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  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
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• • A—HUMAN NECESSITIES
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  • A61M25/00—Catheters; Hollow probes
  • A61M25/0067—Catheters; Hollow probes characterised by the distal end, e.g. tips
  • A61M25/0082—Catheter tip comprising a tool
  • A61M25/0084—Catheter tip comprising a tool being one or more injection needles
  • A61M2025/0093—Catheter tip comprising a tool being one or more injection needles wherein at least one needle is a microneedle
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  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
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  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14171—Demonstrated in vivo effect
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

• Hearing loss can be conductive (arising from the ear canal or middle ear), sensorineural (arising from the inner ear or auditory nerve), or mixed. Most forms of nonsyndromic deafness are associated with permanent hearing loss caused by damage to structures in the inner ear (sensorineural deafness), although some forms may involve changes in the middle ear (conductive hearing loss).
• sensorineural hearing loss is caused by abnormalities in the hair cells of the organ of Corti in the cochlea (poor hair cell function). The hair cells may be abnormal at birth, or may be damaged during the lifetime of an individual (e.g., as a result of noise trauma or infection).
• the present disclosure provides the recognition that diseases or conditions associated with hearing loss can be treated via, e.g., 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 can be useful for treatment of diseases or conditions associated with hair cell and/or supporting cell loss.
• the present disclosure thus provides for the administration of compositions that result in expression of gene products involved in the development, function, and/or maintenance of inner ear cells including supporting cells and hair cell, and/or the use of such compositions in the treatment of hearing loss, or diseases or conditions associated with hearing loss.
• a gene product can be encoded by a gap junction beta-2 (GJB2) gene (the GJB2 gene encodes connexin 26 protein) or a characteristic portion thereof.
• GJB2 gap junction beta-2
• a gene product can be connexin 26 protein (encoded by a GJB2 gene) or a characteristic portion thereof.
• AAV particles can be useful for administration of compositions that result in expression of gene products involved in the development, function, and/or maintenance of inner ear cells, and/or the treatment of hearing loss, or diseases or conditions associated with hearing loss.
• AAV particles comprise (i) a AAV polynucleotide construct (e.g., a recombinant AAV (rAAV) polynucleotide construct), and (ii) a capsid comprising capsid proteins.
• rAAV recombinant AAV
• an AAV polynucleotide construct comprises a GJB2 gene or a characteristic portion thereof.
• compositions comprising polynucleotide constructs comprising a GJB2 gene or a characteristic portion thereof.
• a construct may further include regulatory elements operably attached to a coding sequence.
• included regulatory elements facilitate tissue specific expression at physiologically suitable levels.
• administration involves surgical intervention and the delivery of rAAV particles comprising therapeutic constructs.
• AAV particles may be delivered to the inner ear of a subject in need thereof by surgical introduction through the round window membrane.
• efficacy of an intervention is determined through established tests, and measurements are compared to known control measurements.
• FIG. 1 panel (A) depicts a simplified endogenous AAV genome
• panel (B) depicts a simplified recombinant AAV (rAAV) construct capable of expressing a GJB2 gene.
• FIG. 2A-20 show panels (A)-(O), which depict alternative exemplary rAAV constructs comprising a GJB2 gene.
• FIG. 2A depicts a rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a hGJB2 gene, a bGH poly A, and a 3' ITR.
• FIG. 2B depicts a rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a hGJB2 gene, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 2C depicts a rAAV construct comprising a 5' ITR, a GJB2 promoter, a nucleic acid encoding a hGJB2 gene, a bGH poly A, a C3 domain, and a 5' ITR.
• FIG. 2D depicts a rAAV construct comprising a 5' ITR, a GJB2 promoter, a nucleic acid encoding a hGJB2 gene, a bGH poly A, a D7 domain, and a 3' ITR.
• FIG. 2E depicts a rAAV construct comprising a 5'
• FIG. 2F depicts a rAAV construct comprising a 5' ITR, a CAG promoter, a 5' UTR, a hGJB2 gene, a FLAG tag, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 2F depicts a rAAV construct comprising a 5' ITR, a CAG promoter, a 5' UTR, a hGJB2 gene, a FLAG tag, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 2G depicts a rAAV construct comprising a 5' ITR, a smCBA promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 2H depicts a rAAV construct comprising a 5' ITR, a promoter comprising a CMV promoter and a hGJB2 promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 1 depicts a rAAV construct comprising a 5' ITR, a smCBA promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag
• FIG. 21 depicts a rAAV construct comprising a 5' ITR, a promoter comprising a CMV promoter and a GFAP promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 2J depicts a rAAV construct comprising a 5' ITR, a GFAP inner ear supporting cell-specific promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 1 depicts a rAAV construct comprising a 5' ITR, a promoter comprising a CMV promoter and a GFAP promoter, a 5' UTR, a nucleic acid encoding a
• 2K depicts a rAAV construct comprising a 5' ITR, a CAG promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a destabilization domain, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 1 depicts a rAAV construct comprising a 5' ITR, a CAG promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a destabilization domain, a 3' UTR, a bGH poly A, and a 3' ITR.
• 2L depicts a rAAV construct comprising a 5' ITR, a promoter comprising a hGJB2 enhancer and a hGJB2 promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 1 depicts a rAAV construct comprising a 5' ITR, a promoter comprising a hGJB2 enhancer and a hGJB2 promoter, a 5' UTR, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 2M depicts a rAAV construct comprising a 5' ITR, a CAG promoter, a 5' UTR, a hGJB2 promoter, a hGJB2 gene, a FLAG tag, a microRNA regulatory target site, a 3' UTR, a bGH poly A, and a 3' ITR.
• FIG. 2N depicts a rAAV construct comprising a 5' ITR, a promoter comprising an inner ear supporting cell specific promoter and a hGJB2 minimal promoter, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a 5' UTR, a bGH poly A, and a 3' ITR.
• FIG. 1 depicts a rAAV construct comprising a 5' ITR, a CAG promoter, a 5' UTR, a hGJB2 promoter, a hGJB2 gene, a
• rAAV construct comprising a 5' ITR, a CAG promoter, a nucleic acid encoding a hGJB2 gene, a FLAG tag, a T2A element, a nucleic acid encoding eGFP, a bGH poly A, and a 3' ITR
• FIG. 3 depicts connexin 26 (Cx26)/GJB2 protein expression from HEK293FT cells that have been exposed to exemplary constructs described herein.
• Panel (A) Depicts Cx26 protein expression in HEK293FT cells that have been transfected with exemplary rAAV constructs comprising CAG promoters, bands corresponding to Vinculin and Cx26 are marked.
• Panel (B) Depicts GJB2 protein expression in HEK293FT cells that have been transfected with exemplary constructs comprising hGJB2 coding sequences with GJB2 5’ UTR and 3’UTR sequences, driven by CAG, CMVe-GJB2p, or smCBA promoter/enhancer sequences as noted, bands corresponding to GAPDH and GJB2-FLAG are marked.
• Panel (C) Depicts GJB2 protein expression in HEK293FT cells that have been transduced with exemplary rAAV particles comprising constructs comprising hGJB2 coding sequences with GJB2 5’ UTR and 3’UTR sequences, driven by CAG, CMVe-GJB2p, or smCBA promoter/enhancer sequences as noted, bands corresponding to GAPDH and GJB2-FLAG are marked, positive control is hGJB2 coding sequence driven by CAG promoter/enhancer without GJB2 5’ UTR or 3’ UTR.
• FIG. 4 depicts quantitative PCR (qPCR) results of GJB2 mRNA expression in
• HEK293FT cells and wild type neonatal CD1 explants that have been transduced with exemplary rAAV constructs have been transduced with exemplary rAAV constructs.
• FIG. 5 panels (A) and (B) depict eGFP protein expression in HEK293T cells under the power of various exemplary promoters, cells were sorted and quantified 72 hours after transfection.
• FIG. 6 depicts FLAG protein expression in mouse cochlear explants transduced at
• P2 with exemplary rAAVAnc80 particles comprising constructs driven by CAG, CMVe- GJB2p, or smCBA promoter/enhancer sequences as noted, explants were fixed after 72h, immunostaining for FLAG is noted in green, immunostaining for hair cell marker Myo7a is noted in red, and nuclear marker DAPI is noted in blue.
• Panel (A) depicts exemplary explants transduced with AAVAnc80-CAG.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 82) at 5.8E9 vg/explant.
• Panel (B) depicts exemplary explants transduced with AAVAnc80- smCBA.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 83) at 1.4E10 vg/explant.
• Panel (C) depicts exemplary explants transduced with AAVAnc80- CMVeGJB2p.5UTR.hGJB2.3F.3UTR (SEQ ID NO: 84) at 1.8E10 vg/explant.
• FIG. 7 depicts in vitro expression of GJB2 protein in HEK293FT cells transfected with CAG.5UTR.hGJB2.FLAG.miRTS.3UTR (SEQ ID NO: 87), CAG.5UTR.hGJB2.FLAG.3UTR (SEQ ID NO: 82), or CAG.5UTR.hGJB2.FLAG.GFP constructs.
• CAG.5UTR.hGJB2.FLAG.miRTS.3UTR comprises miRNA targeting sites (miRTS) for miR-182 and miR-183 in the 3UTR to permit exogenous hGJB2 knockdown in the presence of regulatory miR-182 and/or miR-183.
• HEK293FT cells were transfected with hGJB2 comprising plasmids and optionally co-transfected with (+) or without (-) plasmids expressing miR-182 and miR- 183. 72h post transfection the cells were harvested for protein and RNA analysis.
• Panel (A) Depicts exemplary GJB2 protein levels analyzed using western blot;
• panel (B) depicts exemplary GJB2 mRNA levels analyzed using qPCR.
• FIG. 8 illustrates a perspective of a device for delivering fluid to an inner ear, according to aspects of the present disclosure.
• FIG. 9 illustrates a sideview of a bent needle sub-assembly, according to aspects of the present disclosure.
• FIG. 10 illustrates a perspective view of a device for delivering fluid to an inner ear, according to aspects of the present disclosure.
• FIG. 11 illustrates a perspective view of a bent needle sub-assembly coupled to the distal end of a device, according to aspects of the present disclosure.
• FIG. 12 depicts in vivo expression of connexin 26 in wild-type mice (p20) that were administered rAAVAnc80 particles comprising CAG.hGJB2.F.GFP (schematic provided in Fig. 20) to the cochlea.
• Expression of connexin 26 in the supporting cells and inner hair cells was detected 10 days after administration. Immunostaining of actin filaments and hair cell stereocilia bundles by phalloidin is noted in blue, GFP is noted in green, FLAG is noted in purple, and endogenous connexin 26 is noted in red.
• SC supporting cells; IHC - inner hair cells; OHC - outer hair cells.
• a polynucleotide or polypeptide is represented by a sequence of letters (e.g., A, C, G, and T, which denote adenosine, cytidine, guanosine, and thymidine, respectively in the case of a polynucleotide), such polynucleotides or polypeptides are presented in 5’ to 3’ or N-terminus to C-terminus order, from left to right.
• Administration typically refers to administration of a composition to a subject or system to achieve delivery of an agent to a subject or system.
• an agent is, or is included in, a composition; in some embodiments, an agent is generated through metabolism of a composition or one or more components thereof.
• routes may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
• administration may be systematic or local.
• a systematic administration can be intravenous.
• administration can be local. Local administration can involve delivery to cochlear perilymph via, e.g., injection through a round-window membrane or into scala-tympani, a scala-media injection through endolymph, perilymph and/or endolymph following canalostomy.
• administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
• Allele refers to one of two or more existing genetic variants of a specific polymorphic genomic locus.
• Amelioration refers to prevention, reduction or palliation of a state, or improvement of a state of a subject. Amelioration may include, but does not require, complete recovery or complete prevention of a disease, disorder or condition.
• amino acid refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
• an amino acid has a general structure, e.g., H2N-C(H)(R)-COOH.
• an amino acid is a naturally-occurring amino acid.
• an amino acid is a non natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid.
• Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
• Nonstandard amino acid refers to any amino acid, other than standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
• an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide can contain a structural modification as compared with general structure as shown above.
• an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and/or a hydroxyl group) as compared with a general structure.
• such modification may, for example, alter circulating half-life of a polypeptide containing a modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
• such modification does not significantly alter a relevant activity of a polypeptide containing a modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
• the terms “approximately” or “about” may be applied to one or more values of interest, including a value that is similar to a stated reference value.
• the term “approximately” or “about” refers to a range of values that fall within ⁇ 10% (greater than or less than) of a stated reference value unless otherwise stated or otherwise evident from context (except where such number would exceed 100% of a possible value).
• the term “approximately” or “about” may encompass a range of values that within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a reference value.
• association describes two events or entities as “associated” with one another, if the presence, level and/or form of one is correlated with that of the other.
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
• two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
• two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
• biologically active refers to an observable biological effect or result achieved by an agent or entity of interest.
• a specific binding interaction is a biological activity.
• modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity.
• presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.
• cell selective promoter refers to a promoter that is predominately active in certain cell types (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory and/or control proteins that bind to the tissue-specific promoter).
• an inner ear supporting cell selective promoter is a promoter that is predominately active in one or more supporting cells of the inner ear.
• Characteristic portion refers to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance.
• a characteristic portion of a substance is a portion that is found in a given substance and in related substances that share a particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In some embodiments, a characteristic portion shares at least one functional characteristic with the intact substance.
• a “characteristic portion” of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids.
• a characteristic portion of a substance is one that, in addition to a sequence and/or structural identity specified above, shares at least one functional characteristic with the relevant intact substance.
• a characteristic portion may be biologically active.
• Characteristic sequence is a sequence that is found in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.
• 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.
• presence of a characteristic sequence element correlates with presence or level of a particular activity or property of a polymer.
• 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).
• a characteristic sequence element includes 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., contiguously linked monomers).
• a characteristic sequence element includes at least first and second stretches of contiguous monomers spaced apart by one or more spacer regions whose length may or may not vary across polymers that share a sequence element.
• Combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
• two or more agents may be administered simultaneously.
• two or more agents may be administered sequentially.
• two or more agents may be administered in overlapping dosing regimens.
• Comparable refers to two or more agents, entities, situations, sets of conditions, subjects, populations, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
• comparable sets of agents, entities, situations, sets of conditions, subjects, populations, etc. are characterized by a plurality of substantially identical features and one or a small number of varied features.
• a construct refers to a composition including a polynucleotide capable of carrying at least one heterologous polynucleotide.
• a construct can be a plasmid, a transposon, a cosmid, an artificial chromosome (e.g., a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a PI -derived artificial chromosome (PAC)) or a viral construct, and any Gateway® plasmids.
• HAC human artificial chromosome
• YAC yeast artificial chromosome
• BAC bacterial artificial chromosome
• PAC PI -derived artificial chromosome
• a construct can, e.g., include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host primate cell or in an in vitro expression system.
• a construct may include any genetic element (e.g., a plasmid, a transposon, a cosmid, an artificial chromosome, or a viral construct, etc.) that is capable of replicating when associated with proper control elements.
• “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).
• AAV adeno-associated virus
• conservative amino acid substitution refers to instances describing a conservative amino acid substitution, including a substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity).
• a conservative amino acid substitution will not substantially change functional properties of interest of a protein, for example, ability of a receptor to bind to a ligand.
• Examples of groups of amino acids that have side chains with similar chemical properties include: aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (lie, 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 (Gin, 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 methi
• Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine (Val/Leu/Ile, V/L/I), phenylalanine/tyrosine (Phe/Tyr, F/Y), lysine/arginine (Lys/Arg, K/R), alanine/valine (Ala/Val, A/V), glutamate/aspartate (Glu/Asp, E/D), and asparagine/glutamine (Asn/Gln, N/Q).
• a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis.
• a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., 1992, Science 256:1443-1445, which is incorporated herein by reference in its entirety.
• a substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.
• control refers to the art-understood meaning of a “control” being a standard against which results are compared. Typically, controls are used to augment integrity in experiments by isolating variables in order to make a conclusion about such variables.
• a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator.
• a “test” i.e., a variable being tested
• a “control,” the variable being tested is not applied.
• a control is a historical control (e.g., of a test or assay performed previously, or an amount or result that is previously known).
• a control is or comprises a printed or otherwise saved record.
• a control is a positive control. In some embodiments, a control is a negative control.
• determining Determining, measuring, evaluating, assessing, assaying and analyzing.
• the terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” may be used interchangeably to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assaying may be relative or absolute. For example, in some embodiments, “Assaying for the presence of’ can be determining an amount of something present and/or determining whether or not it is present or absent.
• Engineered refers to an aspect of having been manipulated by the hand of man.
• 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 hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
• new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols.
• progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
• Excipient refers to an inactive (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.
• 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.
• expression refers to generation of any gene product (e.g., transcript, e.g., mRNA, e.g., polypeptide, etc.) from a nucleic acid sequence.
• a gene product can be a transcript.
• a gene product can be a polypeptide.
• expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
• flanked referenced nucleic acid sequence has a first sequence or series of nucleotide residues positioned adjacent to the 5' end of the referenced nucleic acid and a second sequence or series of nucleotide residues positioned adjacent to the 3' end of the referenced nucleic acid.
• the upstream and/or downstream flanking sequences are immediately adjacent to the referenced nucleic acid sequence.
• a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
• a functional biological molecule is characterized relative to another biological molecule which is non-functional in that the “non-functional” version does not exhibit the same or equivalent property and/or activity as the “functional” molecule.
• a biological molecule may have one function, two functions (i.e., bifunctional) or many functions (i.e., multifunctional).
• Gene refers to a DNA sequence in a chromosome that codes for a gene product (e.g., an RNA product, e.g., a polypeptide product).
• a gene includes coding sequence (i.e., sequence that encodes a particular product).
• a gene includes non-coding sequence.
• a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequence.
• a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.).
• regulatory sequences e.g., promoters, enhancers, etc.
• intron sequences e.g., cell-type-specific expression, inducible expression, etc.
• the term “gene” generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof; the term may optionally encompass regulatory sequences, as will be clear from context to those of ordinary skill in the art.
• a gene may encode a polypeptide, but that polypeptide may not be functional, e.g., a gene variant may encode a polypeptide that does not function in the same way, or at all, relative to the wild-type gene.
• a gene may encode a transcript which, in some embodiments, may be toxic beyond a threshold level.
• a gene may encode a polypeptide, but that polypeptide may not be functional and/or may be toxic beyond a threshold level.
• hearing loss may be used to a partial or total inability of a living organism to hear.
• hearing loss may be acquired.
• hearing loss may be hereditary.
• hearing loss may be genetic.
• hearing loss may be as a result of disease or trauma (e.g., physical trauma, treatment with one or more agents resulting in hearing loss, etc.).
• hearing loss may be due to one or more known genetic causes and/or syndromes.
• hearing loss may be of unknown etiology.
• hearing loss may or may not be mitigated by use of hearing aids or other treatments.
• heterologous may be used in reference to one or more regions of a particular molecule as compared to another region and/or another molecule.
• heterologous polypeptide domains refers to the fact that polypeptide domains do not naturally occur together (e.g., in the same polypeptide).
• a polypeptide domain from one polypeptide may be fused to a polypeptide domain from a different polypeptide.
• two polypeptide domains would be considered “heterologous” with respect to each other, as they do not naturally occur together.
• Identity refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
• polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
• Calculation of percent identity of two nucleic acid or polypeptide sequences can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
• a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; nucleotides at corresponding positions are then compared.
• 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, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
• nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
• Inhibitory nucleic acid refers to a nucleic acid sequence that hybridizes specifically to a target gene, including target DNA or RNA (e.g., a target mRNA (e.g., a connexin gene product, e.g., a connexin mRNA, e.g., GJB2 mRNA)).
• a target mRNA e.g., a connexin gene product, e.g., a connexin mRNA, e.g., GJB2 mRNA
• an inhibitory nucleic acid inhibits expression and/or activity of a target gene.
• an inhibitory nucleic acid is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an antisense oligonucleotide, a guide RNA (gRNA), or a ribozyme.
• siRNA short interfering RNA
• shRNA short hairpin RNA
• miRNA microRNA
• gRNA guide RNA
• ribozyme ribozyme
• an inhibitory nucleic acid is between about 10 nucleotides to about 30 nucleotides in length (e.g., about 10 nucleotides to about 28 nucleotides, about 10 nucleotides to about 26 nucleotides, about 10 nucleotides to about 24 nucleotides, about 10 nucleotides to about 22 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 18 nucleotides, about 10 nucleotides to about 16 nucleotides, about 10 nucleotides to about 14 nucleotides, about 10 nucleotides to about 12 nucleotides, about 12 nucleotides to about 30 nucleotides, about 12 nucleotides to about 28 nucleotides, about 12 nucleotides to about 26 nucleotides, about 12 nucleotides to about 24 nucleotides, about 12 nucleotides to about 22
• an inhibitory nucleic acid is an inhibitory RNA that targets GJB2.
• an inhibitory GJB2 RNA hybridizes specifically to a target on an RNA molecule comprising GJB2.
• a GJB2 inhibitory RNA includes, e.g., an inhibitory nucleic acid is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an antisense oligonucleotide, a guide RNA (gRNA), or a ribozyme.
• siRNA short interfering RNA
• shRNA short hairpin RNA
• miRNA microRNA
• gRNA guide RNA
• gRNA guide RNA
• an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
• an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
• an appropriate reference is a negative reference; in some embodiments, an appropriate reference is a positive reference.
• Knockdown refers to a decrease in expression of one or more gene products.
• an inhibitory nucleic acid achieve knockdown.
• a genome editing system described herein achieves knockdown.
• Knockout refers to ablation of expression of one or more gene products. In some embodiments, a genome editing system described herein achieve knockout.
• microRNA refers to a class of biomolecules involved in control of gene expression.
• a mature miRNA is typically an 18-25 nucleotide non-coding RNA that regulates expression of an mRNA including sequences complementary to the miRNA.
• These small RNA molecules are known to control gene expression by regulating the stability and/or translation of mRNAs.
• a miRNAs binds to the 3' UTR of target mRNAs and suppress translation.
• MiRNAs can also bind to target mRNAs and mediate gene silencing through the RNAi pathway. MiRNAs can also regulate gene expression by causing chromatin condensation.
• a microRNA is between about 10 nucleotides to about 30 nucleotides in length (e.g., about 10 nucleotides to about 28 nucleotides, about 10 nucleotides to about 26 nucleotides, about 10 nucleotides to about 24 nucleotides, about 10 nucleotides to about 22 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 18 nucleotides, about 10 nucleotides to about 16 nucleotides, about 10 nucleotides to about 14 nucleotides, about 10 nucleotides to about 12 nucleotides, about 12 nucleotides to about 30 nucleotides, about 12 nucleotides to about 28 nucleotides, about 12 nucleotides to about 26 nucleotides, about 12 nucleotides to about 24 nucleotides, about 12 nucleotides to about 22
• microRNA regulatory target site refers to a sequence that directly interacts with a miRNA on the mRNA transcript. Often, the miRTS is present in the 3' untranslated region (UTR) of the mRNA, but it may also be present in the coding sequence, or in the 5' UTR. miRTS are not necessarily perfect complements to miRNAs, usually having only a few bases of complementarity to the miRNA, and often containing one or more mismatches.
• the miRTS may be any sequence capable of being bound by a miRNA sufficiently that the translation of a gene to which the miRTS is operably linked is repressed by a miRNA silencing mechanism such as the RNA-induced silencing complex (RISC).
• RISC RNA-induced silencing complex
• inclusion of a miRTS into a nucleic acid construct comprising a polynucleotide can result in degradation of the therapeutic polynucleotide after transcription.
• inclusion of a miRTS into a nucleic acid construct comprising a polynucleotide can result in decreased expression of the therapeutic polynucleotide in cells expressing the miRNA.
• nucleic acid refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
• a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
• nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues.
• a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5’-N- phosphoramidite linkages rather than phosphodiester bonds.
• a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine).
• adenosine thymidine, guanosine, cytidine
• uridine deoxyadenosine
• deoxythymidine deoxy guanosine
• deoxycytidine deoxycytidine
• a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl- cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases,
• a nucleic acid comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
• a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
• a nucleic acid includes one or more introns.
• nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
• a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
• a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded.
• a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is complementary to a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
• Operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
• a control element “operably linked” to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element.
• “operably linked” control elements are contiguous (e.g., covalently linked) with coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest.
• “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
• a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
• a functional linkage may include transcriptional control.
• a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
• composition refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.
• an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
• a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for, e.g., administration, for example, an injectable formulation that is, e.g., an aqueous or non-aqueous solution or suspension or a liquid drop designed to be administered into an ear canal.
• a pharmaceutical composition may be formulated for administration via injection either in a particular organ or compartment, e.g., directly into an ear, or systemic, e.g., intravenously.
• a formulation may be or comprise drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes, capsules, powders, etc.
• an active agent may be or comprise an isolated, purified, or pure compound.
• composition As used herein, the term “pharmaceutically acceptable” which, for example, may be used in reference to a carrier, diluent, or excipient used to formulate a pharmaceutical composition as disclosed herein, means that a carrier, diluent, or excipient is compatible with other ingredients of a composition and not deleterious to a recipient thereof.
• composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting a subject compound from one organ, or portion of a body, to another organ, or portion of a body.
• Each carrier must be is “acceptable” in the sense of being compatible with other ingredients of a formulation and not injurious to a patient.
• materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
• polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
• mRNA messenger RNA
• a 3’ poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
• a poly(A) tail can be added onto transcripts that contain a specific sequence, the polyadenylation signal or “poly(A) sequence.”
• a poly(A) tail and proteins bound to it aid in protecting mRNA from degradation by exonucleases.
• Polyadenylation can be affect transcription termination, export of the mRNA from the nucleus, and translation. Typically, polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain can be cleaved through the action of an endonuclease complex associated with RNA polymerase.
• the cleavage site can be characterized by the presence of the base sequence AAUAAA near the cleavage site.
• adenosine residues can be added to the free 3’ end at the cleavage site.
• a “poly(A) sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the additional of a series of adenosines to the 3’ end of the cleaved mRNA.
• Polypeptide refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds.
• a polypeptide has an amino acid sequence that occurs in nature.
• a polypeptide has an amino acid sequence that does not occur in nature.
• a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
• a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
• a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at a polypeptide’s N-terminus, at a polypeptide’s C-terminus, or any combination thereof.
• pendant groups or modifications may be acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
• polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art.
• useful modifications may be or include, e.g., terminal acetylation, amidation, methylation, etc.
• a protein may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
• 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.
• a protein is antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
• Polynucleotide refers to any polymeric chain of nucleic acids.
• a polynucleotide is or comprises RNA; in some embodiments, a polynucleotide is or comprises DNA.
• a polynucleotide is, comprises, or consists of one or more natural nucleic acid residues.
• a polynucleotide is, comprises, or consists of one or more nucleic acid analogs.
• a polynucleotide analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone.
• a polynucleotide has one or more phosphorothioate and/or 5’-N-phosphoramidite linkages rather than phosphodiester bonds.
• a polynucleotide is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine).
• a polynucleotide is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases,
• a polynucleotide comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
• a polynucleotide has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
• a polynucleotide includes one or more introns.
• a polynucleotide is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
• a polynucleotide is at least 3, 4,
• a polynucleotide is partly or wholly single stranded; in some embodiments, a polynucleotide is partly or wholly double stranded. In some embodiments, a polynucleotide has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a polynucleotide has enzymatic activity.
• promoter refers to a nucleic acid sequence that functions to control the transcription of one or more coding sequences (e.g., a gene or transgene, e.g., encoding a therapeutic polypeptide), located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence.
• the promoter is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites or other DNA sequence (e.g., a transcription factor binding site, a repressor and/or activator protein binding site, or other sequences of nucleotides that act directly or indirectly to regulate the amount of transcription from the promoter).
• the promoter can comprise a naturally occurring promoter sequence, a functional fragment thereof, or a mutant of the naturally occurring promoter sequence or a functional fragment thereof.
• Protein refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
• Recombinant is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression construct transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encode
• one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc).
• Reference describes a standard or control relative to which a comparison is performed.
• an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
• a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
• a reference or control is a historical reference or control, optionally embodied in a tangible medium.
• a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
• a reference is a negative control reference; in some embodiments, a reference is a positive control reference.
• regulatory element As used herein, the term “regulatory element” or
• regulatory sequence refers to non-coding regions of DNA that regulate, in some way, expression of one or more particular genes. In some embodiments, such genes are apposed or “in the neighborhood” of a given regulatory element. In some embodiments, such genes are located quite far from a given regulatory element. In some embodiments, a regulatory element impairs or enhances transcription of one or more genes. In some embodiments, a regulatory element may be located in cis to a gene being regulated. In some embodiments, a regulatory element may be located in trans to a gene being regulated.
• a regulatory sequence refers to a nucleic acid sequence which is regulates expression of a gene product operably linked to a regulatory sequence. In some such embodiments, this sequence may be an enhancer sequence and other regulatory elements which regulate expression of a gene product.
• sample typically refers to an aliquot of material obtained or derived from a source of interest.
• a source of interest is a biological or environmental source.
• a source of interest may be or comprise a cell or an organism, such as a microbe (e.g., virus), a plant, or an animal (e.g., a human).
• a source of interest is or comprises biological tissue or fluid.
• a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, and/or combinations or component s) thereof.
• a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
• a biological fluid may be or comprise a plant exudate.
• a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchioalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
• a biological sample is or comprises cells obtained from an individual.
• a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
• the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.
• processing e.g., by removing one or more components of and/or by adding one or more agents to
• a primary sample e.g., filtering using a semi-permeable membrane.
• Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.
• “selectively expresses” refers to expression of a coding sequence, gene, transgene, or polynucleotide (e.g., a therapeutic polynucleotide) of interest predominately in certain specific cell types (e.g., inner ear cells, e.g., inner ear supporting cells).
• polynucleotide e.g., a therapeutic polynucleotide
• Subject refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
• the term “substantially” refers to a qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
• One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
• the term “substantially” is therefore used herein to capture a potential lack of completeness inherent in many biological and chemical phenomena.
• Supporting cell refers to cells of the inner ear that maintain the structure of the inner ear and maintain the environment of the sensory epithelium of the inner ear.
• inner ear supporting cells include, but are not limited to, inner phalangeal cells/border cells (IPhC), inner pillar cells (IPC), outer pillar cells (OPC), Deiters' cells rows 1 and 2 (DC 1/2), Deiters' cells row 3 (DC3), Hensen's cells (Hec), Claudius cells/outer sulcus cells (CC/OSC), interdental cells (Idc), inner sulcus cells (ISC), Kolliker’s organ cells (KO), greater ridge epithelial ridge cells (GER) (including lateral greater epithelial ridge cells (LGER)), and OC90+ cells (OC90), fibroblasts, and other cells of the lateral wall.
• IPhC inner phalangeal cells/border cells
• IPC inner pillar cells
• OPC outer pillar cells
• DC 1/2 Deiters' cells row 3
• Hec Hensen's cells
• CC/OSC Claudius cells/outer sulcus cells
• treatment refers to any administration of a therapy that partially or completely alleviates, ameliorates, eliminates, reverses, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
• such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
• such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
• treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of a given disease, disorder, and/or condition.
• variant refers to a version of something, e.g., a gene sequence, that is different, in some way, from another version. To determine if something is a variant, a reference version is typically chosen and a variant is different relative to that reference version.
• a variant can have the same or a different (e.g., increased or decreased) level of activity or functionality than a wild type sequence.
• a variant can have improved functionality as compared to a wild-type sequence if it is, e.g., codon-optimized to resist degradation, e.g., by an inhibitory nucleic acid, e.g., miRNA.
• an inhibitory nucleic acid e.g., miRNA.
• a variant has a reduction or elimination in activity or functionality or a change in activity that results in a negative outcome (e.g., increased electrical activity resulting in chronic depolarization that leads to cell death).
• a GJB2 gene sequence is a wild-type sequence, which encodes a functional protein and exists in a majority of members of species with genomes containing the GJB2 gene.
• a gain-of-function variant can be a gene sequence of GJB2 that contains one or more nucleotide differences relative to a wild-type GJB2 gene sequence.
• a gain-of-function variant is a codon-optimized sequence which encodes a transcript or polypeptide that may have improved properties (e.g., less susceptibility to degradation, e.g., less susceptibility to miRNA mediated degradation) than its corresponding wild type (e.g., non-codon optimized) version.
• 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., decreased function, non functioning) relative to the wild type transcript and/or polypeptide.
• a mutation in a GJB2 sequence results in a non-functional or otherwise defective connexin 26 (Cx26) protein.
• an ear can be described as including: an outer ear, middle ear, inner ear, hearing (acoustic) nerve, and auditory system (which processes sound as it travels from the ear to the brain).
• ears also help to maintain balance.
• disorders of the inner ear can cause hearing loss, tinnitus, vertigo, imbalance, or combinations thereof.
• Hearing loss can be the result of genetic factors, environmental factors, or a combination of genetic and environmental factors.
• phantom noises in their auditory system phantom noises in their auditory system (ringing, buzzing, chirping, humming, or beating)— also have an over-sensitivity to/reduced tolerance for certain sound frequency and volume ranges, known as hyperacusis (also spelled hyperacousis).
• hyperacusis also spelled hyperacousis.
• nonsyndromic and syndromic-related hearing losses will be known to those of skill in the art (e.g., DFNB1 and DFNA3.
• HID hystrix-like ichthyosis with deafness
• KID palmoplantar keratoderma with deafness
• KID keratitis-ichthyosis-deafness
• Vohwinkel syndrome hystrix-like ichthyosis with deafness
• hearing loss can result from noise, ototoxic agents, presbycusis, disease, infection or cancers that affect specific parts of the ear.
• ischemic damage can cause hearing loss via pathophysiological mechanisms.
• intrinsic abnormalities like congenital mutations to genes that play an important role in cochlear anatomy or physiology, or genetic or anatomical changes in supporting and/or hair cells can be responsible for or contribute to hearing loss.
• Hearing loss and/or deafness is one of the most common human sensory deficits, and can occur for many reasons.
• a subject may be bom with hearing loss or without hearing, while others may lose hearing slowly over time.
• Approximately 36 million American adults report some degree of hearing loss, and one in three people older than 60 and half of those older than 85 experience hearing loss.
• Approximately 1.5 in 1,000 children are born with profound hearing loss, and another two to three per 1,000 children are born with partial hearing loss (Smith et al., 2005, Lancet 365:879-890, which is incorporated in its entirety herein by reference). More than half of these cases are attributed to a genetic basis (Di Domenico, et al., 2011, J. Cell. Physiol. 226:2494-2499, which is incorporated in its entirety herein by reference).
• Treatments for hearing loss currently consist of hearing amplification for mild to severe losses and cochlear implantation for severe to profound losses (Krai and O’Donoghue, 2010, N. Engl. J. Med. 363:1438-1450, which is incorporated in its entirety herein by reference).
• Recent research in this arena has focused on cochlear hair cell regeneration, applicable to the most common forms of hearing loss, including presbycusis, noise damage, infection, and ototoxicity.
• nonsyndromic hearing loss and/or deafness is not associated with other signs and symptoms.
• syndromic hearing loss and/or deafness occurs in conjunction with abnormalities in other parts of the body. Approximately 70 percent to 80 percent of genetic hearing loss and/or deafness cases are nonsyndromic; remaining cases are often caused by specific genetic syndromes.
• Nonsyndromic deafness and/or hearing loss can have different patterns of inheritance, and can occur at any age. Types of nonsyndromic deafness and/or hearing loss are generally named according to their inheritance patterns. For example, autosomal dominant forms are designated DFNA, autosomal recessive forms are DFNB, and X-linked forms are DFN.
• DFNA1 was the first described autosomal dominant type of nonsyndromic deafness. Between 75 percent and 80 percent of genetically causative hearing loss and/or deafness cases are inherited in an autosomal recessive pattern, which means both copies of the gene in each cell have mutations. Usually, each parent of an individual with autosomal recessive hearing loss and/or deafness is a carrier of one copy of the mutated gene, but is not affected by this form of hearing loss.
• Another 20 percent to 25 percent of nonsyndromic hearing loss and/or deafness cases are autosomal dominant, which means one copy of the altered gene in each cell is sufficient to result in deafness and/or hearing loss. People with autosomal dominant deafness and/or hearing loss most often inherit an altered copy of the gene from a parent who is deaf and/or has hearing loss. Between 1 to 2 percent of cases of deafness and/or hearing loss show an X-linked pattern of inheritance, which means the mutated gene responsible for the condition is located on the X chromosome (one of the two sex chromosomes).
• X-linked nonsyndromic hearing loss and/or deafness tend to develop more severe hearing loss earlier in life than females who inherit a copy of the same gene mutation.
• a characteristic of X-linked inheritance is that fathers cannot pass X-linked traits to their sons. Mitochondrial nonsyndromic deafness, which results from changes to mitochondrial DNA, occurs in less than one percent of cases in the United States. The altered mitochondrial DNA is passed from a mother to all of her sons and daughters. This type of deafness is not inherited from fathers. The causes of syndromic and nonsyndromic deafness and/or hearing loss are complex.
• deafness and/or hearing loss can be conductive (arising from the ear canal or middle ear), sensorineural (arising from the inner ear or auditory nerve), or mixed.
• nonsyndromic deafness and/or hearing loss is associated with permanent hearing loss caused by damage to structures in the inner ear (sensorineural deafness).
• sensorineural hearing loss can be due to poor hair cell function.
• sensorineural hearing impairments involve the eighth cranial nerve (the vestibulocochlear nerve) or the auditory portions of the brain. In some such embodiments, only the auditory centers of the brain are affected.
• cortical deafness may occur, where sounds may be heard at normal thresholds, but quality of sound perceived is so poor that speech cannot be understood.
• Hearing loss that results from changes in the middle ear is called conductive hearing loss.
• Some forms of nonsyndromic deafness and/or hearing loss involve changes in both the inner ear and the middle ear, called mixed hearing loss.
• Hearing loss and/or deafness that is present before a child learns to speak can be classified as prelingual or congenital.
• Hearing loss and/or deafness that occurs after the development of speech can be classified as postlingual.
• Most autosomal recessive loci related to syndromic or nonsyndromic hearing loss cause prelingual severe-to-profound hearing loss.
• hair cells are sensory receptors for both auditory and vestibular systems of vertebrate ears. Hair cells detect movement in the environment and, in mammals, hair cells are located within the cochlea of the ear, in the organ of Corti. Mammalian ears are known to have two types of hair cells - inner hair cells and outer hair cells. Outer hair cells can amplify low level sound frequencies, either through mechanical movement of hair cell bundles or electrically-driven movement of hair cell soma. Inner hair cells transform vibrations in cochlear fluid into electrical signals that the auditory nerve transmits to the brain. In some embodiments, hair cells may be abnormal at birth, or damaged during the lifetime of an individual. In some embodiments, outer hair cells may be able to regenerate. In some embodiments, inner hair cells are not capable of regeneration after illness or injury. In some embodiments, sensorineural hearing loss is due to abnormalities in hair cells.
• supporting cells may fulfil numerous functions, and include a number of cell types, including but not limited to Hensen’s cells, Deiters’ cells, pillar cells, Claudius cells, inner phalangeal cells, and border cells.
• sensorineural hearing loss is due to abnormalities in supporting cells.
• supporting cells may be abnormal at birth, or damaged during the lifetime of an individual.
• supporting cells may be able to regenerate. In some embodiments, certain supporting cells may not be capable of regeneration.
• the GJB2 gene is highly conserved across the mammalian class and encodes connexin 26 (Cx26) (also referred to as gap junction beta-2 (GJB2) protein).
• Connexin 26 is a member of the gap junction protein family, which is also known as the connexin family.
• Gap junction proteins are specialized proteins, involved in intracellular communication. Mutations in the human GJB2 gene have been associated with hearing loss and deafness (Amorini et ah, Ann. Hum. Genet. 79(5):341-349, 2015; Qing et ah, Genet. Test Mol. Biomarkers 19(l):52-58, 2015).
• the human GJB2 gene is located on chromosome 13ql2. It contains two transcriptional isoforms beginning from alternative transcriptional start sites, both of which contain two exons and a single intron encompassing a total of about 5 kilobases (kb) (approximately 5,469 or 4,675 nucleotides respectively) (NCBI Gene ID 2706, NCBI Reference Sequence: NG 008358.1). Both human GJB2 mRNA isoforms comprise a second exon, which completely encodes a full-length connexin 26 in exon two. This coding sequence is approximately 681 nucleotides, and encodes a connexin 26 that is 226 amino acids in length.
• a monomer of connexin 26 includes four transmembrane helices linked by two extracellular loops and one shorter intracellular loop, with N- and C-termini on the cytosolic side of the plasma membrane. Gap junctions between cells can be formed in a homomeric and/or heteromeric manner. Connexin 26 has been shown to form functional homomeric channels, as well as functional heteromeric channels with at least connexin 30, connexin 32, connexin 46, and connexin 50. In some embodiments, GJB2 gene associated sensorineural hearing loss (e.g., nonsyndromic or syndromic) may be due to compound heterozygous mutations in GJB2 and in an alternative connexin protein encoding gene. The gap junctions created with connexin 26 transport potassium ions and certain other small molecules across cells. Connexin 26 helps maintain the correct level of intracellular potassium ions, and is required for the maturation of certain cells in the cochlea.
• a human GJB2 gene is expressed in a number of tissues, but is known to be involved in important cellular homeostasis functions in the epidermis and inner ear.
• connexin 26 is synthesized by all supporting cell types within the organ of corti, including the inner and outer pillar cells, root cells, interdental cells, fibrocytes from the underlying connective tissue, and basal and intermediate cells from the stria vascularis.
• connexin 26 is known to be present in mesenchymal cells in the lateral wall, and type 1 neurons in the spiral ganglion.
• the human GJB2 gene has a defined 128bp long basal promoter just upstream of the canonical first exon in the most abundant isoform. This sequence includes a TATA box and two GC boxes, which are known to be bound by the Spl and Sp3 TFs.
• GJB2 GJB2
• various mutations in the GJB2 gene have been associated with hearing loss (e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss).
• hearing loss e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss.
• the c.35delG allele was found on 65.5% of patients from Eastern Sicily (Amorini et ak, Ann. Hum. Genet. 79(5):341-349, 2015).
• Human GBJ2 gene mutations which lead to syndromic or nonsyndromic hearing loss vary from large deletions that remove either the entirety of GJB2 or GJB2 gene regulatory regions, to hundreds of small scale alterations including nonsense, missense, indels (leading to phase shifting), and splice-site point mutations.
• GJB2 gene mutations such as Gly59Ser, and Asn52Lys are associated with Bart-Pumphrey syndrome.
• GJB2 gene mutations such as Aspn50Asn are associated with Hystrix-like Ichthyosis with deafness & Keratitis- ichthyosis-deafness syndrome. These syndromes are associated with dry scaly skin, generally congenital profound sensorineural hearing loss, and in Keratitis-ichthyosis- deafness syndrome, additional inflammation of the cornea.
• GJB2 gene missense mutations are associated with
• Palmoplantar keratoderma with deafness A syndrome associated with thick skin on the palms of the hands and soles of the feet, and mild to profound sensorineural hearing loss which begins in early childhood and gets worse over time, affected individuals may have particular trouble hearing high-pitched sounds. While in other embodiments, GJB2 gene missense mutations are associated with Vohwinkel syndrome. A syndrome associated with skin abnormalities (e.g., thick bands of fibrous tissue around their fingers and toes that may cut off the circulation to the digits and result in spontaneous amputation) and sensorineural hearing loss.
• skin abnormalities e.g., thick bands of fibrous tissue around their fingers and toes that may cut off the circulation to the digits and result in spontaneous amputation
• GJB2 gene mutations are associated with nonsyndromic hearing loss, which may be inherited in either a dominant (e.g., DFNA3) or recessive manner (DFNBl).
• loss of function GJB2 gene mutations are associated with nonsyndromic DFNBl which is inherited in an autosomal recessive manner and presents as mild to profound hearing loss that is generally prelingual and does not become more severe over time. It is estimated that DFNBl is present in approximately 14 out of every 100,000 live births in the US and EU5. It has been postulated that an early but not always congenital onset of DFNBl hearing impairment could be followed by a quick progression of the hearing loss.
• DFNB1 patents treatment options include education, hearing aids, and cochlear implants. Patients generally do not have additional symptoms, and live a normal lifespan. It is estimated that DFNB1 accounts for about 50% of congenital severe-to-profound autosomal recessive non-syndromic hearing loss in many first world countries (e.g., US, France, British, and Australia).
• sensorineural hearing loss due to GJB2 gene mutations are inherited in an autosomal dominant manner as nonsyndromic DFNA3. These mutations are generally dominant negative missense mutations that prevent the formation of necessary functional gap junctions. This disease state presents with hearing loss that can be either prelingual or postlingual, ranging from mild to profound, which generally becomes more severe over time.
• the present disclosure provides polynucleotides, e.g., polynucleotides comprising a GJB2 gene or characteristic portion thereof, as well as compositions including such polynucleotides and methods utilizing such polynucleotides and/or compositions.
• a polynucleotide comprising a GJB2 gene or characteristic portion thereof can be DNA or RNA.
• DNA can be genomic DNA or cDNA.
• RNA can be an mRNA.
• a polynucleotide comprises exons and/or introns of a GJB2 gene.
• a gene product is expressed from a polynucleotide comprising a GJB2 gene or characteristic portion thereof.
• expression of such a polynucleotide can utilize one or more control elements (e.g., promoters, enhancers, splice sites, poly-adenylation sites, translation initiation sites, etc.).
• control elements e.g., promoters, enhancers, splice sites, poly-adenylation sites, translation initiation sites, etc.
• a polynucleotide provided herein can include one or more control elements.
• a GJB2 gene is a mammalian GJB2 gene. In some embodiments, a GJB2 gene is a murine GJB2 gene. In some embodiments, a GJB2 gene is a primate GJB2 gene. In some embodiments, a GJB2 gene is a human GJB2 gene.
• An exemplary human GJB2 coding cDNA sequence is or includes the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
• An exemplary human GJB2 spliced cDNA sequence with untranslated regions is or includes the sequence of SEQ ID NO: 3.
• An alternative transcriptional start site exemplary human GJB2 spliced cDNA sequence with untranslated regions is or includes the sequence of SEQ ID NO: 4.
• An exemplary human GJB2 genomic DNA sequence can be found in SEQ ID NO: 5.
• Exemplary spliced Human GJB2 isoform XI cDNA including untranslated regions Sequence SEQ ID NO: 4.
• CTATAATAA Exemplary expanded Human GJB2 Genomic DNA Sequence including certain regulatory regions (SEQ ID NO: 6)
• a polynucleotide comprises a GJB2 gene having one or more silent mutations.
• the disclosure provides a polynucleotide that comprises a GJB2 gene having one or more silent mutations, e.g., a GJB2 gene having a sequence different from SEQ ID NO: 1, 2, 3, 4, 5 or 6 but encoding the same amino acid sequence as a functional GJB2 gene.
• the disclosure provides a polynucleotide that comprises a GJB2 gene having a sequence different from SEQ ID NO: 1, 2, 3, 4, 5 or 6 that encodes an amino acid sequence including one or more mutations (e.g., a different amino acid sequence when compared to that produced from a functional GJB2 gene), where the one or more mutations are conservative amino acid substitutions.
• the disclosure provides a polynucleotide that comprises a GJB2 gene having a sequence different from SEQ ID NO: 1, 2, 3, 4, 5 or 6 that encodes an amino acid sequence including one or more mutations (e.g., a different amino acid sequence when compared to that produced from a functional GJB2 gene), where the one or more mutations are not within a characteristic portion of a GJB2 gene or an encoded connexin 26 protein.
• a polynucleotide in accordance with the present disclosure comprises a GJB2 gene that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a sequence of SEQ ID NO: 1, 2, 3, 4,
• a polynucleotide in accordance with the present disclosure comprises a GJB2 gene that is identical to the sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6.
• SEQ ID NO: 1, 2, 3, 4, 5 or 6 can be optimized (e.g., codon optimized) to achieve increased or optimal expression in an animal, e.g., a mammal, e.g., a human.
• the present disclosure provides polypeptides encoded by a
• GJB2 gene or characteristic portion thereof is a GJB2 gene or characteristic portion thereof.
• a GJB2 gene is a mammalian GJB2 gene.
• a GJB2 gene is a murine GJB2 gene.
• a GJB2 gene is a primate GJB2 gene.
• a GJB2 gene is a human GJB2 gene.
• a polypeptide comprises a connexin 26 protein or characteristic portion thereof.
• a connexin 26 protein or characteristic portion thereof is mammalian connexin 26 protein or characteristic portion thereof, e.g., primate connexin 26 protein or characteristic portion thereof.
• a connexin 26 protein or characteristic portion thereof is a human connexin 26 protein or characteristic portion thereof.
• a polypeptide provided herein comprises post-translational modifications.
• a connexin 26 protein or characteristic portion thereof provided herein comprises post-translational modifications.
• post-translational modifications can comprise but is not limited to glycosylation (e.g., N-linked glycosylation, O-linked glycosylation), phosphorylation, acetylation, amidation, hydroxylation, methylation, ubiquitylation, sulfation, and/or a combination thereof.
• An exemplary human connexin 26 protein sequence is or includes the sequence of SEQ ID NO: 7.
• Exemplary Human Connexin 26 Protein Sequence SEQ ID NO: 7
• a polypeptide described herein e.g., including connexin 26 or a characteristic portion thereof
• a polypeptide includes one or more mutations, where the one or more mutations are conservative amino acid substitutions.
• a polypeptide in accordance with the present disclosure comprises a connexin 26 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 a sequence of SEQ ID NO: 7.
• a polypeptide in accordance with the present disclosure comprises a connexin 26 or a characteristic portion thereof that is identical to the sequence of SEQ ID NO: 7. In some embodiments, a polypeptide in accordance with the present disclosure comprises a connexin 26 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 a sequence of SEQ ID NO: 7. In some embodiments, a polypeptide in accordance with the present disclosure comprises a connexin 26 protein or a characteristic portion thereof that is identical to the sequence of SEQ ID NO: 7.
• polynucleotide constructs include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno-associated viral constructs) that incorporate a polynucleotide comprising a GJB2 gene or characteristic portion thereof.
• cosmids e.g., naked or contained in liposomes
• viral constructs e.g., lentiviral, retroviral, adenoviral, and adeno-associated viral constructs
• a construct is a plasmid (i.e., a circular DNA molecule that can autonomously replicate inside a cell).
• a construct can be a cosmid (e.g., pWE or sCos series).
• a construct is a viral construct.
• a viral construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus construct.
• a construct is an adeno-associated virus (AAV) construct (see, e.g., Asokan et al., Mol. Ther. 20: 699-7080, 2012, which is incorporated in its entirety herein by reference).
• AAV adeno-associated virus
• a viral construct is an adenovirus construct.
• a 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 pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus.
• the genome of such viruses encode nonstructural (e.g., replicon) and structural proteins (e.g., capsid and envelope) that can be translated in the cytoplasm of the host cell.
• Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop viral constructs for coding sequence delivery.
• Pseudotyped viruses may be formed by combining alphaviral envelope glycoproteins and retroviral capsids. Examples of alphaviral constructs can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819; constructs and methods of their making are incorporated herein by reference to each of the publications in its entirety.
• a construct is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8kb, 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.
• a construct is a plasmid and can have a total length in a 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.
• a construct is a viral construct and can have a total number of nucleotides of up to 10 kb. In some embodiments, a viral construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about
• a construct is a lentivirus construct and can have a total number of nucleotides of up to 8 kb.
• a lentivirus 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 4 kb, about 3 kb to about 5 kb, about 2 kb to about 6
• a construct is an adenovirus construct and can have a total number of nucleotides of up to 8 kb.
• an adenovirus construct can have a total number of nucleotides 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 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 4 kb, about 3 kb to about 4 kb, about 3 kb
• any of the constructs described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and/or additional untranslated regions which may house pre- or post-transcriptional regulatory and/or control elements.
• a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.
• control sequences are described herein.
• AAV particles that comprise a construct encoding a GJB2 gene or characteristic portion thereof described herein, and a capsid described herein.
• AAV particles can be described as having a serotype, which is a description of the construct strain and the capsid strain.
• an AAV particle may be described as AAV2, wherein the particle has an AAV2 capsid and a construct that comprises characteristic AAV2 Inverted Terminal Repeats (ITRs).
• ITRs Inverted Terminal Repeats
• an AAV particle may be described as a pseudotype, wherein the capsid and construct are derived from different AAV strains, for example, AAV2/9 would refer to an AAV particle that comprises a construct utilizing the AAV2 ITRs and an AAV9 capsid.
• an AAV capsid is an Anc80 capsid (e.g., an Anc80L65 capsid).
• the present disclosure provides polynucleotide constructs that comprise a GJB2 gene or characteristic portion thereof.
• a polynucleotide comprising a GJB2 gene or characteristic portion thereof can be included in an AAV particle.
• a polynucleotide construct comprises one or more components derived from or modified from naturally occurring AAV genomic construct.
• a sequence derived from an AAV construct is an AAV1 construct, an AAV2 construct, an AAV3 construct, an AAV4 construct, an AAV5 construct, an AAV6 construct, an AAV7 construct, an AAV8 construct, an AAV9 construct, an AAV2.7m8 construct, an AAV8BP2 construct, an AAV293 construct, or AAV Anc80 construct.
• Additional exemplary AAV constructs that can be used herein are known in the art. See, e.g., Kanaan et al., Mol. Ther. Nucleic Acids 8:184-197, 2017; Li et al.,
• provided constructs comprise coding sequence, e.g., a
• the polynucleotide construct may be referred to as a recombinant AAV (rAAV) construct.
• rAAV recombinant AAV
• provided rAAV constructs are packaged into an AAV capsid to form an AAV particle.
• an AAV capsid is an Anc80 capsid (e.g., an Anc80L65 capsid).
• AAV derived sequences typically include the cis-acting 5’ and 3’ ITR sequences (see, e.g., B. J. Carter, in “Handbook of Parvoviruses,” ed., P. Tijsser, CRC Press, pp. 155 168, 1990, which is incorporated herein by reference in its entirety).
• Typical AAV2-derived ITR sequences are about 145 nucleotides in length.
• at least 80% of a typical ITR sequence e.g., at least 85%, at least 90%, or at least 95%) is incorporated into a construct provided herein.
• any of the coding sequences and/or constructs described herein are flanked by 5’ and 3’ AAV ITR sequences.
• the AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types.
• polynucleotide constructs described in accordance with this disclosure and in a pattern known to the art are typically comprised 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).
• ITRs AAV inverted terminal repeats
• provided constructs can be packaged into a capsid to create an AAV particle.
• An AAV particle may be delivered to a selected target cell.
• provided constructs comprise an additional optional coding sequence that is a nucleic acid sequence (e.g., inhibitory nucleic acid sequence), heterologous to the construct sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
• a nucleic acid coding sequence is operatively linked to and/or control components in a manner that permits coding sequence transcription, translation, and/or expression in a cell of a target tissue.
• an unmodified AAV endogenous genome includes two open reading frames, “cap” and “rep,” which are flanked by ITRs.
• exemplary rAAV constructs similarly include ITRs flanking a coding region, e.g., a coding sequence (e.g., a GJB2 gene).
• an rAAV construct also comprises conventional control elements that are operably linked to the coding sequence in a manner that permits its transcription, translation and/or expression in a cell transfected with the plasmid construct or infected with the virus produced by the disclosure.
• an rAAV construct optionally comprises a promoter (shown in Figure 1, panel (B)), an enhancer, an untranslated region (e.g., a 5’ UTR, 3’ UTR), a Kozak sequence, an internal ribosomal entry site (IRES), splicing sites (e.g., an acceptor site, a donor site), a polyadenylation site (shown in FIG.
• an rAAV construct comprises a 5' ITR, a promoter, a hGJB2 gene, a poly A, and a 3' ITR (shown in FIGs. 2A and 2E).
• an rAAV construct comprises a 5' ITR, a promoter, a hGJB2 gene, a 3' UTR, a poly A, and a 3' ITR (shown in FIG. 2B).
• an rAAV construct comprises a 5' ITR, a promoter, a hGJB2 gene, a C3 domain, a poly A, and a 3' ITR (shown in FIG. 2C).
• an rAAV construct comprises a 5' ITR, a promoter, a hGJB2 gene, a D7 domain, a poly A, and a 3' ITR (shown in FIG. 2D).
• an rAAV construct comprises a 5' ITR, a promoter, a 5' UTR, a hGJB2 gene, an optional FLAG tag, a 3'UTR, a poly A, and a 3' ITR (shown in FIGs. 2F-2J, 2L, and 2N).
• an rAAV construct comprises a 5' ITR, a promoter, a 5' UTR, a hGJB2 gene, an optional FLAG tag, a 3' UTR, a microRNA regulatory target site, a poly A, and a 3' ITR (shown in FIGs. 2M).
• additional elements are described further herein.
• a construct is a rAAV construct.
• an rAAV construct can include at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, or at least 4.5 kb.
• an AAV construct can include at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, at most 4 kb, at most 3.5 kb, at most 3 kb, or at most 2.5 kb.
• an AAV construct can include 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 5kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.
• any of the constructs described herein can further include regulatory and/or control sequences, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and/or any combination thereof.
• a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.
• control sequences are described herein.
• AAV derived sequences of a construct typically comprises the cis-acting 5’ and 3’
• ITRs See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990), which is incorporated in its entirety herein by reference).
• ITRs are able to form a hairpin.
• the ability to form a hairpin can contribute to an ITRs ability to self-prime, allowing primase-independent synthesis of a second DNA strand.
• ITRs also play a role in integration of AAV construct (e.g., a coding sequence, e.g., a GJB2 gene) into a genome of a subject’s cell.
• ITRs can also aid in efficient encapsidation of an AAV construct in an AAV particle.
• An rAAV particle (e.g., an AAV2/Anc80 particle) of the present disclosure can comprise a rAAV construct comprising a coding sequence (e.g., GJB2 gene) and associated elements flanked by a 5’ and a 3’ AAV ITR sequences.
• a coding sequence e.g., GJB2 gene
• an ITR is or comprises about 145 nucleic acids.
• an ITR is or comprises about 119 nucleic acids.
• an ITR is or comprises about 130 nucleic acids.
• all or substantially all of a sequence encoding an ITR is used.
• An AAV ITR sequence may be obtained from any known AAV, including presently identified mammalian AAV types.
• an ITR is an AAV2 ITR.
• An example of a construct molecule employed in the present disclosure is a “cis- acting” construct containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5’ or “left” and 3’ or “right” AAV ITR sequences.
• 5’ and left designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
• a 5’ or left ITR is an ITR that is closest to a promoter (as opposed to a polyadenylation sequence) for a given construct, when a construct is depicted in a sense orientation, linearly.
• 3’ and right designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
• a 3’ or right ITR is an ITR that is closest to a polyadenylation sequence (as opposed to a promoter sequence) for a given construct, when a construct is depicted in a sense orientation, linearly.
• ITRs as provided herein are depicted in 5’ to 3’ order in accordance with a sense strand. Accordingly, one of skill in the art will appreciate that a 5’ or “left” orientation ITR can also be depicted as a 3’ or “right” ITR when converting from sense to antisense direction.
• a given sense ITR sequence e.g., a 5 ’/left AAV ITR
• an antisense sequence e.g., 3 ’/right ITR sequence.
• One of ordinary skill in the art would understand how to modify a given ITR sequence for use as either a 5 ’/left or 3 ’/right ITR, or an antisense version thereof.
• an ITR e.g., a 5’ ITR
• an ITR e.g., a 3’ ITR
• an ITR includes one or more modifications, e.g., truncations, deletions, substitutions or insertions, as is known in the art.
• an ITR comprises fewer than 145 nucleotides, e.g., 127, 130, 134 or 141 nucleotides.
• an ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 ,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 144, or 145 nucleotides.
• the ITR comprises about 119 nucleotides.
• the ITR comprises about 130 nucleotides.
• an ITR (e.g., a 5’ ITR) can have a sequence according to SEQ ID NO: 52.
• an ITR e.g., a 3’ ITR
• a non-limiting example of 5’ AAV ITR sequences includes SEQ ID NO: 8 or 52.
• a non-limiting example of 3’ AAV ITR sequences includes SEQ ID NO: 9 or 53.
• the 5’ and a 3’ AAV ITRs flank a portion of a coding sequence, e.g., all or a portion of a GJB2 gene (e.g., SEQ ID NO: 1, 2, 3, 4, 5, or 6).
• the ability to modify these ITR sequences is within the skill of 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 in its entirety herein by reference).
• a 5’ ITR sequence is at least 85%, 90%,
• a 3’ ITR sequence is at least 85%, 90%, 95%, 98% or 99% identical to a 3’ ITR sequence represented by SEQ ID NO: 9.
• a 5’ ITR sequence is at least 85%, 90%, 95%, 98% or 99% identical to a 5’ ITR sequence represented by SEQ ID NO: 52.
• a 3’ ITR sequence is at least 85%, 90%, 95%, 98% or 99% identical to a 3’ ITR sequence represented by SEQ ID NO: 53.
• Exemplary 5’ AAV ITR (SEQ ID NO: 8)
• Exemplary 3’ AAV ITR (SEQ ID NO: 9)
• the disclosure is directed to constructs comprising a cell selective promoter which can be used to regulate (e.g., increase) expression of connexin 26 protein in a cell (e.g., an inner ear cell, e.g., a supporting cell).
• a cell selective promoter which can be used to regulate (e.g., increase) expression of connexin 26 protein in a cell (e.g., an inner ear cell, e.g., a supporting cell).
• the constructs provide reduced toxicity that may be associated with expression of connexin 26 in some cells (e.g., an inner ear cell, e.g., a hair cell).
• a construct (e.g., an rAAV construct) comprises a promoter.
• promoter refers to a DNA sequence recognized by enzymes/proteins that can promote and/or initiate transcription of an operably linked gene (e.g., a GJB2 gene).
• a promoter typically refers to, e.g., a nucleotide sequence to which an RNA polymerase and/or any associated factor binds and from which it can initiate transcription.
• a construct (e.g., an rAAV construct) comprises a promoter operably linked to one of the non-limiting example promoters described herein.
• a 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.
• a promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter.
• a promoter is a RNA polymerase III promoter, including, but not limited to, a HI promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter.
• a promoter will generally be one that is able to promote transcription in an inner ear cell.
• a promoter is a cochlea-specific promoter or a cochlea-oriented promoter.
• a promoter is a hair cell specific promoter, or a supporting cell specific promoter.
• a variety of promoters are known in the art, which can be used herein. Non limiting examples of promoters that can be used herein include: human EFla, human cytomegalovirus (CMV) (US Patent No.
• human ubiquitin C UBC
• mouse phosphogly cerate kinase 1 polyoma adenovirus
• simian virus 40 SV40
• b-globin b-actin
• a-fetoprotein g-globin
• b- interferon g-glutamyl transferase
• mouse mammary tumor virus MMTV
• Rous sarcoma virus rat insulin
• glyceraldehyde-3 -phosphate dehydrogenase metallothionein II (MT II), amylase
• cathepsin MI muscarinic receptor
• retroviral LTR e.g., human T-cell leukemia virus HTLV
• AAV ITR interleukin-2
• collagenase platelet-derived growth factor
• adenovirus 5 E2 stromelysin
• murine MX gene glucose regulated proteins
• a promoter is the CMV immediate early promoter.
• the promoter is a CBA promoter.
• the promoter is a CAG promoter or a CAG/CBA promoter.
• the promoter comprises or consists of SEQ ID NO: 10.
• a promoter comprises or consists of SEQ ID NO: 11.
• a promoter comprises a CMV/CBA enhancer/promoter construct exemplified in SEQ ID NO: 12.
• a promoter comprises a CMV/CBA enhancer/promoter construct exemplified in SEQ ID NO: 13. In certain embodiments, a promoter comprises a CAG promoter or CMV/CBA/SV-40 enhancer/promoter construct exemplified in SEQ ID NO: 14. In certain embodiments, a promoter comprises a CAG promoter or CMV/CBA/SV-40 enhancer/promoter construct exemplified in SEQ ID NO: 15. In some aspects, a promoter comprises a ATOH1 enhance/promoter construct of SEQ ID NO: 16. In some aspects, a promoter comprises a GJB2 enhance/promoter construct of SEQ ID NO: 17.
• a promoter comprises a GJB2 enhance/promoter construct of SEQ ID NO: 61.
• a promoter is an endogenous human SLC26A4 enhancer-promoter sequence comprised within SEQ ID NO: 54.
• a promoter is an endogenous human LGR5 enhancer-promoter sequence comprised within SEQ ID NO: 55.
• a promoter is an endogenous human SYN1 enhancer-promoter sequence comprised within SEQ ID NO: 56.
• a promoter is an endogenous human GFAP enhancer- promoter sequence comprised within SEQ ID NO: 57 or SEQ ID NO: 62.
• a promoter is an endogenous human IGFBP2 enhancer-promoter sequence comprised within SEQ ID NO: 95.
• a promoter is an endogenous human RBP7 promoter as set forth in SEQ ID NO: 98.
• a promoter is an endogenous human GJB6 promoter as set forth in SEQ ID NO: 101.
• a promoter is an endogenous human PARM1 promoter as set forth in SEQ ID NO: 104
• the promoter comprises a GJB6 and a hGJB2 minimal promoter.
• the GJB6 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 91 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the GJB6 has the nucleic acid sequence of SEQ ID NO: 91 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the promoter comprises a IGFBP2 promoter and a hGJB2 minimal promoter.
• the IGFBP2 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 95 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the IGFBP2 has the nucleic acid sequence of SEQ ID NO: 95 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the promoter comprises a RBP7 promoter and a hGJB2 minimal promoter.
• the RBP7 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 98 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the RBP7 has the nucleic acid sequence of SEQ ID NO: 98 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the promoter comprises a GJB6 promoter and a hGJB2 minimal promoter.
• the GJB6 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 101 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the GJB6 has the nucleic acid sequence of SEQ ID NO: 101 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the promoter comprises a PARM1 promoter and a hGJB2 minimal promoter.
• the PARM1 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 104 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the PARM1 has the nucleic acid sequence of SEQ ID NO: 104 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• a promoter sequence is at least 85%, at least 90%, at least
• a promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to the promoter sequences represented by SEQ ID NO: 10.
• a promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to the promoter sequences represented by SEQ ID NO: 11.
• a promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to the promoter sequences represented by SEQ ID NO: 91.
• a promoter is an endogenous human GDF6 promoter sequence comprised within SEQ ID NO: 90.
• an promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to a promoter sequence represented by SEQ ID NO: 95. In some aspects, an promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to a promoter sequence represented by SEQ ID NO: 98. In some aspects, an promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to promoter sequence represented by SEQ ID NO: 101. In some aspects, a promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to promoter sequence represented by SEQ ID NO: 104.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 12. In some aspects, an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 13.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 14. In some aspects, an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer- promoter sequence represented by SEQ ID NO: 15.
• an enhancer- promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 16. In some aspects, an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 17.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer- promoter sequence represented by SEQ ID NO: 61. In some aspects, an enhancer- promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 54.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 55. In some aspects, an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer- promoter sequence represented by SEQ ID NO: 56.
• an enhancer- promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 57 or SEQ ID NO: 62. In some aspects, an promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 90.
• RNA refers to a nucleotide sequence that, when operably linked with a nucleic acid encoding a protein (e.g., a connexin 26 protein), causes RNA to be transcribed from the nucleic acid in a cell under most or all physiological conditions.
• a protein e.g., a connexin 26 protein
• constitutive promoters include, without limitation, the retroviral
• Rous sarcoma virus (RSV) LTR promoter Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41:521-530, 1985, which is incorporated in its entirety herein by reference), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl-alpha promoter (Invitrogen).
• the promoter is a constitutive promoter.
• the constitutive promoter is a CAG promoter, a CBA promoter, a CMV promoter, a CMV/CBA enhancer/promoter, or a CB7 promoter.
• the a CMV/CBA enhancer/promoter comprises a nucleic acid with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NOs:
• the a CMV/CBA enhancer/promoter comprises a nucleic acid of SEQ ID NO: 12. In some aspects, the a CMV/CBA enhancer/promoter comprises a nucleic acid of SEQ ID NO: 13. In some aspects, the CBA promoter comprises a nucleic acid with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NOs: 10 or 11. In some aspects, the CBA promoter comprises a nucleic acid of SEQ ID NO: 10. In some aspects, the CBA promoter comprises a nucleic acid of SEQ ID NO: 11.
• the CMV promoter comprises a nucleic acid with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NOs: 14 or 15. In some aspects, the CMV promoter comprises a nucleic acid of SEQ ID NO: 14. In some aspects, the CMV promoter comprises a nucleic acid of SEQ ID NO: 15.
• Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
• Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art.
• inducible promoters regulated by exogenously supplied compounds 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, which is incorporated in its entirety herein by reference); the ecdysone insect promoter (No et al., Proc. Natl. Acad Sci. US. A. 93:3346- 3351, 1996, which is incorporated in its entirety herein by reference), the tetracycline- repressible system (Gossen et al., Proc. Natl. Acad Sci. US. A.
• tissue-specific promoter refers to a promoter that is active only in certain specific cell types and/or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory and/or control proteins that bind to the tissue-specific promoter).
• regulatory and/or control sequences impart tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory and/or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.
• tissue-specific promoter is a cochlea-specific promoter.
• a tissue-specific promoter is a cochlear hair cell-specific promoter.
• cochlear hair cell-specific promoters include but are not limited to: a ATOH1 promoter, a POU4F3 promoter, a LHX3 promoter, a MY07A promoter, a MY06 promoter, a a9ACHR promoter, and a alOACHR promoter.
• a 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 1: 199-203, 2004; and Ryan et al., Adv. Otorhinolaryngol. 66: 99-115, 2009, each of which is incorporated in their entirety herein by reference.
• tissue-specific promoter is an ear cell specific promoter.
• a tissue-specific promoter is an inner ear cell specific promoter.
• a promoter is a characteristic fragment of a tissue-specific promoter.
• inner ear non-sensory cell-specific promoters include but are not limited to: GJB2, GJB6, SLC26A4, TECTA, DFNA5, COCH, NDP, SYN1, GFAP, PLP, TAK1, IGFBP2, RBP7, GDF6, PARM1, or SOX21.
• a cochlear non-sensory cell specific promoter may 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,
• a cell selective promoter is an ear cell selective promoter. In some aspects, a cell selective promoter is an inner ear cell selective promoter. In some aspects, a promoter is a characteristic fragment of a cell selective promoter. In some aspects, the promoter is a supporting cell selective promoter. In some aspects, the promoter is an inner ear supporting cell selective promoter.
• the promoter is a supporting cell selective promoter.
• the promoter is a hair cell selective promoter.
• the supporting cells are selected from one or more of inner phalangeal cells/border cells (IPhC), inner pillar cells (IPC), outer pillar cells (OPC), Deiters' cells rows 1 and 2 (DC 1/2), Deiters' cells row 3 (DC3), Hensen's cells (Hec), Claudius cells/outer sulcus cells (CC/OSC), interdental cells (Idc), inner sulcus cells (ISC), Kolliker’s organ cells (KO), Lateral greater epithelial ridge cells (LGER), and OC90+ cells (OC90).
• IPhC inner phalangeal cells/border cells
• IPC inner pillar cells
• OPC outer pillar cells
• DC 1/2 Deiters' cells row 3
• Hec Hensen's cells
• CC/OSC Claudius cells/outer sulcus cells
• Idc inner
• supporting cell selective promoters are selected from one or more of GJB6, GDF6, PARM1, RBP7, and IGFBP2.
• the promoter is an inner ear medial support cell selective promoter.
• inner ear medial support cells are selected from one or more of lateral greater epithelial ridge cells and inner sulcus cells.
• inner ear medial support cell selective promoters are selected from one or more of GJB6, IGFBP2, GDF6, PARM1, and GFAP.
• the promoter is an inner ear sensory epithelial support cell selective promoter.
• sensory epithelial support cells are selected from one or more of inner pillar cells, outer pillar cells, dieter cells, and inner phalangeal cells.
• inner ear sensory epithelial support cell selective promoters are selected from one or more of GJB6, IGFBP2, RBP7, GDF6, PARM1, and GFAP.
• the inner ear supporting cell selective promoter is a GJB2 promoter.
• the GJB2 enhance/promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 17.
• the GJB2 enhance/promoter comprises the nucleic acid sequence of SEQ ID NO: 17.
• the GJB2 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 61.
• the GJB2 promoter comprises the nucleic acid sequence of SEQ ID NO: 61.
• the GJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 91.
• the GJB2 minimal promoter comprises the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter is a GJB6 promoter.
• the GJB6 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 101.
• the GJB6 promoter comprises the nucleic acid sequence of SEQ ID NO: 101.
• the inner ear supporting cell selective promoter is a SLC26A4 promoter.
• the SLC26A4 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 54.
• the SLC26A4 promoter comprises the nucleic acid sequence of SEQ ID NO: 54.
• the inner ear supporting cell selective promoter is a GFAP promoter.
• the GFAP promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 57.
• the GFAP promoter comprises the nucleic acid sequence of SEQ ID NO: 57.
• the GFAP promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 62.
• the GFAP promoter comprises the nucleic acid sequence of SEQ ID NO: 62.
• the inner ear supporting cell selective promoter is a IGFBP2 promoter.
• the IGFBP2 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 95.
• the IGFBP2 promoter comprises the nucleic acid sequence of SEQ ID NO: 95.
• the inner ear supporting cell selective promoter is a RBP7 promoter.
• the RBP7 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 98.
• the RBP7 promoter comprises the nucleic acid sequence of SEQ ID NO: 98.
• the inner ear supporting cell selective promoter is a GDF6 promoter.
• the GDF6 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 90.
• the GDF6 promoter comprises the nucleic acid sequence of SEQ ID NO: 90.
• the inner ear supporting cell selective promoter is a PARM1 promoter.
• the PARM1 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 40.
• the PARM1 promoter comprises the nucleic acid sequence of SEQ ID NO: 40.
• the inner ear supporting cell selective promoter is a LGR5 promoter.
• the LGR5 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 55.
• the LGR5 promoter comprises the nucleic acid sequence of SEQ ID NO: 55.
• the inner ear supporting cell selective promoter is a ATOH1 promoter.
• the ATOH1 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 16.
• the ATOH1 promoter comprises the nucleic acid sequence of SEQ ID NO: 16.
• the inner ear supporting cell selective promoter comprises a
• the GJB6 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 91 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the GJB6 has the nucleic acid sequence of SEQ ID NO: 91 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the IGFBP2 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 95 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the IGFBP2 has the nucleic acid sequence of SEQ ID NO: 95 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the RBP7 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 98 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the RBP7 has the nucleic acid sequence of SEQ ID NO: 98 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the GJB6 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 101 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the GJB6 has the nucleic acid sequence of SEQ ID NO: 101 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the PARM1 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO:
• the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the PARM1 has the nucleic acid sequence of SEQ ID NO: 104 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the GJB6 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 91 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the GJB6 has the nucleic acid sequence of SEQ ID NO: 91 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the IGFBP2 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 95 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the IGFBP2 has the nucleic acid sequence of SEQ ID NO: 95 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the RBP7 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 98 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the RBP7 has the nucleic acid sequence of SEQ ID NO: 98 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the GJB6 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO: 101 and the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the GJB6 has the nucleic acid sequence of SEQ ID NO: 101 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• the inner ear supporting cell selective promoter comprises a
• the PARM1 promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identity to SEQ ID NO:
• the hGJB2 minimal promoter comprises a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% to SEQ ID NO: 91.
• the PARM1 has the nucleic acid sequence of SEQ ID NO: 104 and the hGJB2 minimal promoter has the nucleic acid sequence of SEQ ID NO: 91.
• provided AAV constructs comprise a promoter sequence selected from a CAG, a CBA, a CMV, or a CB7 promoter.
• the first or sole AAV construct further includes at least one promoter sequence selected from Cochlea and/or inner ear specific promoters.
• Exemplary CBA promoter (SEQ ID NO: 10)
• a promoter is an endogenous human ATOH1 enhancer- promoter as set forth in SEQ ID NO: 16.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 16.
• a promoter is an endogenous human ATOH1 enhancer-promoter sequence comprised within SEQ ID NO: 16.
• a promoter is an endogenous human GJB2 enhancer- promoter as set forth in SEQ ID NO: 17, or SEQ ID NO: 61.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 17 or SEQ ID NO: 61.
• a promoter is an endogenous human GJB2 enhancer-promoter sequence comprised within SEQ ID NO: 61.
• a promoter is GJB2 minimal promoter of SEQ ID NO: 91.
• a promoter is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 91.
• Exemplary Human GJB2 enhancer-promoter SEQ ID NO: 17
• a promoter is an endogenous human SLC26A4 enhancer- promoter as set forth in SEQ ID NO: 54.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 54.
• a promoter is an endogenous human SLC26A4 enhancer-promoter sequence comprised within SEQ ID NO: 54.
• a promoter is an endogenous human LGR5 enhancer- promoter as set forth in SEQ ID NO: 55.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 55.
• a promoter is an endogenous human LGR5 enhancer- promoter sequence comprised within SEQ ID NO: 55.
• a promoter is an endogenous human SYN1 enhancer- promoter as set forth in SEQ ID NO: 56.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 56.
• a promoter is an endogenous human SYN1 enhancer- promoter sequence comprised within SEQ ID NO: 56.
• a promoter is an endogenous human GFAP enhancer- promoter as set forth in SEQ ID NO: 57, or SEQ ID NO: 62.
• an enhancer-promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to enhancer-promoter sequence represented by SEQ ID NO: 57 or SEQ ID NO: 62.
• a promoter is an endogenous human GFAP enhancer-promoter sequence comprised within SEQ ID NO: 57 or SEQ ID NO: 62.
• a promoter is an endogenous human GDF6 promoter as set forth in SEQ ID NO: 90.
• an promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to a promoter sequence represented by SEQ ID NO: 90.
• a promoter is an endogenous human GDF6 promoter sequence comprised within SEQ ID NO: 90.
• a promoter is an endogenous human IGFBP2 promoter as set forth in SEQ ID NO: 95.
• an promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to a promoter sequence represented by SEQ ID NO: 95.
• a promoter is an endogenous human IGFBP2 enhancer-promoter sequence comprised within SEQ ID NO: 95.
• a promoter is an endogenous human RBP7 promoter as set forth in SEQ ID NO: 98.
• an promoter sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical to a promoter sequence represented by SEQ ID NO: 98.
• a promoter is an endogenous human RBP7 enhancer-promoter sequence comprised within SEQ ID NO:

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