhance the distribution of the administered or introduced composition to the apex, a small 2 mm fenestration can be created in the vestibular window using a surgical laser to allow perilymphatic displacement during the trans-round window membrane injection of the composition. The microinjection device is then primed and brought to the surgical field. The device is steered to the window of the cochlea and the tip is placed in the bony prominence of the window of the cochlea to allow the microneedle(s) to penetrate the membrane. The foot pedal is engaged to allow the composition to be injected in a measured, constant amount. The device is then retracted and the window of the cochlea and the base of the stapes are sealed with a gelfoam patch. Those skilled in the art will understand that other variations or methods of intracochlear administration are available. In some embodiments, any such acceptable method can be used to deliver one or more compositions and / or treat one or more subjects as described herein.

[0337] In some embodiments, an exemplary device for use in any of the methods disclosed herein is depicted in Figures 19-22. Figure 19 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 bonds to a telescoping hypotube needle support 24. The knurled handle 12 (or handle portion) can include curling features and / or grooves to enhance grip. The knurled handle 12 (or handle portion) can 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) can be hollow to allow fluid to pass through the device 10 during use. The device 10 includes a proximal handle adhesive 16 at the proximal end 18 of the knurled handle 12, a needle subassembly 26 (shown in FIG. 20) having a stopper 28 (shown in FIG. 20) 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 bent needle 38 (shown in FIG. 20) disposed therein.

[0338] 20, the stopper 28 may be constructed from a thermoplastic material or a plastic polymer (such as a UV cured polymer), as well as other suitable materials, and may be used to prevent the bent needle 38 from being inserted too far into the ear canal (e.g., to prevent the bent needle 38 from being inserted into the side wall or other inner ear structure). The device 10 may also include a tapered portion 23 disposed between the knurled handle 12 and the distal handle adhesive 14 coupled to the telescoping hypotube needle support 24. The knurled handle 12 (or handle portion) may include a tapered portion 23 at the distal end of the handle portion 12. The device 10 may also include a tube 36 fluidly connected to the proximal end 16 of the device 10, which serves as a fluid inlet line connecting the device to an upstream component (e.g., a pump, a syringe, and / or an upstream component that may be coupled to a control system and / or power source (not shown) in some embodiments). In some embodiments, the bent needle 38 (shown in FIG. 20) 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 bent needle 38 is fluidly connected to the hollow interior of the knurled handle (e.g., via the telescoping hypotube needle support 24), which in turn is fluidly connected to the tubing 36 at the proximal end 16. In embodiments in which the bent needle 38 does not extend completely into the interior of the device 10, the contact areas (e.g., between the overlapping nested hypotubes 42), tolerances, and / or seals between the interface components should be sufficient to prevent leakage of therapeutic fluid 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).

[0339] FIG. 20 illustrates 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 a beveled tip 34 at the distal end 20 of the device 10 for piercing an ear membrane (e.g., RWM). The needle 38, the bent portion 32, and the beveled tip 34 are hollow to allow fluid to flow therethrough. The angle 46 (as shown in FIG. 22) of the bent portion 32 may vary. The shape of the stopper 28 may be cylindrical, disc-shaped, ring-shaped, 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 positioned around the bent portion 32 using an adhesive or a compression fit. Examples of adhesives include UV curable adhesives (such as Dymax 203A-CTH-FT), elastomeric adhesives, thermosetting adhesives (such as epoxy or polyurethane), or emulsion adhesives (such as polyvinyl acetate). The stopper 28 fits concentrically around the bent portion 32 such that the beveled 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.

[0340] FIG. 21 shows a perspective view of an exemplary device 10 for delivering fluid to the inner ear. The length of the tube 36 can be about 1300 mm (dimension 11 in FIG. 21) to about 1600 mm, or about 1400 mm to about 1500 mm, or about 1430 mm to about 1450 mm. The strain relief mechanism 22 can be about 25 mm to about 30 mm in length (dimension 15 in FIG. 21), or about 20 mm to about 35 mm in length. The handle 12 can be about 155.4 mm in length (dimension 13 in FIG. 21), or about 150 mm to about 160 mm, or about 140 mm to about 170 mm. 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. 21) 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 have a length of about 36 mm, or about 25 mm to 45 mm, or about 30 mm to about 40 mm. The three nested hypotubes 42A, 42B, and 42C each have a length of 3.5 mm, 8.0 mm, and 19.8 mm, respectively, ± about 20%. The innermost nested hypotube (or narrowest portion) of the telescoping hypotube needle support 24 may be concentrically disposed around the needle 38 .

[0341] FIG. 22 illustrates a perspective view of the 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. 22, 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 bent to include a 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 ensure that the tube 36 is not kinked or damaged. The needle 38 may be attached to the handle 12 for safe and accurate placement of the needle 38 in the inner ear. As shown in FIG. 22, the bent needle subassembly 26 may also include a stopper 28 disposed around the bent portion 32. FIG. 22 illustrates that the bent portion 32 may include an angled tip 34 to pierce the ear membrane (e.g., RWM). The 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. The 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, the bent portion 32 may have a length greater than 2.0 mm, such that the distance between the distal end of the stopper 28 and the distal end of the beveled 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. 22 illustrates that the stopper 28 may have a shape that is cylindrical, disc-shaped, and / or dome-shaped. One skilled in the art will appreciate that other shapes may be used.

[0342] In some embodiments, delivery approaches disclosed herein include synthetic AAV capsids (e.g., AAV Anc80) for transduction of inner ear cells, and / or devices for targeted delivery directly to the cochlea. In certain embodiments, the present disclosure provides methods and compositions suitable for transduction of inner ear cells.

[0343] 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 oval window membrane of the cochlea. 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 oval window membrane of the cochlea. In some embodiments of any of the methods described herein, the composition is administered with a medical device capable of creating multiple incisions in the oval 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 substantially circular first surface, 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 capable of transporting the composition. In some embodiments, the medical device comprises a means for generating at least a partial vacuum.

[0344] In some embodiments, disclosed herein is a single-use sterile delivery device for intracochlear administration to deliver a composition disclosed herein, e.g., rAAV-CLRN1, to the perilymph of the inner ear through the window window membrane with a pore located in the base of the stapes. In some embodiments, in this intracochlear administration approach, a composition disclosed herein, e.g., rAAV-CLRN1, can be administered to the scala tympani through a window window membrane with a pore at the base of the stapes in the vestibular window, whereby the composition is perfused through the scala tympani and then through the scala vestibuli via a connection at the cochlear foramen, following a fluid path to the pore in the base of the stapes (FIGS. 11A-11B).

[0345] How to treat a subject The present disclosure provides, inter alia, that the technology described herein may be used to treat the underlying disease and / or condition of a subject suffering from or at risk for Usher Syndrome Type III, which is characterized by hearing loss.

[0346] 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 Usher Syndrome Type III, which is characterized by hearing loss.

[0347] In some embodiments, administering a construct (e.g., an rAAV construct), particle (e.g., an rAAV particle), or composition described herein to a subject may alleviate and / or ameliorate one or more symptoms associated with Usher syndrome type III, which is characterized by hearing loss. Symptoms may include, for example, hearing loss, hair cell degeneration, altered biochemical milieu of the inner ear fluid, elevated intralabyrinthic proteins, endolymphatic hydrops, cochlear orifice obstruction, intralabyrinthic hemorrhage, disruption of the cochlear vascular supply, tinnitus, dizziness, intractable headache, facial nerve dysfunction, trigeminal nerve dysfunction, facial paralysis, facial paresthesia, hydrocephalus, cerebellar herniation, and / or death.

[0348] In some embodiments, Usher syndrome type III characterized by hearing loss is associated with a genetic mutation (e.g., a deletion mutation, a frameshift mutation, a nonsense mutation, a hypomorphic mutation, a hypermorphic mutation, a neomorphic mutation, an abnormal overexpression, an abnormal underexpression, et...

Claims

1. An adeno-associated virus (AAV) particle comprising a construct, the construct comprising: (i) 5' inverted terminal repeat (ITR); (ii) cytomegalovirus (CMV) enhancer; (iii) chicken β-actin (CBA) promoter; (iv) chimeric introns; (v) 5' untranslated region (UTR); (vi) a coding sequence comprising the sequence set forth in SEQ ID NO:1, operably linked to the CBA promoter and encoding the Curarin 1 protein; (vii) 3' UTR; (viii) a polyadenylation sequence; and (ix) 3′ ITR, An AAV particle comprising:

2. An adeno-associated virus (AAV) particle comprising a construct, the construct comprising: (i) 5' inverted terminal repeat (ITR); (ii) cytomegalovirus (CMV) enhancer; (iii) chicken β-actin (CBA) promoter; (iv) chimeric introns; (v) 5' untranslated region (UTR); (vi) a coding sequence comprising the sequence set forth in SEQ ID NO: 19, operably linked to the CBA promoter and encoding the Curarin 1 protein; (vii) 3'UTR; (viii) a polyadenylation sequence; and (ix) 3′ ITR, An AAV particle comprising:

3. The following: (i) 5' inverted terminal repeat (ITR); (ii) cytomegalovirus (CMV) enhancer; (iii) chicken β-actin (CBA) promoter; (iv) chimeric introns; (v) 5' untranslated region (UTR); (vi) a coding sequence comprising the sequence set forth in SEQ ID NO:1, operably linked to the CBA promoter and encoding the Curarin 1 protein; (vii) 3'UTR; (viii) a polyadenylation sequence; and (ix) 3′ ITR, A structure, including:

4. The following: (i) 5' inverted terminal repeat (ITR); (ii) cytomegalovirus (CMV) enhancer; (iii) chicken β-actin (CBA) promoter; (iv) chimeric introns; (v) 5' untranslated region (UTR); (vi) a coding sequence comprising the sequence set forth in SEQ ID NO: 19, operably linked to the CBA promoter and encoding the Curarin 1 protein; (vii) 3'UTR; (viii) a polyadenylation sequence; and (ix) 3′ ITR, A structure, including:

5. 5. The AAV particle of claim 1 or 2 or the construct of claim 3 or 4, wherein the 5' ITR and the 3' ITR are flanked by a CMV enhancer and a 3' UTR, respectively.

6. The AAV particle of any one of claims 1, 2 or 5, or the construct of any one of claims 3 to 5, wherein the construct comprises a nucleic acid sequence as set forth in SEQ ID NO: 64 or 68.

7. The AAV particle of any one of claims 1, 2, or 5-6, or the construct of any one of claims 3-6, further comprising an AAV capsid, wherein the AAV capsid is or is derived from an AAV Anc80 capsid.

8. An AAV particle described in any one of claims 1, 2, or 5-7, or a construct described in any one of claims 3-7, wherein the 5'ITR comprises a sequence set forth in SEQ ID NO: 20 or 21.

9. An AAV particle described in any one of claims 1, 2, or 5-8 or a construct described in any one of claims 3-8, wherein the 3'ITR comprises a sequence set forth in SEQ ID NO: 18 or 22.

10. An AAV particle described in any one of claims 1, 2, or 5 to 9 or a construct described in any one of claims 3 to 9, wherein the CMV enhancer comprises the sequence set forth in SEQ ID NO:

38.

11. An AAV particle described in any one of claims 1, 2, or 5 to 10 or a construct described in any one of claims 3 to 10, wherein the CBA promoter comprises a sequence set forth in SEQ ID NO: 23 or 24.

12. An AAV particle described in any one of claims 1, 2, or 5 to 11, or a construct described in any one of claims 3 to 11, wherein the chimeric intron comprises the sequence set forth in SEQ ID NO:

39.

13. An AAV particle described in any one of claims 1, 2, or 5 to 12 or a construct described in any one of claims 3 to 12, wherein the 5'UTR comprises the sequence set forth in SEQ ID NO:

40.

14. An AAV particle described in any one of claims 1, 2, or 5 to 13, or a construct described in any one of claims 3 to 13, wherein the clarin 1 protein comprises the amino acid sequence set forth in SEQ ID NO:

10.

15. An AAV particle described in any one of claims 1, 2, or 5 to 13, or a construct described in any one of claims 3 to 13, wherein the 3'UTR comprises the sequence set forth in SEQ ID NO:

41.

16. An AAV particle described in any one of claims 1, 2, or 5 to 15 or a construct described in any one of claims 3 to 15, wherein the polyadenylation sequence includes a sequence similar to SEQ ID NO:

44.

17. A composition comprising an AAV particle according to any one of claims 1, 2, or 5-16 or a construct according to any one of claims 3-16.

18. 20. The composition of claim 17, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition comprises a pharma- ceutically acceptable carrier.

19. 19. An ex vivo cell comprising the composition of claim 17 or 18.

20. In ex vivo cells, (i) a construct according to any one of claims 3 to 16; and (ii) one or more helper plasmids collectively comprising the AAV Rep, Cap, VA, E2a, and E4 genes; and The method of claim 1, further comprising transfecting

21. A pharmaceutical composition for use in treating hearing loss in a subject suffering from or at risk of hearing loss, comprising a construct described in any one of claims 3 to 16, an AAV particle described in any one of claims 1, 2, or 5 to 16, or a composition described in claim 17 or 18.

22. A pharmaceutical composition for use in treating vision loss in a subject suffering from or at risk of vision loss, comprising a construct described in any one of claims 3 to 16, an AAV particle described in any one of claims 1, 2, or 5 to 16, or a composition described in claim 17 or 18.

23. A pharmaceutical composition for use in treating Usher Syndrome Type III in a subject suffering from or at risk of having Usher Syndrome Type III, comprising a construct described in any one of claims 3 to 16, an AAV particle described in any one of claims 1, 2, or 5 to 16, or a composition described in claim 17 or 18.

24. A pharmaceutical composition for use in treating retinitis pigmentosa in a subject suffering from or at risk of retinitis pigmentosa, comprising a construct described in any one of claims 3 to 16, an AAV particle described in any one of claims 1, 2, or 5 to 16, or a composition described in claim 17 or 18.

25. A kit comprising the composition of claim 17 or 18.

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