CN117098563A

CN117098563A - Variant adeno-associated virus (AAV) capsid polypeptides and gene therapeutic agents for treating hearing loss

Info

Publication number: CN117098563A
Application number: CN202180087595.7A
Authority: CN
Inventors: S·彭诺克; A·M·蒂默斯; M·谢尔曼; C·巴托洛梅; X·王; P·D·马瑟; P·M·乌里贝; S·索博塔; B·E·雅克; A·C·福斯特; F·裴
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2020-11-06
Filing date: 2021-11-05
Publication date: 2023-11-21

Abstract

Variant adeno-associated virus (AAV) capsid polypeptides and gene therapeutics thereof for treating or preventing hearing loss are described herein.

Description

Variant adeno-associated virus (AAV) capsid polypeptides and gene therapeutic agents for treating hearing loss

Cross Reference to Related Applications

The present application claims the benefit of U.S. c. ≡119 (e) from U.S. provisional application nos. 63/110,697 and 63/146,269, each of which is incorporated herein by reference in its entirety, filed on even date 6 at 11/2020 and 5 at 2/2021.

Technical Field

Disclosed herein are variant adeno-associated virus (AAV) capsid polypeptides and gene therapeutic agents for treating or preventing hearing loss.

Disclosure of Invention

According to one aspect, the present disclosure provides a method of treating or preventing hearing loss associated with a deficiency in a gene, the method comprising administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising: (i) A variant AAV capsid polypeptide, optionally, the variant AAV capsid polypeptide exhibiting increased tropism in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide; and (ii) a polynucleotide comprising a nucleic acid sequence encoding said gene.

According to another aspect, the present disclosure provides a method of delivering a nucleic acid sequence encoding a gene associated with hearing loss to inner ear tissue or cells, the method comprising administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising: (i) A variant AAV capsid polypeptide that exhibits increased tropism in inner ear tissue or cells, such as compared to a non-variant AAV capsid polypeptide; and (ii) a polynucleotide comprising a nucleic acid sequence encoding said gene.

According to some embodiments, the inner ear tissue or cell is cochlear tissue or cell, or vestibular tissue or cell. According to certain embodiments, the inner ear tissue or cell is cochlear tissue or cell.

According to some embodiments, the variant AAV capsid polypeptide is any variant AAV capsid polypeptide, optionally selected from the group consisting of: mutating AAV1 capsid polypeptides; mutating AAV2 capsid polypeptides; variant AAV3 capsid polypeptides; variant AAV4 capsid polypeptides; variant AAV5 capsid polypeptides; variant AAV6 capsid polypeptides; variant AAV7 capsid polypeptides; variant AAV8 capsid polypeptides; variant AAV9 capsid polypeptides; variant rh-AAV10 capsid polypeptides; variant AAV10 capsid polypeptides; variant AAV11 capsid polypeptides; variant AAV12 capsid polypeptides; and variant Anc80 capsid polypeptides. According to certain embodiments, the variant AAV capsid polypeptide is a variant AAV2 capsid polypeptide.

According to some embodiments, the variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto, optionally wherein the AAV capsid is selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides.

According to some embodiments, the variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence homology thereto, optionally wherein the AAV capsid is selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides.

According to some embodiments, the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions, insertions and/or deletions relative to the wild type AAV2 capsid polypeptide (SEQ ID NO: 18), optionally wherein the one or more amino acid substitutions, insertions and/or deletions occur at an amino acid residue selected from the group consisting of: q263, S264, Y272, Y444, R487, P451, T454, T455, R459, K490, T491, S492, A493, D494, E499, Y500, T503, K527, E530, E531, Q545, G546, S547, E548, K549, T550, N551, V552, D553, E555, K556, R585, R588, and Y730. According to certain embodiments, the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions with respect to a wild type AAV2 capsid polypeptide (SEQ ID NO: 18), the one or more amino acid substitutions selected from the group consisting of: q263 263 264 272 444 487 451 454 455 459 490 491 492 493 494 499 500 527, 545 546 546 547 548 548 549 550 550 551 552 555 556 585 588T, and Y730F.

According to some embodiments, the variant AAV capsid polypeptide comprises: (i) SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or a sequence of any one of seq id no; (ii) a sequence corresponding to SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity; or (iii) a polypeptide consisting of SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO:32 or SEQ ID NO:34, and an amino acid sequence encoded by the nucleic acid sequence of any one of seq id no. According to certain embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO: 27. According to certain embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO: 29. According to certain embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO: 31. According to certain embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO:33, an amino acid sequence of seq id no. According to certain embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO:35, and a sequence of amino acids.

According to some embodiments, the variant AAV capsid polypeptide results in an increase in rAAV chemotaxis level in inner ear tissue or cells, optionally by at least 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, optionally as compared to a non-variant AAV capsid polypeptide.

According to some embodiments, the variant AAV capsid polypeptide results in an increase in rAAV tropism level in inner ear tissue or cells, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, optionally as compared to a non-variant AAV capsid polypeptide.

According to some embodiments, the variant AAV capsid polypeptide results in an increase in rAAV transduction efficiency in inner ear tissue or cells, optionally by at least 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, optionally as compared to a non-variant AAV capsid polypeptide.

According to some embodiments, the variant AAV capsid polypeptide results in an increase in rAAV transduction efficiency in inner ear tissue or cells, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, optionally as compared to a non-variant AAV capsid polypeptide.

According to some embodiments, the method results in an increase in expression of the gene in inner ear tissue or cells, optionally at least about 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, optionally as compared to normal expression of the gene.

According to some embodiments, the method results in an increase in expression of the gene in inner ear tissue or cells, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, optionally as compared to normal expression of the gene.

According to some embodiments, the method results in overexpression of GJB2 (connexin 26) expression in inner ear tissue or cells.

According to some embodiments, the method results in a reduction in rAAV neutralizing antibody (NAb) titer level, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, optionally as compared to a control level.

According to some embodiments, the method results in a reduction in the inner ear inflammation or toxicity level, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% >, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, optionally as compared to the inner ear inflammation or toxicity level prior to administration. According to certain embodiments, the reduction in inner ear inflammation or toxicity level is compared to a non-variant AAV capsid polypeptide. According to certain embodiments, the reduction in the inner ear inflammation or toxicity level is compared to the inner ear inflammation or toxicity level caused by a disease or condition associated with hearing loss.

According to some embodiments, the method results in a delay of optionally at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% >, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% in the inflammation or toxicity progression of the inner ear, optionally as compared to the inflammation or toxicity progression of the inner ear prior to administration.

According to some embodiments, the method results in a reduction in the level of hair cell loss, degradation, and/or death, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to the level of hair cell loss, degradation, and/or death prior to administration.

According to some embodiments, the method results in a reduction in the level of spiral ganglion neuron loss, degeneration, and/or death, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to the level of spiral ganglion neuron loss, degeneration, and/or death prior to administration.

According to some embodiments, the method results in optionally at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% decrease in Auditory Brainstem Response (ABR) threshold at, for example, 1kHz frequency, 4kHz frequency, 8kHz frequency and/or 16kHz frequency, optionally as compared to ABR threshold level prior to administration.

According to some embodiments, the method results in an improved distortion product otoacoustic emission (DPOAE) profile. According to certain embodiments, the method results in preventing, delaying or slowing the deterioration of DPOAE distribution.

According to some embodiments, the method results in improved speech understanding. According to certain embodiments, the method results in preventing, delaying or slowing the deterioration of speech understanding.

According to some embodiments, the control level is based on: levels obtained from a subject prior to administration of the rAAV, optionally, a sample from the subject.

According to some embodiments, the control level is based on: levels resulting from administration of a rAAV that does not contain a variant AAV capsid polypeptide, optionally, wherein the rAAV that does not contain a variant AAV capsid polypeptide comprises a rAAV capsid polypeptide selected from AAV2 and Anc80L 65.

According to some embodiments, the method results in delivery of a nucleic acid sequence encoding a gene associated with hearing loss (such as GJB 2) to and expression in cells of the lateral wall or spiral ligament, support cells of the organ of kohlrabi, fibroblasts of the spiral ligament, claus cells, burt's cells, cells of the spiral lobe, vestibular support cells, hansen cells, dai Tesi cells, column cells, inner finger cells, outer finger cells, limbic cells, intra-cochlear hair cells, extra-cochlear hair cells, spiral ganglion neurons, vestibular hair cells, vestibular support cells, and/or vestibular ganglion neurons.

According to some embodiments, the method results in delivery of a nucleic acid sequence encoding a gene associated with hearing loss (such as GJB 2) to and expression in at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of cells of the lateral wall or spiral ligament, support cells of the organ of the coti, fibroblasts of the spiral ligament, claus cells, bertschel cells, spiral convex cells, vestibular support cells, hansen cells, dai Tesi cells, columnar cells, inner finger cells, outer finger cells, limbic cells, intracochlear hair cells, extra-cochlear hair cells, spiral ganglion neurons, vestibular hair cells, vestibular support cells and/or vestibular ganglion neurons.

According to some embodiments, the gene is GJB2.

According to some embodiments, the nucleic acid sequence encoding GJB2 is a non-naturally occurring sequence. According to some embodiments, the nucleic acid sequence encoding GJB2 encodes mammalian GJB2. According to some embodiments, the nucleic acid sequence encoding GJB2 encodes human, mouse, non-human primate, or rat GJB2. According to some embodiments, the nucleic acid sequence encoding GJB2 comprises SEQ ID NO:10. according to some embodiments, the nucleic acid sequence encoding GJB2 is codon optimized for mammalian expression. According to some embodiments, the nucleic acid sequence encoding GJB2 comprises SEQ ID NO: 11. SEQ ID NO:12 or SEQ ID NO:13. according to some embodiments, the nucleic acid sequence encoding GJB2 is codon optimized for expression in human, rat, non-human primate, guinea pig, minipig, pig, cat, sheep, or mouse cells. According to some embodiments, the nucleic acid sequence encoding GJB2 is a cDNA sequence. According to some embodiments, the nucleic acid sequence encoding GJB2 further comprises an operably linked C-terminal tag or N-terminal tag. According to some embodiments, the tag is a FLAG tag or an HA tag.

According to some embodiments, the nucleic acid sequence encoding GJB2 is operably linked to a promoter. According to some embodiments, the promoter is a ubiquitously active CBA, small CBA (smCBA), EF1a, CASI promoter, cochlear support cell promoter, GJB2 expression specific GFAP promoter, small GJB2 promoter, medium GJB2 promoter, large GJB2 promoter, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoter. According to some embodiments, the promoter is optimized to drive sufficient GJB2 expression to treat or prevent hearing loss.

According to some embodiments, the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region. According to some embodiments, the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

According to some embodiments, the nucleic acid sequence encoding GJB2 further comprises an operably linked polyadenylation signal. According to some embodiments, the polyadenylation signal is an SV40 polyadenylation signal. According to some embodiments, the polyadenylation signal is a human growth hormone (hGH) polyadenylation signal.

According to some embodiments, the polynucleotide further comprises a 27 nucleotide C-terminal tag of hemagglutinin or a 24 nucleotide Flag tag; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) Ubiquitously active CBA, small CBA (smCBA), EF1a or CASI promoters; (b) Cochlear support cells or GJB2 expression specific 1.68kb GFAP, small/medium/large GJB2 promoters, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoters; operably linked to a 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal.

According to some embodiments, the polynucleotide further comprises an AAV genome cassette, optionally wherein: (i) The AAV genome cassette is flanked by two sequence-regulating inverted terminal repeats, preferably about 143 bases in length; or (ii) an AAV genome cassette flanked by self-complementary AAV (scAAV) genome cassettes consisting of two inverted identical repeat sequences, preferably no longer than 2.4kb, separated by about 113 base-enabling scAAV ITRs (itrΔtrs) and flanked on both ends by about 143 base-sequence-regulated ITRs.

According to some embodiments, the polynucleotide comprises a codon/sequence optimized human GJB2 cDNA with or without a hemagglutinin C-terminal tag, preferably about 27 nucleotides in length, optionally a tag of about 0.68 kilobases (kb) size or a Flag tag of 24 nucleotides; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) A ubiquitously active CBA, preferably about 1.7kb in size, a small CBA (smCBA), preferably about 0.96kb in size, EF1a, preferably about 0.81kb in size, or a CASI promoter, preferably about 1.06kb in size; (b) A cochlear support cell or GJB2 expression specific GFAP promoter preferably about 1.68kb in size, a small GJB2 promoter preferably about 0.13kb in size, a medium GJB2 promoter preferably about 0.54kb in size, a large GJB2 promoter preferably about 1.0kb in size, or a sequential combination of 2-3 individual GJB2 expression specific promoters; operably linked to a 0.9kb 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal and further comprising two about 143 base sequence regulatory Inverted Terminal Repeats (ITRs) flanking an AAV genome cassette, or a self-complementary AAV (scAAV) genome cassette consisting of two inverted identical repeats, preferably no longer than 2.4kb, separated by about 113 base scAAV-enabled ITRs (ITR Δtrs) and flanked on both ends by about 143 base sequence regulatory ITRs.

According to some embodiments, the hearing loss is hereditary hearing loss. According to some embodiments, the hearing loss is DFNB1 hearing loss. According to some embodiments, the hearing loss is caused by a mutation in GJB 2. According to some embodiments, the hearing loss is caused by an autosomal recessive GJB2 mutant (DFNB 1). According to some embodiments, the hearing loss is caused by an autosomal dominant GJB2 mutant (DFNA 3A).

According to some embodiments, the administering is to the cochlea or vestibular system, optionally, wherein the delivering comprises directly administering into the cochlea or vestibular system via a Round Window Membrane (RWM), oval window, or semicircular canal. According to some embodiments, the direct administration is injection. According to some embodiments, the administration is intravenous, intraventricular, intracochlear, intrathecal, intramuscular, subcutaneous, or a combination thereof.

According to another aspect, the present disclosure provides a composition for use in a method of treating or preventing hearing loss associated with a deficiency in a gene, the composition comprising a recombinant adeno-associated virus (rAAV) virion comprising: (i) A variant AAV capsid polypeptide, optionally, the variant AAV capsid polypeptide exhibiting increased tropism in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide; and (ii) a polynucleotide comprising a nucleic acid sequence encoding said gene.

According to another aspect, the present disclosure provides a composition for use in a method of delivering a nucleic acid sequence encoding a gene associated with hearing loss to inner ear tissue or cells, the method comprising administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising: (i) A variant AAV capsid polypeptide, optionally, the variant AAV capsid polypeptide exhibiting increased tropism in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide; and (ii) a polynucleotide comprising a nucleic acid sequence encoding gap junction protein β2 (GJB 2).

According to some embodiments of the foregoing compositions, the inner ear tissue or cell is cochlear tissue or cell, or vestibular tissue or cell. According to some embodiments, the inner ear tissue or cell is cochlear tissue or cell.

According to some embodiments of the foregoing composition, the variant AAV capsid polypeptide is selected from the group consisting of: mutating AAV1 capsid polypeptides; mutating AAV2 capsid polypeptides; variant AAV3 capsid polypeptides; variant AAV4 capsid polypeptides; variant AAV5 capsid polypeptides; variant AAV6 capsid polypeptides; variant AAV7 capsid polypeptides; variant AAV8 capsid polypeptides; variant AAV9 capsid polypeptides; variant rh-AAV10 capsid polypeptides; variant AAV10 capsid polypeptides; variant AAV11 capsid polypeptides; and variant AAV12 capsid polypeptides. According to some embodiments, the variant AAV capsid polypeptide is a variant AAV2 capsid polypeptide. According to some embodiments, the variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto, optionally wherein the AAV capsid is selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides. According to some embodiments, the variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence homology thereto, optionally wherein the AAV capsid is selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides. According to some embodiments, the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions, insertions and/or deletions relative to the wild type AAV2 capsid polypeptide (SEQ ID NO: 1), optionally wherein the one or more amino acid substitutions, insertions and/or deletions occur at an amino acid residue selected from the group consisting of: q263, S264, Y272, Y444, R487, P451, T454, T455, R459, K490, T491, S492, A493, D494, E499, Y500, T503, K527, E530, E531, Q545, G546, S547, E548, K549, T550, N551, V552, D553, E555, K556, R585, R588, and Y730. According to some embodiments, the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions with respect to a wild type AAV2 capsid polypeptide (SEQ ID NO: 1), the one or more amino acid substitutions selected from the group consisting of: q263 263 264 272 444 487 451 454 455 459 490 491 492 493 494 499 500 527, 545 546 546 547 548 548 549 550 550 551 552 555 556 585 588T, and Y730F. According to some embodiments, the variant AAV capsid polypeptide comprises: (i) SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or a sequence of any one of seq id no; (ii) a sequence corresponding to SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity; or (iii) a polypeptide consisting of SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO:32 or SEQ ID NO:34, and an amino acid sequence encoded by the nucleic acid sequence of any one of seq id no. According to some embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO: 27. According to some embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO: 29. According to some embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO: 31. According to some embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO:33, an amino acid sequence of seq id no. According to some embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO:35, and a sequence of amino acids.

According to some embodiments of the foregoing compositions, the gene is GJB2. According to some embodiments, the nucleic acid sequence encoding GJB2 is a non-naturally occurring sequence. According to some embodiments, the nucleic acid sequence encoding GJB2 encodes mammalian GJB2. According to some embodiments, the nucleic acid sequence encoding GJB2 encodes human, mouse, non-human primate, or rat GJB2. According to some embodiments, the nucleic acid sequence encoding GJB2 comprises SEQ ID NO:10. according to some embodiments, the nucleic acid sequence encoding GJB2 is codon optimized for mammalian expression. According to some embodiments, the nucleic acid sequence encoding GJB2 comprises SEQ ID NO: 11. SEQ ID NO:12 or SEQ ID NO:13. according to some embodiments, the nucleic acid sequence encoding GJB2 is codon optimized for expression in human, rat, non-human primate, guinea pig, minipig, pig, cat, sheep, or mouse cells. According to some embodiments, the nucleic acid sequence encoding GJB2 is a cDNA sequence. According to some embodiments of the foregoing compositions, the nucleic acid sequence encoding GJB2 further comprises an operably linked C-terminal tag or N-terminal tag. According to some embodiments, the tag is a FLAG tag or an HA tag.

According to some embodiments of the foregoing compositions, the nucleic acid sequence encoding GJB2 is operably linked to a promoter. According to some embodiments, the promoter is a ubiquitously active CBA, small CBA (smCBA), EF1a, CASI promoter, cochlear support cell promoter, GJB2 expression specific GFAP promoter, small GJB2 promoter, medium GJB2 promoter, large GJB2 promoter, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoter. According to some embodiments, the promoter is optimized to drive sufficient GJB2 expression to treat or prevent hearing loss.

According to some embodiments of the foregoing compositions, the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region. According to some embodiments, the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

According to some embodiments of the foregoing compositions, the nucleic acid sequence encoding GJB2 further comprises an operably linked polyadenylation signal. According to some embodiments, the polyadenylation signal is an SV40 polyadenylation signal. According to some embodiments, the polyadenylation signal is a human growth hormone (hGH) polyadenylation signal.

According to some embodiments of the foregoing compositions, the polynucleotide further comprises a 27 nucleotide C-terminal tag of hemagglutinin or a 24 nucleotide Flag tag; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) Ubiquitously active CBA, small CBA (smCBA), EF1a or CASI promoters; (b) Cochlear support cells or GJB2 expression specific 1.68kb GFAP, small/medium/large GJB2 promoters, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoters; operably linked to a 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal.

According to some embodiments of the foregoing compositions, the polynucleotide further comprises an AAV genome cassette, optionally wherein: (i) The AAV genome cassette is flanked by two sequence-regulating inverted terminal repeats, preferably about 143 bases in length; or (ii) an AAV genome cassette flanked by self-complementary AAV (scAAV) genome cassettes consisting of two inverted identical repeat sequences, preferably no longer than 2.4kb, separated by about 113 base-enabling scAAV ITRs (itrΔtrs) and flanked on both ends by about 143 base-sequence-regulated ITRs.

According to some embodiments of the foregoing composition, the polynucleotide comprises a codon/sequence optimized human GJB2 cDNA with or without a hemagglutinin C-terminal tag of preferably about 27 nucleotides in length, optionally a tag of about 0.68 kilobases (kb) in size, or a Flag tag of 24 nucleotides; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) A ubiquitously active CBA, preferably about 1.7kb in size, a small CBA (smCBA), preferably about 0.96kb in size, EF1a, preferably about 0.81kb in size, or a CASI promoter, preferably about 1.06kb in size; (b) A cochlear support cell or GJB2 expression specific GFAP promoter preferably about 1.68kb in size, a small GJB2 promoter preferably about 0.13kb in size, a medium GJB2 promoter preferably about 0.54kb in size, a large GJB2 promoter preferably about 1.0kb in size, or a sequential combination of 2-3 individual GJB2 expression specific promoters; operably linked to a 0.9kb 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal and further comprising two about 143 base sequence regulatory Inverted Terminal Repeats (ITRs) flanking an AAV genome cassette, or a self-complementary AAV (scAAV) genome cassette consisting of two inverted identical repeats, preferably no longer than 2.4kb, separated by about 113 base scAAV-enabled ITRs (ITR Δtrs) and flanked on both ends by about 143 base sequence regulatory ITRs.

According to another aspect, the present disclosure provides a method of treating or preventing hearing loss, the method comprising administering to a subject in need thereof an effective amount of a composition as described herein.

According to another aspect, the present disclosure provides a method of delivering a nucleic acid sequence encoding a gene associated with hearing loss to inner ear tissue or cells, the method comprising administering to a subject in need thereof an effective amount of a composition as described herein.

According to another aspect, the present disclosure provides a method of delivering a nucleic acid sequence encoding GJB2 to inner ear tissue or cells, the method comprising administering to a subject in need thereof an effective amount of a composition as described herein.

According to another aspect, the present disclosure provides a kit comprising a composition as described herein and instructions for use.

Drawings

Fig. 1A is a schematic view of cochlear anatomy and cell type.

FIG. 1B shows a close-up of the support cells. Outer hair cells (01, 02, 03), inner Hair Cells (IHC), hansen cells (h 1, h2, h3, h 4), dai Tesi cells (d 1, d2, d 3), column cells (p), inner finger cells (IPC), outer finger cells/limbic cells (bc) are shown.

Fig. 1C is a schematic representation of cochlear anatomy and cell type, indicating the GJB2 expression region.

Figure 2 shows a schematic representation of the GJB2 vector (genome) construct single stranded (ss) AAV-GJB2 and self-complementing scAAV-GJB 2.

FIG. 3 shows the nucleic acid sequence of the CBA promoter (SEQ ID NO. 1).

FIG. 4 shows the nucleic acid sequence of the EF1a promoter (SEQ ID NO. 2).

FIG. 5 shows the nucleic acid sequence of the CASI promoter (SEQ ID NO. 3).

FIG. 6 shows the nucleic acid sequence of the smCBA promoter (SEQ ID NO. 4).

FIG. 7 shows the nucleic acid sequence of the GFAP promoter (SEQ ID NO. 5).

FIG. 8 shows the nucleic acid sequence of the GJB2 promoter (SEQ ID NO. 6). Such promoters may have three different iterations: underlined sequence (128 bp), green shaded region (539 bp), and whole sequence (1000 bp).

FIG. 9 shows the following nucleic acid sequences for ITR (AAV 2): 5'-3': for single stranded (ss) and self complementary (sc) AAV genome (SEQ ID NO. 7); 3'-5': for single stranded (ss) AAV genome only (SEQ ID No. 8); 3'-5': targeting only the self-complementary (sc) AAV genome (SEQ ID NO. 9).

FIG. 10 shows the nucleic acid sequence (SEQ ID NO. 10) of human wild-type GJB2 (hGJB 2 wt).

FIG. 11 shows the nucleic acid sequence of human codon optimized GJB2 (hGJB 2co 3) (SEQ ID NO. 11).

FIG. 12 shows the nucleic acid sequence of human codon optimized GJB2 (hGJB 2co 6) (SEQ ID NO. 12).

FIG. 13 shows the nucleic acid sequence (SEQ ID NO. 13) of human codon optimized GJB2 (hGJB 2co 9).

FIG. 14 shows the nucleic acid sequence of the HA tag (SEQ ID NO. 14).

FIG. 15 shows the nucleic acid sequence of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (SEQ ID NO. 15).

FIG. 16 shows the nucleic acid sequence of the SV40poly (A) terminator sequence (SEQ ID NO. 16).

FIG. 17 shows the nucleic acid sequence of the bGH poly (A) terminator sequence (SEQ ID NO. 17).

FIG. 18 shows the FLAG tagged nucleic acid sequence (SEQ ID NO. 18).

FIG. 19 shows bar graphs comparing GFP coverage of the helical edges normalized to OMY-906 (gray bars) OMY-903, OMY-907, OMY-911, OMY-912, OMY-913, OMY-914, OMY-915, OMY-916, OMY-917, AAV2, and Anc 80.

FIG. 20 shows bar graphs comparing GFP coverage of OMY-903, OMY-907, OMY-911, OMY-912, OMY-913, OMY-914, OMY-915, OMY-916, OMY-917, AAV2, and Anc80 normalized to OMY-906 (gray bars) in the Cotinia organ.

FIG. 21 shows bar graphs comparing GFP coverage of spiral ligaments normalized to OMY-906 (grey bars) OMY-903, OMY-907, OMY-911, OMY-912, OMY-913, OMY-914, OMY-915, OMY-916, OMY-917, AAV2, and Anc 80.

FIGS. 22A-22B show representative Z-stack images of GFP reporter gene expression in the mid-cochlear region, including the spiraling ligament, the organ of Kotinia, and the spiral rim, following treatment with OMY-903, anc80, OMY-912, and wild-type AAV 2.

Fig. 23 shows fluorescence images comparing coverage of OMY-912 GFP at the following two doses: 2e9vg and 2e10 vg.

Fig. 24 shows fluorescence images comparing coverage of OMY-915 GFP at the following two doses: 2e9vg and 2e10 vg.

FIG. 25 shows a bar graph comparing GFP coverage for the following two doses in the helical margin, OMY-912 and OMY-915: 2e9vg and 2e10 vg.

FIG. 26 shows a bar graph comparing GFP coverage in the Cotinia organs for the following two doses, OMY-912 and OMY-915: 2e9vg and 2e10 vg.

Fig. 27 shows fluorescence images of FLAG antibody staining in support cells of rat cochlear explants, indicating AAV-induced expression of connexin 26. The FLAG staining clearly overlaps with the expression region of connexin 26, indicating that the FLAG-tagged protein targets the normal connexin 26 expression site. FLAG antibodies are shown in green, connexin 26 in magenta, and DAPI nuclear staining in blue. The left panel shows all colors combined, the middle panel shows only connexin 26 staining, and the right panel shows FLAG staining.

Fig. 28 shows fluorescent images of Flag antibody staining in supporting cells of cochlea of mice 2-6 weeks after intra-cochlear injection of AAV-GJB2-Flag, indicating AAV-induced expression of connexin 26. Representative images of the basal (basal), middle (middle) and apical (top) rings of the cochlea are shown. FLAG antibodies are shown in red and DAPI nuclear staining is shown in blue.

Fig. 29 shows fluorescent images of Flag antibody staining in supporting cells of adult mice cochlea following intra-cochlear injection of AAV-GJB2-Flag, indicating AAV-induced expression of connexin 26. Representative images of the basal (basal), middle (middle) and apical (top) rings of the cochlea are shown. FLAG antibodies are shown in red and DAPI nuclear staining is shown in blue.

Fig. 30 shows images of non-human primate (NHP) cochlear slices assessed by immunohistochemistry 12 weeks after intra-cochlear injection OMY-913. DAB staining expressed by GFP was pseudo red. Fig. 30 (top panel) shows a low magnification image of the entire cochlea and demonstrates that consistent expression from basal to apical can be observed in the entire cochlea after a single OMY-913 injection administered near the basal via a round window membrane injection. FIG. 30 (bottom panel) shows OMY-913 expression observed in the areas associated with GJB2 rescue, including the side wall (LW), the organ of Koroti (OC) supporting cells and the spiral edge (SL).

Fig. 31 shows a line graph with hearing thresholds measured by Auditory Brainstem Response (ABR) at different frequencies in Cx26 expressing Wild Type (WT) mice and induced cre mice with Cx26 Knockdown (KO) as measured 30 days postnatal and 60 days postnatal.

Fig. 32 shows a line graph with hearing thresholds measured by Auditory Brainstem Response (ABR) at different frequencies in a wild-type (WT) mouse expressing Cx26 and a constitutive cre mouse with Cx26 Knockout (KO) as measured 30 days after birth.

Fig. 33A shows a line graph with hearing threshold values measured by Auditory Brainstem Response (ABR) at different frequencies in a constitutive cre mouse with Cx26 Knockout (KO) or a Wild Type (WT) mouse expressing Cx26 and treated with vehicle (green line) treated with vehicle (black line) or therapeutic agent-a (blue line) as measured by 30 days after birth.

FIG. 33B shows a bar graph of Cx26 expression in inducible cre mice with Cx26 Knockout (KO) treated with vehicle or therapeutic-A.

FIG. 33C shows images of Cx26 expression in inducible cre mice with Cx26 Knockout (KO) treated with vehicle or therapeutic-A.

Fig. 33D shows a bar graph of cochlear injury (flat epithelium) in induced cre mice with Cx26 Knockdown (KO) treated with vehicle or therapeutic agent-a.

Fig. 34 shows the time line of photobleaching and image capture for each FRAP test (top panel), and a graph showing that therapeutic a and therapeutic a-FLAG restored fluorescence faster than the non-transduced HeLa cells (bottom panel), indicating that the transgene-driven protein may be forming a functional gap junction.

Fig. 35A-35C are a series of images showing that therapeutic agent a-FLAG expression is present at high levels throughout the cochlea length and forms membranous plaque-like structures in the sulcus (fig. 35A), claus cells (fig. 35B), and other supporting cell types (fig. 35C).

Fig. 36 is a series of images showing that intra-cochlear injection of therapeutic agent a or therapeutic agent a-FLAG via round window membrane with windowing in the posterior semicircular canal of an adult C57BL/6J mouse of age P30 was safe and did not cause damage to inner or outer hair cells 42 days after surgery.

FIG. 37 is a series of images showing CX26-FLAG transduction (green) in sulcus, claus cells and lateral wall fibroblasts 14 days post-surgery following administration of therapeutic A-FLAG in adult (P30) C57BL/6J mice.

FIG. 38 is a series of images showing CX26-FLAG transduction (green) in sulcus, claus cells and lateral wall fibroblasts 14 days post-surgery following administration of therapeutic A-FLAG in adult (P30) C57BL/6J mice.

Fig. 39A-39B show an induced mouse model of GJB2 congenital hearing loss (Rosa-cre).

Fig. 39C shows that intra-cochlear injection of therapeutic agent a-FLAG into wild-type mice during postnatal period provided broad cochlear coverage, including all cell types that naturally express CX 26. Intra-cochlear injections were performed at the age at which rescue studies were performed in the Rosa-cre animal model.

Fig. 39D-39E show that therapeutic a in the Rosa-cre animal model exhibited substantial rescue of ABR thresholds across multiple frequencies, restoration of CX26 expression, and preservation of cochlear morphology.

FIGS. 40A-40B illustrate the process by which Cx26 is processed ^loxp/loxp Mice were crossed with mice expressing Cre driven by the inner ear specific promoter P0 (P0-Cre) to have a mouse model of inner ear GJB2 deletion.

Fig. 40C shows that intra-cochlear injection of therapeutic agent a-FLAG into wild-type mice during postnatal period provided broad cochlear coverage, including all cell types that naturally express CX 26. Intra-cochlear injections were performed at the age at which rescue studies were performed in the P0-cre animal model.

Figures 40D-40E show that therapeutic a in the P0-cre animal model exhibited substantial rescue of ABR thresholds across multiple frequencies, restoration of CX26 expression, and preservation of cochlear morphology.

Fig. 41 shows that intra-cochlear injection of therapeutic agent a-FLAG shows high transduction and good chemotaxis in non-human primate (NHP).

Detailed Description

Non-symptomatic hearing loss and deafness (DFNB 1; also known as connexin 26 deafness) are autosomally recessive and are characterized by congenital non-progressive mild to severe sensorineural hearing impairment. The GJB2 gene encodes connexin-26, which connexin-26 is expressed in cochlear support cells, forming gap junctions that are involved in intercellular communication important for cochlear homeostasis, including control of potassium gradients that play an important role in hair cell survival and function and normal hearing. Mutations in GJB2 impair the gap junction and cochlear homeostasis, resulting in hair cell dysfunction and hearing loss.

According to NIDCD, 2 to 3 children out of every 1,000 children in the united states have some degree of hearing loss at birth, more than half of which is due to genetic factors. Mutations in the GJB2 gene encoding gap junction protein 26 (CX 26) are the most common form of hereditary hearing loss responsible for > 50% of cases across each population. Although in most subjects the onset of hearing loss is premalignant and moderate to severe, in some subjects hearing loss due to CX26 loss is mild and progressive. In the inner ear, expression of CX26 is critical for the function of various non-sensory cell types including supporting cells and fibroblasts.

Genetic testing can be used to diagnose DFNB1 by identifying bi-allelic pathogenicity variations in GJB2, which cover sequence variations that alter the expression of gap junction β -2 protein (connexin 26) and variations in upstream cis-regulatory elements. When a GJB2 pathogenic variation leading to DFNB1 is detected in the affected family members, carrier testing for at-risk relatives, prenatal testing for pregnant women with increased risk, and pre-embryo implantation genetic diagnosis can be performed. Smith and Jones. Nonsyndromic Hearing Loss and Deafness, DFNB1.1998: adam et al, genereviews, university of Washington, seattle; kemperman et al Journal of the Royal Society of Medicine 2002 95:171-177. The present disclosure recognizes that cochlear access is possible surgically and topical application to a relatively immune-protected environment, and that gene therapy using viral vectors can be used to treat hearing loss. The present disclosure also recognizes that gene therapy capable of increasing chemotaxis and transduction in inner ear tissues and cells can effectively treat hearing loss associated with a deficiency of a certain gene.

The present disclosure relates to variant adeno-associated virus (AAV) capsid polypeptides that exhibit an increased propensity to become in inner ear tissues or cells, e.g., as compared to non-variant AAV capsid polypeptides. The variant AAV capsid polypeptides described herein can be incorporated into rAAV vectors and/or rAAV virions by which the genes can be packaged for targeted delivery to patients suffering from hearing loss associated with a deficiency in the genes, including patients having autosomal mutations, recessive mutant dominant mutations in the genes. In a specific embodiment, the gene is gap junction protein β2 (GJB 2). GJB2 mutates back to impair the gap junction and cochlear homeostasis, resulting in destruction of cochlear structures, hair cell dysfunction, and hearing loss. The goal of GJB2 gene therapy as described herein is to restore functional gap junctions and protect hair cells to improve hearing.

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. The following references provide the skilled artisan with a general definition of many of the terms used in the present disclosure. Singleton et al Dictionary of Microbiology and Molecular Biology (2 nd edition 1994); the Cambridge Dictionary of Science and Technology (Walker editions, 1988); the Glossary of Genetics, 5 th edition, R.Rieger et al (eds.), springer Verlag (1991); and Hale and Marham, the Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings given thereto unless otherwise indicated.

The article "a" or "an" as used herein refers to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

The term "comprising" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to.

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or" unless the context clearly indicates otherwise.

The term "such as" is used herein to mean, and is used interchangeably with, the phrase "such as, but not limited to".

As used herein, the term "administration" or the like is intended to refer to a method for enabling the delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological action.

As used herein, the term "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof, such as AAV vectors, AAV virions, and/or AAV virions. Unless otherwise required, the term encompasses all subtypes as well as both naturally occurring and recombinant forms. In some embodiments, AAV may refer to its capsid polypeptide, e.g., to its variant capsid polypeptide. For example, an AAV comprising a OMY-913 variant capsid polypeptide can be referred to herein as "OMY-913".

As used herein, the term "AAV virion" or "AAV virus" or "AAV virion" or "AAV vector particle" is intended to broadly refer to an intact virion, such as, for example, a wild-type AAV virion, comprising single stranded genomic DNA packaged into an AAV capsid protein. The single stranded nucleic acid molecule is either the sense strand or the antisense strand, as both strands are equally infectious. The term "rAAV virion" refers to recombinant AAV virions, i.e., particles that are infectious but replication defective. The rAAV virions comprise single stranded genomic DNA packaged into AAV capsid proteins. In certain embodiments, the AAV capsid protein is a variant AAV capsid protein that exhibits an increased propensity in inner ear tissue or cells, e.g., as compared to a non-variant AAV capsid protein. The amino acid sequences and nucleotide sequences of exemplary variant AAV capsid proteins are provided in table 1.

As used herein, the term "bioreactor" is intended to broadly refer to any device that can be used for the purpose of culturing cells.

As used herein, the term "gene" or "coding sequence" is intended to broadly refer to a DNA region (transcribed region) encoding a protein. When the coding sequence is placed under the control of a suitable regulatory region, such as a promoter, the coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide. The gene may comprise several operably linked fragments, such as a promoter, a5 '-leader, a coding sequence and a 3' -untranslated sequence, including polyadenylation sites. The phrase "gene expression" refers to the process by which a gene is transcribed into RNA and/or translated into an active protein.

As used herein, the term "gene of interest (GOI)" as used herein broadly refers to a heterologous sequence introduced into an AAV expression vector, and generally refers to a nucleic acid sequence encoding a protein having therapeutic use in humans or animals. In some embodiments, the GOI is a gene associated with hearing loss. In some embodiments, the gene is gap junction protein β2 (GJB 2). Other genes associated with hearing loss that may be used according to the methods described herein (e.g., hearing loss associated with gene deficiency) are known in the art and are described, for example, in Shearer et al, "Hereditary Hearing Loss and Deafness Overview",2017, which is incorporated herein by reference in its entirety. Examples of genes associated with hearing loss (e.g., hearing loss associated with gene deficiency) include, but are not limited to ACTG1, ADCY1, ADGRV1, AIFM1, BDP1, BSND, BTD, CABP2, CCDC50, CD164, CDC14A, CDH, CEACAM16, CIB2, CLDN14, CLIC5, CLRN1, COCH, COL11A1, COL11A2, COL2A1, COL4A3, COL4A4, COL4A5, COL4A6, COL9A1, COL9A2, COL9A3, DCDC2, DIAPH1, DMXL2, DSPP, EDN3, EDNRB, ELMOD3, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM189A2, GIPC3, GJB2, GJB3, GJB6, GPSM2, GRHL2, GRXCR1, GRXCR2, GSDME, HADR 1, HGF, HOMER2, ILRS 1, KARS1, KCQ 1, NQ 4; LHFPL5, LOXHD1, LRTOMT, MARVELD, MCM2, MET, MIR96, MITF, MSRB3, MT-CO1, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, MYO7A, NARS2, NF2, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PEX7, PHYH, PJVK, PNPT1, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, RIPOR, ROR1, S1PR2, SERPINB6, SIX1, SIX5, SLC17A8, SLC22A4, SLC26A5, SMPX, SOX10, STRC, SYNE4, TBC1D24, TECTA, TIMM8A, TJP2, TMC1, TMEM132E, TMIE, TMPRSS, TPRN, TRIOBP, TSPEAR, USH C, USH1G, USH2A, WBP, WFS1, and WHON.

As used herein, the term "hearing loss" is intended to refer to a reduced sensitivity to sounds normally heard by a subject. The severity of hearing loss is categorized according to the required volume increase above a normal level before the listener can perceive it. According to some embodiments, the hearing loss may be characterized by an increase in threshold volume when the individual perceives different frequency tones. In certain embodiments, hearing may be measured in decibels (dB). In certain embodiments, the threshold or 0dB signature for each frequency refers to the level at which a normal subject (e.g., a normal human subject) perceives a tone burst 50% of the time. In certain embodiments, hearing is considered normal if the subject's threshold is within 15dB of the normal threshold. In certain embodiments, the severity of the hearing loss is graded as follows: the light weight is 26-40dB, the moderate is 41-55dB, the moderate is 56-70dB, the heavy weight is 71-90dB, and the extremely heavy weight is 90dB. In certain embodiments, the methods described herein can reduce and/or slow the progression of hearing loss in a subject from one level (e.g., mild) to another level (e.g., moderate, moderately severe, and/or extremely severe). In certain embodiments, the methods described herein can improve and/or reverse progression of hearing loss in a subject from one level (e.g., moderate, moderately severe, and/or extremely severe) to another level (e.g., mild). In certain embodiments, hearing loss is associated with a gene deficiency. In certain embodiments, hearing loss is associated with a deficiency in a gene selected from the group consisting of: ACTG1, ADCY1, ADGRV1, AIFM1, BDPI, BSND, BTD, CABP2, CCDC50, CD164, CDC14A, CDH, CEACAM16, CIB2, CLDN14, CLIC5, CLRN1, COCH, COL11A1, COL11A2, COL2A1, COL4A3, COL4A4, COL4A5, COL4A6, COL9A1, COL9A2, COL9A3, DCDC2, DIAPH1, DMXL2, DSPP, EDN3, EDNRB, ELMOD3, EPS8L2, ESPN, ESRRB, EYA, EYA4, FAM189A2, GIPC3, GJB2, GJB3, GJB6, SM2, GRHL2, GRXCR1, GRXCR2, GSDME, HARS1, HGF, HOMER2, ILR 1, KARS1, KCQ 1, KCNQ1, KANQ 4; LHFPL5, LOXHD1, LRTOMT, MARVELD, MCM2, MET, MIR96, MITF, MSRB3, MT-CO1, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, MYO7A, NARS2, NF2, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PEX7, PHYH, PJVK, PNPT1, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, RIPOR, ROR1, S1PR2, SERPINB6, SIX1, SIX5, SLC17A8, SLC22A4, SLC26A5, SMPX, SOX10, STRC, SYNE4, TBC1D24, TECTA, TIMM8A, TJP2, TMC1, TMEM132E, TMIE, TMPRSS, TPRN, TRIOBP, TSPEAR, USH C, USH1G, USH2A, WBP, WFS1, and WHON. In certain embodiments, hearing loss is associated with mutations, such as substitutions, deletions, insertions, and/or duplications, in the genes described herein. In certain embodiments, hearing loss is associated with two or more mutations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations) in the genes described herein. In some embodiments, two or more mutations may occur in the same gene or in different genes. In some embodiments, two or more mutations can occur in the same allele or different alleles of a gene. In some embodiments, hearing loss may be associated with heterozygous mutations in the genes described herein. In some embodiments, hearing loss may be associated with homozygous mutations in the genes described herein. In certain embodiments, hearing loss is symptomatic, involving other manifestations that accompany hearing impairment. In certain embodiments, hearing loss is non-symptomatic, which occurs when there are no other problems associated with the individual other than hearing loss. In certain embodiments, dominant and recessive hearing loss is caused by allelic mutations in some genes, both symptomatic and non-symptomatic hearing loss is caused by mutations in the same gene, and recessive hearing loss may be caused by a combination of two mutations in different genes from the same functional group. In certain embodiments, the hearing loss is hereditary. In certain embodiments, the hearing loss is autosomal dominant non-symptomatic hearing loss. In certain embodiments, the hearing loss is autosomal recessive non-symptomatic hearing loss. In certain embodiments, the hearing loss is X-linked non-syndrome hearing loss. In certain embodiments, the hearing loss is mitochondrial syndrome hearing impairment. In certain embodiments, the hearing loss is acquired hearing loss. In certain embodiments, the hearing loss is progressive hearing loss. In certain embodiments, hearing loss of a subject may be associated with a potential disease or condition, such as, for example, waldenstrom's syndrome (Waardenburg syndrome, WS), a branchia kidney lineage disorder, neurofibromatosis 2 (NF 2), shi Dike lux syndrome, type I Wu Xieer syndrome, type II Wu Xieer syndrome, type III Wu Xieer syndrome, peng Delai syndrome, nielsen syndrome, biotin enzyme deficiency, raffmm disease, alport syndrome, and/or deafness-dystonia-optic neuropathy syndrome (Mohr-Tranebjaerg syndrome).

As used herein, the term "herpesvirus" or "herpesviridae" is intended to broadly refer to the general family of enveloped double-stranded DNA viruses having a relatively large genome. This family replicates in a wide range of vertebrate and invertebrate hosts, in preferred embodiments mammalian hosts, such as nuclei in humans, horses, cattle, mice and pigs. Exemplary members of the herpesviridae family include Cytomegalovirus (CMV), herpes simplex virus types 1 and 2 (HSV 1 and HSV 2), varicella Zoster Virus (VZV) and epstein-barr virus (EBV).

As used herein, the term "heterologous" means derived from an entity that differs in genotype from the remaining entities to which it is compared or to which it is introduced or incorporated. For example, polynucleotides introduced into different cell types by genetic engineering techniques are heterologous polynucleotides (and may encode heterologous polypeptides when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a nucleotide sequence that is heterologous with respect to the vector.

As used herein, the term "infection" is intended to broadly refer to the delivery of heterologous DNA into a cell by a virus. The term "co-infection" as used herein means "simultaneous infection", "double infection", "multiple infection" or "sequential infection" with two or more viruses. Infection of a producer cell with two (or more) viruses will be referred to as "co-infection". The term "transfection" refers to the process of delivering heterologous DNA (such as plasmid DNA) to a cell by physical or chemical means, which is transferred into the cell by electroporation, calcium phosphate precipitation, or other methods known in the art.

As used herein, the term "inner ear cells" or "cells of the inner ear" refers to Inner Hair Cells (IHCs) and Outer Hair Cells (OHCs), spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, support cells, and cells in the vascular striations, spiral ligaments, or spiral rims. Support cells refer to cells in the ear that are not excitable, e.g., cells that are not hair cells or neurons. In some embodiments, the tissue and cells of the inner ear include cells of the lateral wall or helical ligament, support cells of the coti's organ, fibroblasts of the helical ligament, claus cells, burt's cells, spiraling cells, vestibular support cells, hansen cells, dai Tesi cells, column cells, inner finger cells, outer finger cells, and/or limbic cells.

As used herein, the term "inverted terminal repeat" or "ITR" sequence means a relatively short sequence found at the end of an oppositely oriented viral genome. An "AAV Inverted Terminal Repeat (ITR)" sequence is a term well known in the art and is a sequence of about 145 nucleotides present at both ends of the native single stranded AAV genome. The outermost nucleotide of the ITR can exist in one of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome.

As used herein, the term "isolated" molecule (e.g., an isolated nucleic acid or protein or cell) means that the molecule has been identified and isolated and/or recovered from components of its natural environment.

As used herein, the term "middle ear" means the space between the tympanic membrane and the inner ear.

As used herein, the term "minimal regulatory element" means a regulatory element necessary for efficient expression of a gene in a target cell, and thus should be included in a transgenic expression cassette. Such sequences may include, for example, promoter or enhancer sequences, polylinker sequences that facilitate insertion of DNA fragments within plasmid vectors, and sequences responsible for intron splicing and polyadenylation of mRNA transcripts.

As used herein, the term "non-naturally occurring" is intended to broadly refer to proteins, nucleic acids, ribonucleic acids, or viruses that do not exist in nature. For example, it may be a genetically modified variant, such as a cDNA or a codon optimized nucleic acid.

As used herein, "nucleic acid" or "nucleic acid molecule" is intended to refer to a molecule composed of a single nucleotide chain, such as, for example, a DNA molecule (e.g., cDNA or genomic DNA). The nucleic acid may encode, for example, a promoter, a gene of interest or a portion thereof (e.g., a GJB2 gene or a portion thereof), or a regulatory element. The nucleic acid molecule may be single-stranded or double-stranded. "GJB2 nucleic acid" refers to a nucleic acid comprising the GJB2 gene or part thereof, or a functional variant of the GJB2 gene or part thereof. Functional variants of a gene include variants of a gene with minor changes such as silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly alter the function of the gene.

The asymmetric ends of DNA and RNA strands are referred to as 5 '(five prime) and 3' (three prime) ends, with the 5 'end having a terminal phosphate group and the 3' end having a terminal hydroxyl group. The five prime number (5') terminal has the fifth carbon in the sugar ring of deoxyribose or ribose at its terminal. Nucleic acids are synthesized in vivo in the 5' to 3' direction, as the polymerase used to assemble the new strand attaches each new nucleotide to a 3' -hydroxyl (-OH) group via a phosphodiester bond.

As used herein, the term "operably linked" or "coupled" can refer to the juxtaposition of genetic elements wherein the elements are in a relationship permitting them to operate in their intended manner. For example, a promoter may be operably linked to a coding region if the promoter helps to initiate transcription of the coding sequence. So long as this functional relationship is maintained, there may be intervening residues between the promoter and the coding region.

As used herein, "percent (%) sequence identity" with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and without regard to any conservative substitutions that are part of the sequence identity. Alignment for the purpose of determining percent amino acid or nucleic acid sequence identity can be accomplished in a variety of ways within the skill of those in the art, for example using publicly available computer software programs, e.g., described in Current Protocols in Molecular Biology (Ausubel et al, 1987), journal 30, section 7.7.18, table 7.7.1, and includes BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. An example of an alignment program is ALIGN Plus (Scientific and Educational Software, pennsylvania). One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences. For purposes herein, the% amino acid sequence identity of a given amino acid sequence a to, with or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence a having or comprising a specific% amino acid sequence identity to, with or against a given amino acid sequence B) is calculated as follows: 100 by a score X/Y, where X is the number of amino acid residues scored by the sequence alignment program as identical matches in the alignment of sequence alignment program pairs a and B, and where Y is the total number of amino acid residues in B. It will be appreciated that in the case where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. For purposes herein, the% nucleic acid sequence identity of a given nucleic acid sequence C with, with or for a given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C having a specific% nucleic acid sequence identity with, with or for a given nucleic acid sequence D) is calculated as follows: 100 by a fraction W/Z, where W is the number of nucleotides scored by the sequence alignment program as identical matches in the alignment of the program pair C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the% nucleic acid sequence identity of C to D will not be equal to the% nucleic acid sequence identity of D to C.

Similarly, "sequence homology" as used herein also refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above, and gaps are introduced as necessary. However, in contrast to "sequence identity," conservative amino acid substitutions are counted as matches in determining sequence homology. In other words, in order to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 95% of the amino acid residues or nucleotides in the reference sequence must be matched or contain conservative substitutions with another amino acid or nucleotide, or up to 5% of the total amino acid residues or nucleotides in the reference sequence may be inserted into the reference sequence without including the conservative substitutions.

As used herein, the term "pharmaceutical composition" or "composition" is intended to refer to a composition or agent (e.g., recombinant adeno-associated (rAAV) expression vector and/or rAAV virion) as described herein, optionally in admixture with at least one pharmaceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, and the like.

As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such amino acid residue polymers may comprise natural or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of this disclosure, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions, and substitutions (typically conservative in nature), so long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or occasional, such as by mutation of the host producing the protein or by error due to PCR amplification.

As used herein, "promoter" is intended to refer to a region of DNA that promotes transcription of a particular gene. As part of the transcription process, an enzyme that synthesizes RNA (referred to as RNA polymerase) is attached to DNA near the gene. The promoter comprises a specific DNA sequence and response elements that provide an initial binding site for RNA polymerase and transcription factors that recruit RNA polymerase. According to some embodiments, the promoter is highly specific for supporting cell expression in the cochlea. According to some embodiments, the promoter is an endogenous GJB2 promoter. According to some embodiments, the promoter is a synthetic promoter. According to some embodiments, the promoter is selected from the group consisting of: CBA promoter, smCBA promoter, CASI promoter, GFAP promoter and elongation factor-1 a (EF 1 a) promoter. "chicken beta-actin (CBA) promoter" refers to a polynucleotide sequence derived from a chicken beta-actin gene (e.g., chicken (Gallus) beta actin, represented by GenBank Entrez Gene ID 396526). The "smCBA" promoter refers to a small version of the hybrid CMV-chicken beta-actin promoter. "CASI" promoter refers to a promoter comprising a portion of the CMV enhancer, a portion of the chicken beta-actin promoter, and a portion of the UBC enhancer.

As used herein, the term "recombinant" may refer to a biomolecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or part of a polynucleotide to which the gene is naturally present, (3) is operably linked to a polynucleotide to which it is not naturally linked, or (4) is not naturally present. The term "recombinant" may be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biosynthesized by heterologous systems, as well as proteins and/or mrnas encoded by such nucleic acids.

As used herein, the terms "recombinant HSV," "rHSV," and "rHSV vector" are intended to broadly refer to an isolated, genetically modified form of Herpes Simplex Virus (HSV) type 1 that contains a heterologous gene incorporated into the viral genome. The term "rHSV-rep2cap2" or "rHSV-rep2cap1" means an rHSV in which AAV rep and cap genes from AAV serotypes 1 or 2 have been incorporated into the rHSV genome, in certain embodiments the DNA sequence encoding the therapeutic gene of interest has been incorporated into the viral genome.

As used herein, "subject" or "patient" or "individual" to be treated by the methods of the present disclosure is intended to refer to a human or non-human animal. According to some embodiments, the subject is a child. According to some embodiments, the subject is an infant. "non-human animal" includes any vertebrate or invertebrate. In some embodiments, the subject suffers from hearing loss associated with a deficiency in a gene (such as the GJB2 gene).

As used herein, the term "transgene" is intended to refer to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally translated and/or expressed under appropriate conditions. In various aspects, it imparts desirable properties to the cell into which it is introduced, or otherwise results in a desirable therapeutic or diagnostic outcome. In certain embodiments, introducing a GJB2 transgene into a cell results in the formation of a functional gap junction.

As used herein, a "transgenic expression cassette" or "expression cassette" comprises a gene sequence that a nucleic acid vector will deliver to a target cell. These sequences include a gene of interest (e.g., a GJB2 nucleic acid or variant thereof), one or more promoters, and minimal regulatory elements.

As used herein, the term "treating" a disease or condition is intended to refer to alleviating one or more signs or symptoms of the disease or condition, attenuating the extent of the disease or condition, stabilizing (e.g., not worsening) the disease or condition, preventing the spread of the disease or condition, delaying or slowing the progression of the disease or condition, ameliorating or alleviating the disease or condition, and alleviating (whether partial or total), whether detectable or undetectable. For example, a gene of interest, such as GJB2, when expressed in an effective amount (or dose), is sufficient to prevent, correct, and/or normalize an abnormal physiological response, e.g., sufficient to reduce a clinically significant feature of a disease or disorder by at least about 30%, more preferably at least 50%, and most preferably at least 90% of the therapeutic effect. "treatment" may also refer to an extended survival period compared to an expected survival period if not treated.

As used herein, the term "vector" is intended to refer to a recombinant plasmid or virus comprising a nucleic acid to be delivered into a host cell in vitro or in vivo.

As used herein, the term "recombinant viral vector" is intended to refer to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid is flanked by at least one Inverted Terminal Repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two ITRs.

As used herein, the term "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., a nucleic acid sequence that is not AAV-derived) flanked by at least one AAV inverted terminal repeat sequence (ITR). Such rAAV vectors can be replicated and packaged into infectious virions when present in host cells that have been infected with a suitable helper virus (or express a suitable helper function) and express AAV Rep and Cap gene products (i.e., AAV Rep and Cap proteins). When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid for cloning or transfection), then the rAAV vector may be referred to as a "pro-vector," which may be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. The rAAV vector can be in any of a variety of forms, including, but not limited to, a plasmid, a linear artificial chromosome, complexed with a lipid, encapsulated within a liposome, and encapsulated in a viral particle (e.g., AAV particle). The rAAV vector can be packaged into an AAV viral capsid to produce a "recombinant adeno-associated viral particle (rAAV particle)". In certain embodiments, the AAV viral capsid is a variant AAV capsid described herein.

As used herein, the term "rAAV virus" or "rAAV virion" is intended to refer to a virion consisting of at least one AAV capsid protein and a encapsidated rAAV vector genome. In certain embodiments, the AAV capsid protein is a variant AAV capsid protein, such as a variant AAV capsid protein that exhibits an increased propensity in inner ear tissue or cells as compared to a non-variant AAV capsid protein. The amino acid sequences and nucleotide sequences of exemplary variant AAV capsid proteins that can be used according to the methods described herein are provided in table 1.

The term "variant" in reference to a polypeptide, such as a capsid polypeptide, refers to a polypeptide sequence that differs from a parent polypeptide sequence (also referred to as a non-variant polypeptide sequence) by at least one amino acid. In some embodiments, the polypeptide is a capsid polypeptide and the variants differ by at least one amino acid substitution. Amino acids also include naturally occurring and non-naturally occurring amino acids and derivatives thereof. Amino acids also include both D and L forms.

The terms "tropism" and "transduction" are interrelated, but also different. The term "tropism" as used herein refers to the ability of an AAV vector or virion to infect one or more specific cell types, but can also encompass how the vector functions to transduce cells of one or more specific cell types; that is, trending refers to the preferential entry of an AAV vector or virion into certain cell or tissue types and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression (e.g., transcription and optionally translation) in a cell of a sequence carried by the AAV vector or virion, e.g., expression of a heterologous nucleotide sequence for a recombinant virus. As used herein, the term "transduction" refers to the ability of an AAV vector or virion to infect one or more specific cell types; that is, transduction refers to the entry of an AAV vector or virion into a cell and the transfer of genetic material contained in the AAV vector or virion into the cell to obtain expression from the vector genome. In some cases, but not in all cases, transduction and trending may be relevant. In certain embodiments, for example, a variant AAV capsid polypeptide described herein exhibits an increased propensity in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide. In certain embodiments, for example, a variant AAV capsid polypeptide described herein exhibits increased transduction in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide. In certain embodiments, for example, a variant AAV capsid polypeptide described herein exhibits increased tropism and/or transduction in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide. In certain embodiments, for example, exhibit increased tropism and/or increased propensity in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide

The transduced variant AAV capsid polypeptides are provided in table 1.

Nucleic acid

The present disclosure provides promoters, expression cassettes, vectors, kits and methods useful for treating hearing loss, such as hearing loss associated with a deficiency in a gene. In some embodiments, the hearing loss is hereditary hearing impairment. Certain aspects of the disclosure relate to delivering heterologous nucleic acids to tissues and cells of the inner ear of a subject, including administration of recombinant adeno-associated virus (rAAV) vectors and/or virions. According to some aspects, the present disclosure provides methods of treating or preventing hearing loss, e.g., hearing loss associated with a deficiency in a gene, comprising delivering to the subject a composition comprising a rAAV vector and/or rAAV virion as described herein, wherein the rAAV vector and/or rAAV virion comprises a heterologous nucleic acid (e.g., a nucleic acid encoding GJB 2).

In certain embodiments, the rAAV vector comprises a heterologous nucleic acid encoding a gene associated with hearing loss. Examples of genes associated with hearing loss (e.g., hearing loss associated with gene deficiency) include, but are not limited to ACTG1, ADCY1, ADGRV1, AIFM1, BDP1, BSND, BTD, CABP2, CCDC50, CD164, CDC14A, CDH, CEACAM16, CIB2, CLDN14, CLIC5, CLRN1, COCH, COL11A1, COL11A2, COL2A1, COL4A3, COL4A4, COL4A5, COL4A6, COL9A1, COL9A2, COL9A3, DCDC2, DIAPH1, DMXL2, DSPP, EDN3, EDNRB, ELMOD3, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM189A2, GIPC3, GJB2, GJB3, GJB6, GPSM2, GRHL2, GRXCR1, GRXCR2, GSDME, HADR 1, HGF, HOMER2, ILRS 1, KARS1, KCQ 1, NQ 4; LHFPL5, LOXHD1, LRTOMT, MARVELD, MCM2, MET, MIR96, MITF, MSRB3, MT-CO1, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, MYO7A, NARS2, NF2, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PEX7, PHYH, PJVK, PNPT1, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, RIPOR, ROR1, S1PR2, SERPINB6, SIX1, SIX5, SLC17A8, SLC22A4, SLC26A5, SMPX, SOX10, STRC, SYNE4, TBC1D24, TECTA, TIMM8A, TJP2, TMC1, TMEM132E, TMTE, TMPRSS, TPRN, TRIOBP, TSPEAR, USH C, USH1G, USH2A, WBP, WFS1, and WHON. In certain embodiments, the rAAV vector and/or rAAV virion comprises a heterologous nucleic acid encoding GJB 2.

The gene GJB2, which is the most commonly mutated in subjects with hereditary Hearing Impairment (HI), encodes the connexin-26 (Cx 26) gap junction channel protein, which is the basis for supporting intercellular communication between cells and cochlear fluid, endolymph and perilymph homeostasis. GJB2 is located at the DFNB1 locus on 13q 12. GJB2 is 5513bp long and contains two exons (193 bp and 2141bp long, respectively) separated by an intron of 3179bp (Kiang et al, 1997). Transcription starts from a single start site and results in the synthesis of 2334 nucleotide mRNA (GenBank nm_ 004004.5), which is considered canonical.

According to some embodiments, the gene of interest (e.g., GJB 2) is optimized to be superior to the wild-type gene (e.g., wild-type GJB 2) in expression (and/or function), and further has the ability to distinguish (at the DNA/RNA level) from the wild-type (e.g., wild-type GJB 2).

Fig. 2 shows a schematic diagram of exemplary GJB2 vector (genome) constructs single stranded (ss) AAV-GJB2 and self-complementary scAAV-GJB 2.

Human wild-type GJB2 is an important element that encodes the major gap junction protein required for normal hearing. Loss of GJB2 results in massive cell death of various cell types in the inner ear after hearing begins. "GJB2 nucleic acid" refers to a nucleic acid comprising the GJB2 gene or part thereof, or a functional variant of the GJB2 gene or part thereof. Functional variants of a gene include variants of a gene with minor changes such as silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly alter the function of the gene.

According to some embodiments, the present disclosure provides nucleic acids encoding mammalian GJB2 proteins. According to some embodiments, the present disclosure provides nucleic acids encoding wild-type GJB2 proteins. According to some embodiments, the present disclosure provides nucleic acids encoding wild-type human, mouse, non-human primate, or rat GJB2 proteins. According to some embodiments, the present disclosure provides nucleic acids encoding human wild-type GJB2 proteins. According to some embodiments, the nucleic acid sequence encoding the human wild-type GJB2 protein is 678bp in length. According to one embodiment, the nucleic acid encoding a human wild-type GJB2 protein comprises SEQ ID NO:10. according to one embodiment, the nucleic acid hybridizes to SEQ ID NO:10 is at least 85% identical. According to one embodiment, the nucleic acid is at least 90% identical to SEQ ID NO:10. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:10 is at least 95% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:10 is at least 99% identical. According to one embodiment, the nucleic acid consists of SEQ ID NO:10.

According to some embodiments, the present disclosure provides nucleic acids encoding a GJB2 protein, wherein the nucleic acid sequence is codon optimized for mammalian expression. According to certain embodiments, the present disclosure provides nucleic acids encoding a GJB2 protein, wherein the nucleic acid sequence is codon optimized for expression in human, rat, non-human primate, guinea pig, minipig, pig, cat, sheep, or mouse cells. Human codon-optimized GJB2 is an important element of encoding the major gap junction protein required for normal hearing. Codon optimization was performed to enhance protein expression of GJB 2.

According to some embodiments, the present disclosure provides nucleic acids encoding a GJB2 protein, wherein the nucleic acid sequence encoding the GJB2 protein is a non-naturally occurring sequence.

According to some embodiments, the present disclosure provides nucleic acids encoding human codon optimized GJB2 proteins.

According to some embodiments, the nucleic acid sequence encoding the human codon optimized GJB2 protein is 678bp in length. According to one embodiment, the nucleic acid encoding the human codon optimized GJB2 protein comprises SEQ ID NO:11. according to one embodiment, the nucleic acid hybridizes to SEQ ID NO:11 is at least 85% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:11 is at least 90% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:11 is at least 95% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:11 is at least 99% identical. According to one embodiment, the nucleic acid consists of SEQ ID NO:11.

According to some embodiments, the nucleic acid sequence encoding the human codon optimized GJB2 protein is 678bp in length. According to one embodiment, the nucleic acid encoding the human codon optimized GJB2 protein comprises SEQ ID NO:12. according to one embodiment, the nucleic acid hybridizes to SEQ ID NO:12 is at least 85% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:12 is at least 90% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:12 is at least 95% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:12 is at least 99% identical. According to one embodiment, the nucleic acid consists of SEQ ID NO:12.

According to some embodiments, the nucleic acid sequence encoding the human codon optimized GJB2 protein is 678bp in length. According to one embodiment, the nucleic acid encoding the human codon optimized GJB2 protein comprises SEQ ID NO:13. according to one embodiment, the nucleic acid hybridizes to SEQ ID NO:13 is at least 85% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:13 is at least 90% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:13 is at least 95% identical. According to one embodiment, the nucleic acid hybridizes to SEQ ID NO:13 is at least 99% identical. According to one embodiment, the nucleic acid consists of SEQ ID NO:13.

In certain embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide. According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide, e.g., that exhibits an increased propensity in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide.

According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide selected from the group consisting of: mutating AAV1 capsid polypeptides; mutating AAV2 capsid polypeptides; variant AAV3 capsid polypeptides; variant AAV4 capsid polypeptides; variant AAV5 capsid polypeptides; variant AAV6 capsid polypeptides; variant AAV7 capsid polypeptides; variant AAV8 capsid polypeptides; variant AAV9 capsid polypeptides; variant rh-AAV10 capsid polypeptides; variant AAV10 capsid polypeptides; variant AAV11 capsid polypeptides; and variant AAV12 capsid polypeptides. According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV2 capsid polypeptide.

According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide listed in table 1, or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity to said nucleic acid sequence.

According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising an amino acid sequence listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity to said amino acid sequence.

According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding an AAV capsid selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides.

According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising an amino acid sequence having one or more amino acid substitutions, insertions, and/or deletions relative to a wild type AAV2 capsid polypeptide (SEQ ID NO: 18), optionally wherein the one or more amino acid substitutions, insertions, and/or deletions occur at an amino acid residue selected from the group consisting of: q263, S264, Y272, Y444, R487, P451, T454, T455, R459, K490, T491, S492, A493, D494, E499, Y500, T503, K527, E530, E531, Q545, G546, S547, E548, K549, T550, N551, V552, D553, E555, K556, R585, R588, and Y730.

According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising an amino acid sequence having one or more amino acid substitutions with respect to a wild type AAV2 capsid polypeptide (SEQ ID NO: 18), the one or more amino acid substitutions selected from the group consisting of: q263 263 264 272 444 487 451 454 455 459 490 491 492 493 494 499 500 527, 545 546 546 547 548 548 549 550 550 551 552 555 556 585 588T, and Y730F.

According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising: (i) SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or a sequence of any one of seq id no; (ii) a sequence corresponding to SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity; or (iii) a polypeptide consisting of SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO:32 or SEQ ID NO:34, and an amino acid sequence encoded by the nucleic acid sequence of any one of seq id no. According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 27. According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 29. According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 31. According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NO:33, an amino acid sequence of seq id no. According to some embodiments, the rAAV vector comprises a nucleic acid sequence encoding a variant AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NO:35, and a sequence of amino acids.

Promoters

Various promoters are contemplated for use in the present disclosure.

According to some embodiments, the promoter is an endogenous GJB2 promoter. The GJB2 promoter is a supporting cell specific promoter and can transduce inner ear cells expressing the GJB2 gene; in view of its short length, this promoter can be used for the production of scAAV. According to some embodiments, the promoter comprises SEQ ID NO:6. according to some embodiments, the promoter consists of SEQ ID NO:6. FIG. 8 shows the nucleic acid sequence of the GJB2 promoter (SEQ ID NO. 6).

According to some embodiments, the promoter is a CBA promoter. CBA promoters are powerful ubiquitous promoters that transduce multiple cell types in the inner ear. According to some embodiments, the promoter comprises SEQ ID NO:1. according to some embodiments, the promoter consists of SEQ ID NO:1. FIG. 3 shows the nucleic acid sequence of the CBA promoter (SEQ ID NO. 1).

According to some embodiments, the promoter is an EF1a promoter. The EF1a promoter is a potent promoter of mammalian origin, which can transduce a variety of cell types in the inner ear and, due to its short length, can be used for the production of scaAAV. According to some embodiments, the promoter comprises SEQ ID NO:2. according to some embodiments, the promoter consists of SEQ ID NO:2. FIG. 4 shows the nucleic acid sequence of the EF1a promoter (SEQ ID NO. 2).

According to some embodiments, the promoter is a CASI promoter. The CASI promoter is a ubiquitous promoter that can transduce a variety of cell types in the inner ear and, due to its short length, can be used to produce scAAV. According to some embodiments, the promoter comprises SEQ ID NO:3. according to some embodiments, the promoter consists of SEQ ID NO:3. FIG. 5 shows the nucleic acid sequence of the CASI promoter (SEQ ID NO. 3).

According to some embodiments, the promoter is a smCBA promoter. The smCBA promoter is a ubiquitous promoter that can transduce a variety of cell types in the inner ear and is useful for production of scAAV due to its short length. According to some embodiments, the promoter comprises SEQ ID NO:4. according to some embodiments, the promoter consists of SEQ ID NO:4. FIG. 6 shows the nucleic acid sequence of the smCBA promoter (SEQ ID NO. 4).

According to some embodiments, the promoter is a GFAP promoter. The GFAP promoter is cell specific and active in the supporting cells of the inner ear. According to some embodiments, the promoter comprises SEQ ID NO:5. according to some embodiments, the promoter consists of SEQ id no:5. FIG. 7 shows the nucleic acid sequence of the GFAP promoter (SEQ ID NO. 5).

According to some embodiments, the promoter is a synthetic promoter. In certain embodiments, a synthetic promoter is a DNA sequence that does not exist in nature and has been designed to control gene expression of a target gene (e.g., GJB 2).

Inverted terminal repeat sequence

The Inverted Terminal Repeat (ITR) sequence is required for efficient proliferation of the AAV genome due to its ability to form a hairpin structure that allows synthesis of the second DNA strand. The scAAV shortened ITR (TRS) forms an intramolecular double stranded DNA template, thereby eliminating the rate limiting step of second strand synthesis.

FIG. 9 shows the following nucleic acid sequences for ITR (AAV 2): 5'-3': for single stranded (ss) and self-complementary (sc) AAV genome (SEQ ID NO. 7); 3'-5': for single stranded (ss) AAV genome only (SEQ ID NO. 8); 3'-5': targeting only the self-complementary (sc) AAV genome (SEQ ID NO. 9).

Gene therapy for hearing loss

The present disclosure generally provides methods for producing recombinant adeno-associated virus (AAV) virions comprising a gene construct (e.g., a GJB2 gene construct), and uses thereof in gene therapy methods for hearing loss (e.g., hearing loss associated with a deficiency in a gene). AAV vectors and AAV virions as described herein are particularly effective in delivering nucleic acids (e.g., GJB2 gene constructs) to inner ear tissues and cells. Described herein are methods for creating, evaluating, and utilizing recombinant adeno-associated virus (rAAV) therapeutic vectors that are capable of efficiently delivering genes, such as GJB2, into cells for expression and subsequent secretion. Described herein are optimized modified gene of interest (GOI) cdnas and associated genetic elements for use in recombinant adeno-associated virus (rAAV) -based gene therapies for hearing loss (e.g., hearing loss associated with a deficiency in a gene). More specifically, described herein are optimized modified GJB 2/connexin 26 (Cx 26) cdnas and associated genetic elements for use in recombinant adeno-associated virus (rAAV) -based gene therapies for hereditary hearing loss, including the treatment and/or prevention of congenital deafness associated with DFNB1 and DFNA 3A.

Recombinant adeno-associated virus (rAAV) vectors can efficiently accommodate both target genes (e.g., GJB2 target genes) and associated genetic elements. Furthermore, such vectors may be designed to specifically express genes (e.g., GJB 2) in treatment-related inner ear tissues and cells, such as support cells of the cochlea. The present disclosure describes methods of creating, evaluating, and utilizing rAAV therapy vectors and rAAV virions that are capable of efficiently delivering functional genes of interest (e.g., the GJB2 gene) to a patient.

In some embodiments, the GJB2 gene construct may comprise: (1) Codon/sequence optimized 0.68kb human GJB2 cDNA with or without a 27 nucleotide Hemagglutinin (HA) C-terminal tag; (2) One of the following promoter elements optimized to drive high GJB2 expression: (a) The ubiquitously active 1.7kb CBA, 0.96kb small CBA (smCBA), 0.81kb EF1a or 1.06kb CASI promoter; (b) Cochlear support cells or GJB2 expression specific 1.68kb GFAP, 0.13/0.54/1.0kb small/medium/large GJB2 promoter, or sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoters; (3) A 0.9kb 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal, (4) two 143 base sequence regulatory Inverted Terminal Repeats (ITRs) flanking the AAV genome box, or a self-complementary AAV (scAAV) genome box consisting of two inverted identical repeats (each no longer than 3.0 kb) separated by a 113 base scAAV-enabled ITR (itrΔtrs) and flanked on both ends by 143 base sequence regulatory ITRs; and (5) protein capsid variants best suited for cochlear delivery. In some embodiments, the nucleic acid sequence encoding GJB2 may comprise an operably linked C-terminal tag or N-terminal tag, such as a FLAG tag or HA tag.

HA tags are human influenza hemagglutinin, a surface glycoprotein, used as a general epitope tag in expression vectors to aid in the detection of proteins of interest. FLAG tag (peptide sequence dykdddk) is a short hydrophilic protein tag that is commonly used as a general epitope tag in expression vectors to aid in the detection of proteins of interest. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is a DNA sequence that enhances the expression of a protein of interest by generating tertiary structures that stabilize its mRNA. According to certain embodiments, other regulatory sequences may be used. In certain embodiments, regulatory sequences useful in accordance with the present disclosure comprise DNA sequences that, when transcribed, produce a tertiary structure that enhances the expression of a target gene (such as GJB 2). poly (a) sequences are important elements that promote RNA processing and transcript stability. The SV40 and bGH polyA sequences are terminator sequences that signal the end of the transcription unit. According to certain embodiments, other polyA terminator sequences may be used.

According to some embodiments, the AAV vectors and AAV virions described herein are particularly suitable for delivering and expressing genes, such as GJB2, in inner ear tissue or cells (including cochlear support cells). According to some embodiments, the AAV vectors and AAV virions described herein are particularly suitable for delivering and expressing genes, such as GJB2, in one or more of external support cells and/or support cells of the organ of coti. According to some embodiments, the AAV vectors and AAV virions described herein are particularly suitable for delivering and expressing genes, such as GJB2, in one or more of outer hair cells, inner hair cells, hansen cells, dai Tesi cells, columnar cells, inner finger cells, and/or outer finger/limbal cells.

Adeno-associated virus (AAV)

Adeno-associated virus (AAV) is a nonpathogenic single-stranded DNA parvovirus. The capsid diameter of AAV is about 20nm. Each end of the single stranded DNA genome contains an Inverted Terminal Repeat (ITR), which is the only cis-acting element required for genome replication and packaging. AAV genomes carry two viral genes: rep and cap. The virus utilizes two promoters and alternative splicing to generate the four proteins (Rep 78, rep 68, rep 52, and Rep 40) required for replication. The third promoter generates transcripts of three structural viral capsid proteins 1, 2 and 3 (VP 1, VP2 and VP 3) by a combination of alternative splicing and alternate translation initiation codons. Berns and Linden Bioessays 1995;17:237-45. Three capsid proteins share the same C-terminal 533 amino acids, while VP2 and VP1 contain additional N-terminal sequences with 65 amino acids and 202 amino acids, respectively. AAV virions contain a total of 60 copies of VP1, VP2 and VP3 arranged in a ratio of 1:1:20 in T-1 icosahedral symmetry. Rose et al JVirol.1971;8:766-70.AAV requires adenovirus (Ad), herpes Simplex Virus (HSV), or other viruses as helper viruses to complete its lytic life cycle. Atchison et al Science,1965;149:754-6; hoggan et al Proc Natl Acad Sci USA,1966;55:1467-74. In the absence of helper virus, wild-type AAV establishes latency by integrating with the help of Rep proteins via its interactions with chromosomes. Berns and Linden (1995). In certain embodiments, an AAV described herein comprises a variant AAV capsid polypeptide, e.g., that exhibits increased tropism and/or transduction in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide. Exemplary variant AAV capsid polypeptides are provided in table 1.

AAV serotypes

There are many different AAV serotypes including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, anc80L65, and variants or hybrids thereof. In vivo studies have shown that various AAV serotypes exhibit different tissue or cell chemotaxis. For example, AAV1 and AAV6 are two serotypes that are effective for skeletal muscle transduction. Gao et al Proc Natl Acad Sci USA,2002;99:11854-11859; xiao et al J virol 1999;73:3994-4003; chao et al Mol Ther.2000;2:619-623.AAV-3 has proven to be advantageous in megakaryocyte transduction. Handa et al J Gen Virol.2000;81:2077-2084.AAV5 and AAV6 effectively infect apical airway cells. Zabner et al J Virol'.2000;74:3852-3858; halbert et al J Virol.2001;75:6615-6624.AAV2, AAV4 and AAV5 transduce different types of cells in the central nervous system. Davidson et al Proc Natl Acad Sci usa.2000;97:3428-3432.AAV8 and AAV5 can transduce hepatocytes better than AAV-2. AAV-5 based vectors transduce certain cell types (cultured airway epithelial cells, cultured striated muscle cells, and cultured human umbilical vein endothelial cells) with greater efficiency than AAV2, whereas AAV2 and AAV5 both exhibit poor transduction efficiency for NIH 3T3, skbr3, and T-47D cell lines. Gao et al Proc Natl Acad Sci USA.2002;99:11854-11859; mingozzi et al J Virol.2002;76:10497-10502.WO 99/61601. AAV4 was found to transduce the rat retina most effectively, followed by AAV5 and AAV1.Rabinowitz et al J Virol.2002;76:791-801; weber et al Mol ter 2003;7:774-781. Overall, AAV1, AAV2, AAV4, AAV5, AAV8 and AAV9 show chemotaxis for CNS tissues. AAV1, AAV8 and AAV9 show a chemotaxis towards heart tissue. AAV2 exhibits chemotaxis for kidney tissue. AAV7, AAV8 and AAV9 exhibit a tendency toward liver tissue. AAV4, AAV5, AAV6 and AAV9 exhibit a tendency toward lung tissue. AAV8 shows a tendency towards pancreatic cells. AAV3, AAV5 and AAV8 show chemotaxis for photoreceptor cells. AAV1, AAV2, AAV4, AAV5 and AAV8 exhibit a tropism for Retinal Pigment Epithelial (RPE) cells. AAV1, AAV6, AAV7, AAV8 and AAV9 show a chemotaxis for skeletal muscle.

Further modifications to the virus may be performed to increase the efficiency of gene transfer, for example by improving the tropism of each serotype. One approach is to swap the domains from one serotype capsid to another, creating hybrid vectors with the desired quality from each parent. Since the viral capsid is responsible for cellular receptor binding, it is important to understand the viral capsid domain that is critical for binding. Mutation studies on viral capsids (mainly on AAV 2) performed before the crystal structure is available are mainly based on capsid surface functionalization by adsorption of exogenous moieties, insertion of peptides at random positions or comprehensive mutagenesis at amino acid level. Choi et al Curr Gene Ther.2005, month 6; 5 (3): 299-310 describes different methods and precautions for hybridizing serotypes.

Capsids from other AAV serotypes provide advantages over rAAV vectors based on AAV2 capsids in certain in vivo applications. First, proper use of rAAV vectors with specific serotypes can increase the efficiency of gene delivery in vivo to specific target cells that are poorly infected or not infected at all by AAV 2-based vectors. Second, if re-administration of the rAAV vector is clinically required, it may be advantageous to use rAAV vectors based on other AAV serotypes. Re-administration of the same rAAV vector with the same capsid has proven to be ineffective, possibly due to the generation of neutralizing antibodies raised against the vector. Xiao et al 1999; halbert et al 1997. This problem can be avoided by administering rAAV particles whose capsids are composed of proteins from different AAV serotypes, unaffected by the presence of neutralizing antibodies to the first rAAV vector. Xiao et al 1999. For the reasons described above, there is a need for recombinant AAV vectors constructed using cap genes from serotypes including AAV2 and that are complements of AAV 2. It will be appreciated that construction of recombinant HSV vectors similar to rHSV but encoding cap genes from other AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV5 to AAV 9) can be accomplished using the methods described herein to produce rHSV. In certain preferred embodiments of the present disclosure as described herein, recombinant AAV vectors constructed using cap genes from different AAV are preferred. The significant advantage of constructing these additional rHSV vectors is simplicity and time saving compared to alternative methods for large scale production of rAAV. In particular, the difficult process of constructing new rep and cap induced cell lines for each different capsid serotype is avoided.

In certain preferred embodiments of the disclosure as described herein, recombinant AAV vectors constructed using cap genes encoding variant AAV capsid polypeptides that exhibit increased tropism and/or transduction in inner ear tissues or cells, e.g., as compared to non-variant AAV capsids, are preferred. Such variant AAV capsid polypeptides are described herein. Exemplary variant AAV capsid polypeptides are provided in table 1.

Variant AAV capsid polypeptides

The present disclosure generally provides variant adeno-associated virus (AAV) capsid polypeptides that exhibit increased tropism and/or transduction in inner ear tissue or cells, e.g., as compared to non-variant AAV capsid polypeptides; and methods for treating or preventing hearing loss, such as that associated with a deficiency in a gene.

According to some embodiments, the variant AAV capsid polypeptide is selected from the group consisting of: mutating AAV1 capsid polypeptides; mutating AAV2 capsid polypeptides; variant AAV3 capsid polypeptides; variant AAV4 capsid polypeptides; variant AAV5 capsid polypeptides; variant AAV6 capsid polypeptides; variant AAV7 capsid polypeptides; variant AAV8 capsid polypeptides; variant AAV9 capsid polypeptides; variant rh-AAV10 capsid polypeptides; variant AAV10 capsid polypeptides; variant AAV11 capsid polypeptides; and variant AAV12 capsid polypeptides. According to some embodiments, the variant AAV capsid polypeptide is a variant AAV2 capsid polypeptide.

According to some embodiments, the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions, insertions and/or deletions relative to the wild type AAV2 capsid polypeptide (SEQ ID NO: 18), optionally wherein the one or more amino acid substitutions, insertions and/or deletions occur at an amino acid residue selected from the group consisting of: q263, S264, Y272, Y444, R487, P451, T454, T455, R459, K490, T491, S492, A493, D494, E499, Y500, T503, K527, E530, E531, Q545, G546, S547, E548, K549, T550, N551, V552, D553, E555, K556, R585, R588, and Y730.

According to some embodiments, the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions with respect to a wild type AAV2 capsid polypeptide (SEQ ID NO: 18), the one or more amino acid substitutions selected from the group consisting of: q263 263 264 272 444 487 451 454 455 459 490 491 492 493 494 499 500 527, 545 546 546 547 548 548 549 550 550 551 552 555 556 585 588T, and Y730F.

According to some embodiments, the variant AAV capsid polypeptide comprises: (i) SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or a sequence of any one of seq id no; (ii) a sequence corresponding to SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity; or (iii) a polypeptide consisting of SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO:32 or SEQ ID NO:34, and an amino acid sequence encoded by the nucleic acid sequence of any one of seq id no. According to some embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO: 27. According to some embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO: 29. According to some embodiments, the variant AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 31. According to some embodiments, the variant AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 33. According to some embodiments, the variant AAV capsid polypeptide comprises SEQ ID NO:35, and a sequence of amino acids.

According to some embodiments, the variant AAV capsid polypeptide results in an increase in rAAV chemotaxis level in inner ear tissue or cells, optionally at least 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, as compared to a non-variant AAV capsid polypeptide. In certain embodiments, mutating the AAV capsid polypeptide results in an increased level of rAAV tropism in inner ear tissue or cells selected from the group consisting of: cells of the outer wall or helix ligament, support cells of the coti organ, fibroblasts of the helix ligament, claus cells, burt's cells, spiraling cells, vestibular support cells, hansen cells, dai Tesi cells, column cells, inner finger cells, outer finger cells, and/or limbal cells.

According to some embodiments, the variant AAV capsid polypeptide results in an increase in rAAV transduction efficiency in inner ear tissue or cells, optionally by at least 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, optionally as compared to a non-variant AAV capsid polypeptide. In certain embodiments, mutating the AAV capsid polypeptide results in an increased level of rAAV transduction efficiency in inner ear tissue or cells selected from the group consisting of: cells of the outer wall or spiral ligament, support cells of the organ of kotinia, fibroblasts of the spiral ligament, claus cells, burchet cells, spiral bulge cells, vestibular support cells, hansen cells, dai Tesi cells, column cells, inner finger cells, outer finger cells, and/or limbic cells, intra-cochlear hair cells, extra-cochlear hair cells, spiral ganglion neurons, vestibular hair cells, vestibular support cells, and/or vestibular ganglion neurons.

Production of recombinant AAV (rAAV) vectors

The production, purification, and characterization of the rAAV vectors of the present disclosure can be performed using any of a number of methods known in the art. According to some embodiments, the rAAV vector encodes an AAV variant capsid polypeptide as described herein (e.g., table 1), e.g., the AAV variant capsid polypeptide exhibits increased tropism and/or transduction in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide. For comments on laboratory scale production methods, see, for example, clark RK, recent advances in recombinant adeno-associated virus vector production. Kidney int.61s:9-15 (2002); choi VW et al, Production of recombinant adeno-associated viral vectors for in vitro and in vivo use current Protocols in Molecular Biology 16.25.1-16.25.24 (2007) (hereinafter Choi et al); grieger JC and Samulski RJ, adeno-associated virus as a gene therapy vector: vector development, production, and clinical applications, adv Biochem Engin/Biotechnol 99:119-145 (2005) (hereinafter Grieger and Samulski); heilbronn R and Weger S, viral Vectors for Gene Transfer: current Status of Gene Therapeutics, in M.Korting (edit), drug Delivery, handbook of Experimental Pharmacology,197:143-170 (2010) (hereinafter Heilbronn); howarth JL et al Using viral vectors as gene transfer tools.cell Biol Toxicol 26:1-10 (2010) (hereinafter Howarth). The production methods described below are intended as non-limiting examples.

AAV vector production can be accomplished by cotransfection of the packaging plasmid. Heilbronn. The cell line supplies the deleted AAV genes rep and cap and the required helper functions. Adenovirus helper genes VA-RNA, E2A and E4 are transfected with AAV rep and cap genes together on two separate plasmids or a single helper construct. Recombinant AAV vector plasmids in which the AAV capsid gene is replaced by an ITR-surrounded transgene expression cassette (comprising the gene of interest, e.g., GJB2 nucleic acid; promoter; and minimal regulatory elements) are also transfected. These packaging plasmids are typically transfected into 293 cells, a human cell line constitutively expressing the remaining essential Ad helper genes E1A and E1B. This results in the amplification and packaging of AAV vectors carrying the gene of interest.

A number of AAV serotypes have been identified, including 12 human serotypes and greater than 100 serotypes from non-human primates. Howarth et al Cell Biol Toxicol: 1-10 (2010). AAV vectors of the present disclosure may comprise capsid sequences derived from AAV of any known serotype. As used herein, "known serotypes" encompass capsid mutants that can be produced using methods known in the art. Such methods include, for example, genetic manipulation of viral capsid sequences, domain exchange of exposed surfaces of capsid regions of different serotypes, and generation of AAV chimeras using techniques such as marker rescue. See Bowles et al Marker rescue of adeno-associated viruses (AAV) capsid variants: a novel approach for chimeric AAV production. Journal of Virology,77 (1): 423-432 (2003), and references cited therein. Furthermore, AAV vectors of the present disclosure may comprise ITRs derived from AAV of any known serotype. Preferably, the ITRs are derived from one of the human serotypes AAV1 to AAV 12. In some embodiments of the disclosure, a pseudotyping method is employed in which the genome of one ITR serotype is packaged into a different serotype capsid.

According to some embodiments, the capsid sequence is derived from one of human serotypes AAV1 to AAV 12. According to some embodiments, the capsid sequence is derived from serotype AAV2. According to some embodiments, the capsid sequence is derived from an AAV2 variant having a high tropism for targeting inner ear tissue or cells, such as supporting cells (e.g., outer hair cells, inner hair cells, hansen cells, dai Tesi cells, columnar cells, inner finger cells, outer finger/limbic cells, intra-cochlear hair and outer hair cells, spiral ganglion neurons, vestibular hair cells, vestibular supporting cells, and vestibular ganglion neurons). In certain embodiments, mutating the AAV capsid polypeptide results in an increased level of rAAV tropism and/or transduction efficiency in inner ear tissue or cells selected from the group consisting of: cells of the outer wall or spiral ligament, support cells of the organ of coti, fibroblasts of the spiral ligament, claus cells, burchet cells, spiral bulge cells, vestibular support cells, hansen cells, dai Tesi cells, column cells, inner finger cells, outer finger cells, limbic cells, intra-cochlear hair cells, extra-cochlear hair cells, spiral ganglion neurons, vestibular hair cells, vestibular support cells, and/or vestibular ganglion neurons. Capsids suitable for this purpose include AAV2 and AAV2 variants, including AAV2-tYF, AAV2-MeB, AAV2-P2V2, AAV2-MeBtYFTV, AAV2-P2V6; and AAV5, AAV8, and Anc80L65. According to some embodiments, the variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity to said amino acid sequence.

According to some embodiments, the recombinant AAV vector may be directly targeted by: genetic manipulation of viral capsid sequences, particularly in the circular region of AAV three-dimensional structures; or domain exchange of exposed surfaces of capsid regions of different serotypes; or AAV chimeras are generated using techniques such as marker rescue. See Bowles et al Marker rescue of adeno-associated viruses (AAV) capsid variants: a novel approach for chimeric AAV production. Journal of Virology,77 (1): 423-432 (2003), and references cited therein.

A possible protocol for the production, purification and characterization of recombinant AAV (rAAV) vectors is provided in Choi et al. Generally, the following steps are involved: design of transgenic expression cassettes, design of capsid sequences for targeting specific receptors, generation of adenovirus-free rAAV vectors, purification and titers. These steps are summarized below and described in detail in Choi et al.

The transgene expression cassette may be a single stranded AAV (ssav) vector or a "dimer" or self-complementary AAV (scAAV) vector packaged as a pseudo-double stranded transgene. Choi et al; howarth et al. The use of traditional ssav vectors typically results in slow onset of gene expression (from days to weeks until a platform of transgene expression is reached) due to the need to convert single stranded AAV DNA to double stranded DNA. In contrast, scAAV vectors show that gene expression begins within hours and reaches a plateau within days after resting cell transduction. Heilbronn. According to some embodiments, a scAAV is used, wherein the scAAV has a rapid onset of transduction and increased stability compared to single stranded AAV. Alternatively, the transgene expression cassette may be split between two AAV vectors, which allows for longer constructs to be delivered. See, e.g., daya s. And Berns, k.i., gene therapy using adeno-associated viruses v.clinical Microbiology Reviews,21 (4): 583-593 (2008) (hereinafter Daya et al). ssAAV vectors can be constructed by digesting a suitable plasmid (such as, for example, a plasmid containing the GJB2 gene) with a restriction endonuclease to remove the rep fragment and cap fragment, and gel purifying the AAVwt-ITR-containing plasmid backbone. Choi et al. Subsequently, the desired transgene expression cassette can be inserted between appropriate restriction sites to construct a single stranded rAAV vector plasmid. scAAV vectors can be constructed as described in Choi et al.

Then, large scale plasmid preparations (at least 1 mg) of rAAV vectors and suitable AAV helper plasmids and pXX6Ad helper plasmids can be purified by double CsCl gradient fractionation. Choi et al. Suitable AAV helper plasmids may be selected from the pXR series pXR1 to pXR5, which allow cross-packing of AAV2ITR genomes into capsids of AAV serotypes 1 to 5, respectively. The appropriate capsid may be selected based on the efficiency with which the capsid targets the cells of interest (e.g., inner ear tissue and cells). Known methods of altering genome (i.e., transgene expression cassette) length and AAV capsids can be employed to improve expression and/or gene transfer to a particular cell type (e.g., cone cells). See, e.g., yang GS, viruses-mediated transduction of murine retina with adeno-associated viruses: effects of viral capsid and genome size. Journal of Virology,76 (15): 7651-7660. According to some embodiments, the variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity to said amino acid sequence.

Next, 293 cells were transfected with pXX6 helper plasmid, rAAV vector plasmid and AAV helper plasmid. Choi et al. Subsequently, the fractionated cell lysate is subjected to a multi-step process of rAAV purification followed by CsCl gradient purification or heparin sepharose column purification. Production and quantification of rAAV virions can be determined using a dot blot assay. In vitro transduction of rAAV in cell culture can be used to verify viral infectivity and expression cassette functionality.

In addition to the methods described in Choi et al, various other transfection methods for producing AAV may also be used in the context of the present disclosure. For example, transient transfection methods are available, including methods that rely on calcium phosphate precipitation protocols.

In addition to laboratory scale methods for producing rAAV vectors, the present disclosure may also utilize techniques known in the art for bioreactor scale production of AAV vectors, including, for example, heilbronn; clement, N. et al, large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufactu ring for clinical publications. Human Gene Therapy,20:796-606.

Advances toward achieving the desired goal of scalable production systems that can produce large amounts of clinical grade rAAV vectors have been mainly achieved in production systems that utilize transfection as a means of delivering genetic elements required for rAAV production in cells. For example, the removal of contaminating adenovirus helper has been circumvented by replacing adenovirus infection with plasmid transfection in a three-plasmid transfection system in which the third plasmid contains a nucleic acid sequence encoding adenovirus helper protein (Xiao et al, 1998), and improvements in the two-plasmid transfection system have also simplified the production process and improved rAAV vector production efficiency (Grimm et al, 1998).

Several strategies for increasing rAAV yields from cultured mammalian cells are based on the development of specialized production cells created by genetic engineering. In one approach, large-scale production of rAAV is achieved by using genetically engineered "proviral" cell lines in which the inserted AAV genome can be "rescued" by infecting the cells with helper adenovirus or HSV. Proviral cell lines can be rescued by simple adenovirus infection, providing increased efficiency relative to transfection protocols.

The second cell-based approach to increasing rAAV yield from cells involves the use of genetically engineered "packaging" cell lines that have AAV rep and cap genes, or both rep-cap and ITR genes of interest, in their genomes (Qiao et al 2002). In the former method, to produce a rAAV, a packaging cell line is infected or transfected with helper functions and with AAV ITR-GOI elements. The latter method requires infection or transfection of cells with only helper functions. Generally, rAAV production using packaging cell lines is initiated by infecting cells with wild-type adenovirus or recombinant adenovirus. Since the packaging cell contains the rep gene and cap gene, it is not necessary to supply these elements exogenously.

rAAV yields from packaging cell lines have been shown to be higher than those obtained by proviral cell line rescue or transfection protocols.

The yields of rAAV have been improved using methods based on the delivery of helper functions from Herpes Simplex Virus (HSV) using recombinant HSV amplification subsystems. Although initial transfer (repotted) provided a modest level of rAAV vector yield of about 150-500 viral genomes (vg) per cell (Conway et al, 1997), recent improvements to the rHSV amplicon-based system have provided significantly higher rAAV vg yields and infectious particles (ip)/cells (Feudner et al, 2002). The amplification subsystem is replication defective in nature; however, the use of "hollowed" vectors, replication competent (rcHSV) or replication defective rHSV will still introduce immunogenic HSV components into the rAAV production system. Thus, appropriate assays for these components and corresponding purification schemes for removal of the compounds were performed.

In addition to these methods, described herein are methods of producing recombinant AAV virions in mammalian cells, comprising co-infecting mammalian cells capable of suspension growth with a first recombinant herpesvirus comprising a nucleic acid sequence encoding an AAV rep gene and an AAV cap gene each operably linked to a promoter, and a second recombinant herpesvirus comprising a gene (e.g., a GJB2 gene) and a promoter operably linked to the gene (e.g., a GJB2 gene) flanked by AAV inverted terminal repeats to facilitate packaging of the gene of interest, and allowing the virus to infect mammalian cells, thereby producing recombinant AAV virions in mammalian cells. In some embodiments, an AAV cap gene encodes an AAV variant capsid polypeptide as described herein (e.g., table 1), e.g., the AAV variant capsid polypeptide exhibits an increased propensity in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide.

Any type of mammalian cell capable of supporting replication of the herpes virus is suitable for use in accordance with the methods of the present disclosure described herein. Thus, mammalian cells may be considered host cells for replication of herpes viruses as described in the methods herein. The present disclosure contemplates any cell type that is used as a host cell, so long as the cell is capable of supporting replication of the herpes virus. Examples of suitable genetically unmodified mammalian cells include, but are not limited to, cell lines such as HEK-293 (293), vero, RD, BHK-21, HT-1080, A549, cos-7, ARPE-19 and MRC-5.

The host cells used in various embodiments of the present disclosure may be derived from, for example, mammalian cells, such as human embryonic kidney cells or primate cells. Other cell types may include, but are not limited to, BHK cells, vero cells, CHO cells, or any eukaryotic cell for which tissue culture techniques have been established, so long as the cell is herpes virus-accepting. The term "herpes virus-received" means that the herpes virus or herpes virus vector is capable of completing the whole intracellular viral life cycle within the cellular environment. In certain embodiments, the methods are performed in a suspension grown mammalian cell line BHK. The host cells may be derived from existing cell lines, for example from BHK cell lines, or developed de novo.

The methods for producing a rAAV gene construct described herein further comprise producing a recombinant AAV virion in a mammalian cell by a method comprising co-infecting a mammalian cell capable of suspension growth with a first recombinant herpesvirus comprising nucleic acid encoding an AAV rep gene and an AAV cap gene each operably linked to a promoter, and (ii) a second recombinant herpesvirus comprising a gene (e.g., GJB 2) and a promoter operably linked to the gene (e.g., GJB2 gene); and allowing the virus to infect the mammalian cell, thereby producing recombinant AAV virions in the mammalian cell. As described herein, a herpes virus is a virus selected from the group consisting of: cytomegalovirus (CMV), herpes Simplex Virus (HSV), varicella Zoster Virus (VZV) and epstein-barr virus (EBV). Recombinant herpes viruses are replication-defective. According to some embodiments, the AAV cap gene has a serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, anc80L65, including variants or hybrids (e.g., capsid hybrids of two or more serotypes). According to some embodiments, an AAV cap gene encodes an AAV variant capsid polypeptide as described herein (e.g., table 1), e.g., the AAV variant capsid polypeptide exhibits an increased propensity in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide.

U.S. patent application publication No. 2007/0202587, the entire contents of which are incorporated herein by reference, describes the required elements of a rAAV production system. Recombinant AAV is produced in vitro by introducing the genetic construct into cells called producer cells. Known systems for producing rAAV employ three basic elements: (1) a gene cassette comprising a gene of interest, (2) a gene cassette comprising an AAV rep gene and a cap gene, and (3) a source of "helper" viral proteins.

The first gene cassette was constructed with the gene of interest flanked by Inverted Terminal Repeats (ITRs) from AAV. The function of the ITR is to integrate the gene of interest directly into the host cell genome and to play an important role in encapsidation of the recombinant genome. Hermonat and Muzyczka,1984; samulski et al 1983. The second gene cassette comprises AAV genes rep and cap encoding proteins required for replication and packaging of the rAAV. The Rep gene encodes four proteins (Rep 78, rep 68, rep 52, and Rep 40) required for DNA replication. The cap gene encodes three structural proteins (VP 1, VP2, and VP 3) that make up the viral capsid. Muzyczka and Berns,2001.

The third element is necessary because AAV does not replicate itself. Helper functions are protein products from helper DNA viruses that create a cellular environment that facilitates efficient replication and packaging of rAAV. Traditionally, adenoviruses (Ad) have been used to provide helper functions for rAAV, but herpesviruses may also provide these functions as described herein.

The production of rAAV vectors for gene therapy is performed in vitro using a suitable producer cell line, such as BHK cells grown in suspension. Other cell lines suitable for use in the present disclosure include HEK-293 (293), vero, RD, BHK-21, HT-1080, A549, cos-7, ARPE-19 and MRC-5.

Any cell type may be used as a host cell, provided that the cell is capable of supporting replication of the herpes virus. Those skilled in the art will be familiar with a wide range of host cells that can be used to produce herpesviruses from host cells. Examples of suitable genetically unmodified mammalian host cells may include, for example, but are not limited to, cell lines such as HEK-293 (293), vero, RD, BHK-21, HT-1080, A549, cos-7, ARPE-19 and MRC-5.

The host cell may be adapted to be grown in suspension culture. The host cell may be a Baby Hamster Kidney (BHK) cell. The suspension grown BHK cell lines were derived from adaptation of adherent BHK cell lines. Both cell lines are commercially available.

One strategy to provide all the necessary elements for rAAV production is to use two plasmids and a helper virus. This method relies on transfecting producer cells with a plasmid containing a gene cassette encoding the necessary gene product, and infecting cells with Ad to provide helper functions. This system employs a plasmid with two different gene cassettes. The first plasmid is a proviral plasmid encoding the recombinant DNA to be packaged as a rAAV. The second plasmid is a plasmid encoding the rep gene and cap gene. To introduce these various elements into cells, the prize cells were infected with Ad and transfected with two plasmids. The gene products provided by Ad are encoded by the genes E1a, E1b, E2a, E4orf6 and Va. Samulski et al 1998: hauswirth et al 2000; muzyczka and Burns,2001. Alternatively, in a more recent approach, the Ad infection step can be replaced by transfection of an adenovirus "helper plasmid" containing the VA gene, E2A gene and E4 gene. Xiao et al 1998; matsushita et al 1998.

While Ad is typically used as a helper virus for rAAV production, other DNA viruses, such as herpes simplex virus type 1 (HSV-1), may also be used. The minimal HSV-1 gene set required for AAV2 replication and packaging has been identified and includes early genes UL5, UL8, UL52 and UL29.Muzyczka and Burns,2001. These genes encode components of the HSV-1 core replication mechanism, namely helicases, primases, primase helper proteins and single stranded DNA binding proteins. Knipe,1989; weller,1991. This rAAV helper property of HSV-1 has been used to design and construct recombinant herpes virus vectors capable of providing helper virus gene products required for rAAV production. Conway et al 1999.

The host cell may be adapted to be grown in suspension culture. In certain embodiments of the present disclosure, the host cell is a Baby Hamster Kidney (BHK) cell. The suspension grown BHK cell lines were derived from adaptation of adherent BHK cell lines. Both cell lines are commercially available.

rHSV-based rAAV manufacturing process

Described herein are methods of producing recombinant AAV virions in suspension-grown cells. According to some embodiments, an AAV particle comprises an AAV variant capsid polypeptide as described herein (e.g., table 1), e.g., the AAV variant capsid polypeptide exhibits increased tropism and/or transduction in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide. Suspension or non-anchorage dependent cultures from continuously established cell lines are the most widely used means for mass production of cells and cell products. Large scale suspension culture based on fermentation technology has significant advantages for the production of mammalian cell products. Homogeneous conditions may be provided in the bioreactor that allow for accurate monitoring and control of temperature, dissolved oxygen and pH, and ensure that a representative sample of the culture may be collected. The rHSV vector used readily propagated to high titers on the recipient cell lines in both tissue culture flasks and bioreactors and provided a production regimen suitable for scaling up the level of virus production required for clinical and market production.

Cell culture in stirred tank bioreactors provides a very high volume specific culture surface area and has been used to produce viral vaccines (Griffiths, 1986). Furthermore, stirred tank bioreactors have proven to be scalable in industry. One example is a multi-plate CELL CUBE CELL culture system. The ability to produce infectious viral vectors is of increasing importance to the pharmaceutical industry, especially in the context of gene therapy.

The cell growth according to the methods described herein can be performed in a bioreactor that allows for large scale production of fully bioactive cells capable of being infected with the herpesvirus vectors of the present disclosure. Bioreactors have been widely used to produce biological products from both suspension and anchorage dependent animal cell cultures. Most large scale suspension cultures operate as batch or fed-batch processes because they are most intuitive for operation and scale-up. However, continuous processes based on chemostat or perfusion principles are available. The bioreactor system may be configured to include a system that allows for exchange of media. For example, a filter may be incorporated into the bioreactor system to allow separation of cells from the used media to facilitate media exchange. In some embodiments of the methods of the invention for producing a herpes virus, the medium exchange and infusion is initiated on the day of cell growth. For example, medium exchange and infusion may begin on day 3 of cell growth. The filter may be external to the bioreactor or internal to the bioreactor.

Methods for producing recombinant AAV virions can include: co-infecting the suspension cells with a first recombinant herpesvirus comprising nucleic acids encoding an AAV rep gene and an AAV cap gene each operably linked to a promoter, and a second recombinant herpesvirus comprising a gene construct (e.g., a GJB2 gene construct), and a promoter operably linked to the gene of interest; and allowing the cell to produce the recombinant AAV virion, thereby producing the recombinant AAV virion. The cells may be HEK-293 (293), vero, RD, BHK-21, HT-1080, A549, cos-7, ARPE-19 and MRC-5. According to some embodiments, the cap gene may be selected from AAV having a serotype selected from the group consisting of: AAV1, AAV2, AAV-, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, anc80L65, including variants or hybrids thereof (e.g., capsid hybrids of two or more serotypes). According to some embodiments, an AAV cap gene encodes an AAV variant capsid polypeptide as described herein (e.g., table 1), e.g., the AAV variant capsid polypeptide exhibits increased tropism and/or transduction in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide. The cells may be infected at a combined multiplicity of infection (MOI) of between 3 and 14. The first herpesvirus and the second herpesvirus may be viruses selected from the group consisting of: cytomegalovirus (CMV), herpes Simplex Virus (HSV), varicella Zoster Virus (VZV), and epstein-barr virus (EBV). The herpes virus may be replication defective. Co-infection may be simultaneous.

Methods for producing recombinant AAV virions in mammalian cells can include co-infecting suspension cells with a first recombinant herpesvirus comprising nucleic acids encoding an AAV rep gene and an AAV cap gene each operably linked to a promoter and a second recombinant herpesvirus comprising a gene construct (e.g., a GJB2 gene construct) and a promoter operably linked to the gene construct (e.g., a GJB2 gene construct); and allowing the cells to proliferate, thereby producing recombinant AAV virions, whereby the number of virions produced is equal to or greater than the number of virions grown in the same number of cells under adherent conditions. The cells may be HEK-293 (293), vero, RD, BHK-21, HT-1080, A549, cos-7, ARPE-19 and MRC-5. The cap gene may be selected from AAV having a serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, anc80L65, including variants or hybrids thereof (e.g., capsid hybrids of two or more serotypes). According to some embodiments, an AAV cap gene encodes an AAV variant capsid polypeptide as described herein (e.g., table 1), e.g., the AAV variant capsid polypeptide exhibits increased tropism and/or transduction in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide. The cells may be infected at a combined multiplicity of infection (MOI) of between 3 and 14. The first herpesvirus and the second herpesvirus may be viruses selected from the group consisting of: cytomegalovirus (CMV), herpes Simplex Virus (HSV), varicella Zoster Virus (VZV), and epstein-barr virus (EBV). The herpes virus may be replication defective. Co-infection may be simultaneous.

Disclosed is a method for delivering a nucleic acid sequence encoding a therapeutic protein to a suspension cell, the method comprising: co-infecting a BHK cell with a first recombinant herpesvirus comprising nucleic acids encoding an AAV rep gene and an AAV cap gene each operably linked to a promoter, and a second herpesvirus comprising a gene construct (e.g., a GJB2 gene construct), wherein the gene of interest comprises a therapeutic protein coding sequence, and a promoter operably linked to the gene (e.g., a GJB2 gene); and wherein the cells can be infected at a combined multiplicity of infection (MOI) of between 3 and 14; and allowing the virus to infect the cell and express the therapeutic protein, thereby delivering a nucleic acid sequence encoding the therapeutic protein to the cell. The cells may be HEK-293 (293), vero, RD, BHK-21, HT-1080, A549, cos-7, ARPE-19 and MRC-5. See, for example, U.S. patent No. 9,783,826. According to some embodiments, an AAV cap gene encodes an AAV variant capsid polypeptide as described herein (e.g., table 1), e.g., the AAV variant capsid polypeptide exhibits increased tropism and/or transduction in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide.

Therapeutic method

AAV and gene therapy

Gene therapy refers to the treatment of genetic or acquired diseases by replacing, altering or supplementing the genes responsible for the disease. This is achieved by introducing one or more correction genes into the host cell, typically by means of a carrier or vector. According to some embodiments, a rAAV described herein comprises an AAV variant capsid polypeptide (e.g., table 1), e.g., that exhibits increased tropism and/or transduction in inner ear tissue or cells as compared to a non-variant AAV capsid polypeptide.

According to some embodiments, the GJB2 AAV construct provides a gene therapy vehicle for treating the DFNB1 deafness phenotype. The GJB2 AAV gene therapy constructs and methods of use described herein provide therapies for DFNB1 deafness, a long unmet need because no gene therapy-based therapies are available for patients.

Method for treating hearing loss

Provided herein are methods useful for treating a hearing disorder or preventing hearing loss (or further hearing loss) in a subject. Delivery of one or more of the nucleic acids described herein to cells within the inner ear (e.g., in the cochlea) (or cells of the cochlea or cochlear cells) can be used to treat hearing disorders, which are generally defined as partial hearing loss or complete deafness.

According to some embodiments, provided herein are methods of treating non-symptomatic hearing loss and deafness characterized by congenital progressive and non-progressive mild to severe sensorineural hearing impairment using GJB2 AAV-based gene therapy. The GJB2 AAV gene therapy constructs and methods of use described herein provide examples of long-term (e.g., lifelong) therapies for correcting congenital deafness by gene supplementation. Importantly, the GJB2 AAV gene therapy constructs and methods of use described herein will preserve natural hearing, whereas cochlear implants cannot.

The methods described herein allow for the production of recombinant AAV virions in mammalian cells, including co-infecting mammalian cells capable of suspension growth with a first recombinant herpesvirus and a second recombinant herpesvirus comprising a GJB2 gene construct that is therapeutically valuable in treating hereditary hearing loss.

GJB2 encodes the major gap junction protein connexin 26 (Cx 26), which Cx26 associates with other gap junction proteins, providing a broad network, allowing intercellular coupling between non-sensory cells in the cochlea. In addition, GJB2/Cx26 can play an important role in the formation of gap junction networks required for normal hearing by maintaining potassium gradient homeostasis in the coti's organ. Individuals with autosomal recessive mutations in GJB2 exhibit a DFNB1 deafness phenotype and this accounts for nearly half of all cases of hereditary hearing loss, with prevalence of about 2-3 people per 1000 newborns. These appear as homozygous or complex heterozygous mutations (del castullo and del castullo, front Mol neurosci.2017; 10:428). Furthermore, heterozygous carriers at risk of accelerated age-related hearing loss exist (del casttillo and del casttillo, front Mol neurosci.2017; 10:428).

In one aspect, the disclosure relates to a novel rAAV-based gene therapy for treating or preventing hereditary hearing loss (about 45% of all congenital deafness cases) caused by GJB2 mutations. Furthermore, the present disclosure relates to the treatment or prevention of hearing loss associated with heterozygous mutations. The rAAV constructs detailed in this disclosure will correspond to pre-or post-speech therapies for preventing or treating both the autosomal recessive GJB2 mutant (DFNB 1) and the autosomal dominant GJB2 mutant (DFNA 3A) and are administered by any method necessary for intra-cochlear delivery. The genetic constructs described herein may be used in methods and/or compositions for treating and/or preventing DFNB1 deafness.

According to some embodiments, the GJB2 AAV gene therapy is administered to a subject who has developed severe hearing loss. According to some embodiments, the GJB2 AAV gene therapy is administered prior to the subject developing hearing loss. According to some embodiments, the subject is diagnosed with DFNB1 by a molecular genetic test for identifying a GJB2 mutation that causes deafness. According to some embodiments, the subject has family members with non-syndromic hearing loss and deafness. According to some embodiments, the subject is a child. According to some embodiments, the subject is an infant.

The rAAV constructs described herein transduce inner ear cells and tissues, e.g., cochlear cells, with greater efficiency than conventional AAV vectors. According to some embodiments, the compositions and methods described herein enable efficient delivery of nucleic acids to inner ear cells, e.g., cochlear cells. According to some embodiments, the compositions and methods described herein enable delivery of and expression of transgenes in at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of inner ear cells (e.g., cochlear cells). According to some embodiments, the compositions and methods described herein enable delivery of and expression of transgenes in at least 50% (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of inner ear cells (e.g., cochlear cells). According to some embodiments, the compositions and methods described herein enable delivery of and expression of transgenes in at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of inner ear cells (e.g., cochlear cells). According to some embodiments, the compositions and methods described herein enable delivery of and expression of transgenes in at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of inner ear cells (e.g., cochlear cells).

The rAAV constructs described herein transduce auditory hair cells, e.g., inner hair cells and/or outer hair cells, with greater efficiency than conventional AAV vectors. According to some embodiments, the compositions and methods described herein enable efficient delivery of nucleic acids to auditory hair cells, e.g., inner hair cells and/or outer hair cells. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to or expression of the transgene in at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the inner hair cells, or delivery of the transgene to or expression of the transgene in at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the outer hair cells. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to or expression of at least 50% (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of an inner hair cell, or delivery of a transgene to or expression of at least 50% (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of an outer hair cell. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to or expression of at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of an inner hair cell or delivery of a transgene to or expression of at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of an outer hair cell. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to or expression of at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of inner hair cells or delivery of a transgene to or expression of at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of outer hair cells.

The rAAV constructs described herein transduce inner ear support cells with greater efficiency than conventional AAV vectors. According to some embodiments, the compositions and methods described herein enable efficient delivery of nucleic acids to inner ear supporting cells. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to and expression of a transgene in at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of inner ear support cells. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to and expression of a transgene in at least 50% (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the inner ear support cells. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to and expression of the transgene in at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the inner ear support cells. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to and expression of the transgene in at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the inner ear support cells.

According to some embodiments, the nucleic acid sequences described herein are introduced directly into the cell prior to in vivo administration of the resulting recombinant cells, with the nucleic acid sequences expressed to produce the encoded product. This can be accomplished by any of a number of methods known in the art, for example, by methods such as electroporation, lipofection, calcium phosphate mediated transfection.

Accordingly, provided herein are methods for treating or preventing hearing loss associated with a deficiency in a gene (such as GJB 2). In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising: (i) A variant AAV capsid polypeptide, optionally, the variant AAV capsid polypeptide exhibiting increased tropism in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide; and (ii) a polynucleotide comprising a nucleic acid sequence encoding said gene.

In addition, provided herein are methods for delivering a nucleic acid sequence encoding a gene associated with hearing loss (such as GJB 2) to inner ear tissue or cells. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising: (i) A variant AAV capsid polypeptide, optionally, the variant AAV capsid polypeptide exhibiting increased tropism in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide; and (ii) a polynucleotide comprising a nucleic acid sequence encoding said gene.

The methods provided herein can result in increased expression of the gene in inner ear tissue or cells. According to some embodiments, the methods described herein optionally increase the expression of a gene (e.g., GJB 2) by at least about 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold as compared to the normal expression of the gene. In some embodiments, the methods can result in overexpression of a gene (e.g., GJB 2) in inner ear tissue or cells.

The methods provided herein can result in reduced levels of rAAV neutralizing antibodies (NAb). According to some embodiments, the methods described herein reduce rAAVNab titer levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, optionally as compared to a control level.

The methods provided herein can result in reduced inflammation and/or toxicity levels of the inner ear. According to some embodiments, the methods described herein reduce the inner ear inflammation or toxicity level by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% >, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, optionally as compared to the inner ear inflammation or toxicity level prior to administration. In some embodiments, optionally, the methods provided herein can result in a delay in progression of inner ear inflammation or toxicity, optionally, of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to progression of inner ear inflammation or toxicity prior to administration. In certain embodiments, the inner ear inflammation and/or toxicity level is an inner ear inflammation and/or toxicity level associated with administration of an AAV virion comprising a non-variant AAV capsid. In certain embodiments, the inner ear inflammation and/or toxicity level is an inner ear inflammation and/or toxicity level associated with a potential disease and/or disorder characterized by hearing loss in a subject.

The methods provided herein may result in reduced hair cell loss, degradation, and/or death levels. According to some embodiments, the methods described herein result in at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% reduction in the level of hair cell loss, degradation and/or death, optionally as compared to the level of hair cell loss, degradation and/or death prior to administration.

The methods provided herein can result in reduced levels of spiral ganglion neuron loss, degeneration, and/or death. According to some embodiments, the methods described herein result in at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% reduction in the spiral ganglion neuron loss, degeneration and/or death level, optionally as compared to the spiral ganglion neuron loss, degeneration and/or death level prior to administration.

The methods provided herein may result in various improvements in hearing. The improvement in hearing can be assessed in a number of ways known in the art. For example, physiological testing may be used to objectively determine the functional state of the auditory system, and may be performed at any age. Exemplary physiological tests include the following: auditory brainstem response tests (ABR, also known as BAER, BSER), auditory steady state response tests (ASSR), evoked otoacoustic emissions (EOAE), and immittance tests (tympanostomy, acoustic reflex threshold, acoustic reflex decay).

Auditory brainstem response tests (ABR, also known as BAER, BSER) use stimuli (ticks or pure tones) to evoke electrophysiological responses that originate in the eighth cranial nerve and auditory brainstem and are recorded with surface electrodes.

Auditory steady state response test (ASSR) is similar to ABR in that both are auditory evoked potentials, and they are measured in a similar manner. ASSR uses objective, statistical-based mathematical detection algorithms to detect and define hearing thresholds. ASSR may be obtained using broadband or frequency specific stimuli and may provide hearing threshold differentiation in the severe to very severe range. It is often used to give frequency specific information not given by ABR. Test frequencies of 500Hz, 1000Hz, 2000Hz and 4000Hz are typically used. In some embodiments, the methods provided herein result in improved ASSR responses.

Otoacoustic emissions (EOAE) are sounds originating in the cochlea, which are measured in the external auditory canal using a probe with a microphone and transducer. EOAE reflects primarily the activity of extra-cochlear hair cells over a wide frequency range and is found in ears with hearing sensitivity better than 40-50dB HL. In some embodiments, the methods provided herein result in improved EOAE reactions.

The immittance test (tympanostomy, acoustic reflex threshold, acoustic reflex decay) evaluates the surrounding auditory system, including middle ear pressure, tympanic membrane mobility, eustachian tube function, and mobility of middle ear ossicles. In some embodiments, the methods provided herein result in improved immittance test reactions.

The methods provided herein can result in improved distortion product otoacoustic emission (DPOAE) distribution. For example, DPOAEs may be generated in the cochlea in response to two tones of given frequency and sound pressure level presented in the ear canal. In certain embodiments, DPOAEs can be used as an objective indicator of normally functioning extra-cochlear hair cells. According to some embodiments, the methods described herein result in preventing, delaying or slowing the deterioration of DPOAE distribution.

The methods provided herein can result in improved speech understanding. In some embodiments, the method results may result in preventing, delaying, or slowing the deterioration of speech understanding.

As used herein, a control level may be based on, for example, a level obtained from a subject, optionally a sample from the subject, prior to administration of the rAAV. In some embodiments, the control level is based on: levels resulting from administration of a rAAV that does not contain a variant AAV capsid polypeptide, optionally, wherein the rAAV that does not contain a variant AAV capsid polypeptide comprises a rAAV capsid polypeptide selected from AAV2 and Anc80L 65.

The methods provided herein can result in delivery of a nucleic acid sequence encoding a gene of interest (such as GJB 2) to and expression in cells of the lateral wall or spiral ligament, support cells of the coti organ, fibroblasts of the spiral ligament, claus cells, burt schart cells, spiral bulge cells, vestibular support cells, hansen cells, dai Tesi cells, column cells, inner finger cells, outer finger cells, limbal cells, intra-cochlear hair cells and/or extra-cochlear hair cells, spiral ganglion neurons, vestibular hair cells, vestibular support cells and/or vestibular ganglion neurons. In certain embodiments, the method results in delivery of a nucleic acid sequence encoding a gene of interest (such as GJB 2) to and expression in at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of cells of the lateral wall or spiral ligament, support cells of the organ of kolie, fibroblasts of the spiral ligament, claus cells, burcher cells, spiral convex cells, vestibular support cells, hansen cells, dai Tesi cells, columnar cells, inner finger cells, outer finger cells, limbic cells, intra-cochlear hair cells, extra-cochlear hair cells, spiral ganglion neurons, vestibular hair cells, vestibular ganglion neurons, and/or vestibular ganglion neurons.

Pharmaceutical composition

According to some aspects, the present disclosure provides pharmaceutical compositions comprising any of the AAV described herein, optionally in a pharmaceutically acceptable excipient. For example, the present disclosure provides various compositions comprising an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising: (i) A variant AAV capsid polypeptide, optionally, the variant AAV capsid polypeptide exhibiting increased tropism in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide; and (ii) a polynucleotide comprising a nucleic acid sequence encoding a gene of interest (such as GJB 2).

As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmacologically active substance and may be supplied as a liquid solution or suspension, as an emulsion, or as a solid form suitable for dissolution or suspension in a liquid prior to use. For example, the excipient may be administered in form or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts for altering osmotic pressure, encapsulating agents, pH buffering substances and buffers. Such excipients include any agent suitable for direct delivery to the ear (e.g., inner ear), which can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of a variety of TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Which may include pharmaceutically acceptable salts, e.g., inorganic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and organic acid salts such as acetates, propionates, malonates, benzoates, and the like. An exhaustive discussion of pharmaceutically acceptable excipients is available in REMINGTON' S PHARMACEUTICAL SCIENCES (Mack Pub.Co., N.J.1991).

According to some embodiments, the pharmaceutical composition comprises one or more of bst, PBS, or BSS.

According to some embodiments, the pharmaceutical composition further comprises a histidine buffer.

Although not required, the compositions may optionally be supplied in unit dosage forms suitable for administration of precise amounts.

According to some embodiments, the composition is administered to the subject prior to cochlear implantation.

Application method

Generally, the compositions described herein are formulated for application to the ear. According to some embodiments, the composition is formulated for administration to cells in the Organ of Coti (OC) in the cochlea. Cells in the OC include hansen cells, dai Tesi cells, column cells, inner finger cells, and/or outer finger cells/limbal cells. OC includes two classes of sensory hair cells: an Inner Hair Cell (IHC) that converts mechanical information carried by sound into an electrical signal that is transmitted to a neuronal structure; and Outer Hair Cells (OHCs) for amplifying and tuning cochlear responses, a process required for complex hearing functions. According to some embodiments, the composition is formulated for application to IHC and/or OHC.

Injection into the cochlear canal filled with high potassium endolymphatic fluid may provide direct access to the hair cells. However, changing this fragile fluid environment may destroy intra-cochlear potentials, increasing the risk of injection-related toxicity. Peri-scala tympani and scala vestibuli can be accessed through an oval or round window membrane to fill the peri-scala cochlear space with perilymph. Round window membranes are non-bony openings into the inner ear, are accessible in many animal models, and use of this route to administer viral vectors is well tolerated. In humans, cochlear implant placement conventionally relies on surgical electrode insertion through a round window membrane. According to some embodiments, the composition is administered by injection through a round window membrane. According to some embodiments, the composition is administered by injection into the scala tympani or mid-scala. According to some embodiments, the composition is administered during surgery, for example during a cochlear ostomy or during an ear canal ostomy (canalostomy).

According to some embodiments, the composition is administered to the cochlea or vestibular system, optionally, wherein the delivering comprises administration directly into the cochlea or vestibular system via Round Window Membranes (RWMs), oval windows, or semicircular canals. In some embodiments, the direct administration is by injection. In some embodiments, the administration is intravenous, intraventricular, intracochlear, intrathecal, intramuscular, subcutaneous, or a combination thereof.

By safely and effectively transducing earworm cells as described herein, the methods of the present disclosure can be used to treat an individual, e.g., a human, wherein the transduced cells produce an amount of GJB2 sufficient to restore hearing or vestibular function for an extended period of time (e.g., months, years, decades, life-time)

According to the methods of treatment of the present disclosure, the volume of the carrier delivered may be determined based on characteristics of the subject being treated, such as the age of the subject and the volume of the area to which the carrier is to be delivered. According to some embodiments, the volume of the injected composition is between about 10 μl and about 1000 μl, or between about 10 μl and about 50 μl, or between about 25 μl and about 35 μl, or between about 100 μl and about 1000 μl, or between about 100 μl and about 500 μl, or between about 500 μl and about 1000 μl. According to some embodiments, the volume of the injected composition is greater than about any of the following: 1. Mu.l, 2. Mu.l, 3. Mu.l, 4. Mu.l, 5. Mu.l, 6. Mu.l, 7. Mu.l, 8. Mu.l, 9. Mu.l, 10. Mu.l, 15. Mu.l, 20. Mu.l, 25. Mu.l, 30. Mu.l, 35. Mu.l, 40. Mu.l, 45. Mu.l, 50. Mu.l, 75. Mu.l, 100. Mu.l, 200. Mu.l, 300. Mu.l, 400. Mu.l, 500. Mu.l, 600. Mu.l, 700. Mu.l, 800. Mu.l, 900. Mu.l, or 1mL, or any amount therebetween. According to some embodiments, the volume of the injected composition is at least about any of the following: 1. Mu.l, 2. Mu.l, 3. Mu.l, 4. Mu.l, 5. Mu.l, 6. Mu.l, 7. Mu.l, 8. Mu.l, 9. Mu.l, 10. Mu.l, 15. Mu.l, 20. Mu.l, 25. Mu.l, 30. Mu.l, 35. Mu.l, 40. Mu.l, 45. Mu.l, 50. Mu.l, 75. Mu.l, 100. Mu.l, 200. Mu.l, 300. Mu.l, 400. Mu.l, 500. Mu.l, 600. Mu.l, 700. Mu.l, 800. Mu.l, 900. Mu.l, or 1mL, or any amount therebetween. According to some embodiments, the volume of the injected composition is about any of the following: 1. Mu.l, 2. Mu.l, 3. Mu.l, 4. Mu.l, 5. Mu.l, 6. Mu.l, 7. Mu.l, 8. Mu.l, 9. Mu.l, 10. Mu.l, 15. Mu.l, 20. Mu.l, 25. Mu.l, 30. Mu.l, 35. Mu.l, 40. Mu.l, 45. Mu.l, 50. Mu.l, 75. Mu.l, 100. Mu.l, 200. Mu.l, 300. Mu.l, 400. Mu.l, 500. Mu.l, 600. Mu.l, 700. Mu.l, 800. Mu.l, 900. Mu.l, or 1mL, or any amount therebetween.

According to the treatment methods of the present disclosureThe concentration of the carrier administered may vary depending on the method of manufacture, and may be selected or optimized based on the concentration determined to be therapeutically effective for the particular route of administration. According to some embodiments, the concentration per milliliter of vector genome (vg/ml) is selected from the group consisting of: about 10 ⁸ vg/ml, about 10 ⁹ vg/ml, about 10 ¹⁰ vg/ml, about 10 ¹¹ vg/ml, about 10 ¹² vg/ml, about 10 ¹³ vg/ml and about 10 ¹⁴ vg/ml. In a preferred embodiment, the concentration is at 10 ¹⁰ vg/ml-10 ¹³ In the range of vg/ml.

The effectiveness of the compositions described herein may be monitored by several criteria. For example, after a subject is treated using the methods of the present disclosure, improvement and/or stabilization and/or delay in progression of one or more signs or symptoms, e.g., a disease state, of the subject can be assessed by one or more clinical parameters, including parameters described herein. Examples of such tests are known in the art and include objective as well as subjective (e.g., subject reported) metrics. According to some embodiments, these tests may include, but are not limited to, subjective assessment of Auditory Brainstem Response (ABR) measurements, speech perception, communication patterns, and auditory response recognition.

According to some embodiments, subjects exhibiting non-symptomatic hearing loss and deafness (DFNB 1) are first tested to determine their threshold hearing sensitivity in the hearing range. The subject is then treated with a rAAV composition described herein. A change in threshold hearing level as a function of frequency measured in dB is determined. According to some embodiments, the improvement in hearing is determined as a 5dB to 50dB improvement in threshold hearing sensitivity of at least one ear at any frequency. According to some embodiments, the improvement in hearing is determined as a 10dB to 30dB improvement in threshold hearing sensitivity of at least one ear at any frequency. According to some embodiments, the improvement in hearing is determined as a 10dB to 20dB improvement in threshold hearing sensitivity of the at least one ear at any frequency.

Non-limiting embodiments

1. A method of treating or preventing hearing loss associated with a deficiency in a gene, the method comprising administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising:

(i) A variant AAV capsid polypeptide, optionally, the variant AAV capsid polypeptide exhibiting increased tropism in inner ear tissue or cells, as compared to a non-variant AAV capsid polypeptide; and

(ii) A polynucleotide comprising a nucleic acid sequence encoding said gene.

2. A method of delivering a nucleic acid sequence encoding a gene associated with hearing loss to inner ear tissue or cells, the method comprising administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising:

(ii) A polynucleotide comprising a nucleic acid sequence encoding said gene.

3. The method according to embodiment 1 or 2, wherein the inner ear tissue or cell is cochlear tissue or cell, or vestibular tissue or cell.

4. The method of embodiment 1 or 2, wherein the inner ear tissue or cell is cochlear tissue or cell.

5. The method of any one of the preceding embodiments, wherein the variant AAV capsid polypeptide is any variant AAV capsid polypeptide, optionally selected from the group consisting of: mutating AAV1 capsid polypeptides; mutating AAV2 capsid polypeptides; variant AAV3 capsid polypeptides; variant AAV4 capsid polypeptides; variant AAV5 capsid polypeptides; variant AAV6 capsid polypeptides; variant AAV7 capsid polypeptides; variant AAV8 capsid polypeptides; variant AAV9 capsid polypeptides; variant rh-AAV10 capsid polypeptides; variant AAV10 capsid polypeptides; variant AAV11 capsid polypeptides; variant AAV12 capsid polypeptides; and variant Anc80 capsid polypeptides.

6. The method of any one of the preceding embodiments, wherein the variant AAV capsid polypeptide is a variant AAV2 capsid polypeptide.

7. The method according to any of the preceding embodiments, wherein:

(i) The variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto, optionally wherein the AAV capsid is selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides; and/or

(ii) The variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence homology thereto, optionally wherein the AAV capsid is selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides.

8. The method of any one of the preceding embodiments, wherein the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions, insertions, and/or deletions relative to a wild type AAV2 capsid polypeptide (SEQ ID NO: 18), optionally wherein the one or more amino acid substitutions, insertions, and/or deletions occur at an amino acid residue selected from the group consisting of: q263, S264, Y272, Y444, R487, P451, T454, T455, R459, K490, T491, S492, A493, D494, E499, Y500, T503, K527, E530, E531, Q545, G546, S547, E548, K549, T550, N551, V552, D553, E555, K556, R585, R588, and Y730.

9. The method of embodiment 8, wherein the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions with respect to a wild type AAV2 capsid polypeptide (SEQ ID NO: 18), the one or more amino acid substitutions selected from the group consisting of: q263 263 264 272 444 487 451 454 455 459 490 491 492 493 494 499 500 527, 545 546 546 547 548 548 549 550 550 551 552 555 556 585 588T, and Y730F.

10. The method of any one of the preceding embodiments, wherein the variant AAV capsid polypeptide comprises:

(i) SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or a sequence of any one of seq id no;

(ii) And SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity; or alternatively

(iii) Consists of SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO:32 or SEQ ID NO:34, or a nucleic acid sequence encoding any one of the nucleic acid sequences.

11. The method of any one of embodiments 1-10, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 27.

12. The method of any one of embodiments 1-10, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 29.

13. The method of any one of embodiments 1-10, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 31.

14. The method of any one of embodiments 1-10, wherein the variant AAV capsid polypeptide comprises SEQ ID NO:33, an amino acid sequence of seq id no.

15. The method of any one of embodiments 1-10, wherein the variant AAV capsid polypeptide comprises SEQ ID NO:35, and a sequence of amino acids.

16. The method of any one of the preceding embodiments, wherein the variant AAV capsid polypeptide results in an increase in rAAV chemotaxis level in inner ear tissue or cells, optionally by at least 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, optionally as compared to a non-variant AAV capsid polypeptide.

17. The method of any one of the preceding embodiments, wherein the variant AAV capsid polypeptide results in an increase in rAAV transduction efficiency in inner ear tissue or cells, optionally by at least 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, optionally as compared to a non-variant AAV capsid polypeptide.

18. The method of any one of the preceding embodiments, wherein optionally the method results in an increase in expression of the gene in inner ear tissue or cells, optionally by at least about 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, as compared to normal expression of the gene.

19. The method of any one of the preceding embodiments, wherein the method results in overexpression of GJB2 expression in the inner ear tissue or cells.

20. The method of any one of the preceding embodiments, wherein the method results in a reduction in rAAV neutralizing antibody (NAb) titer level, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, optionally as compared to a control level.

21. The method of any one of the preceding embodiments, wherein the method results in a reduction in the inner ear inflammation or toxicity level, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to the inner ear inflammation or toxicity level prior to administration.

22. The method of any one of the preceding embodiments, wherein the method results in a delay of optionally at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% in inner ear inflammation or toxicity progression, optionally as compared to inner ear inflammation or toxicity progression prior to administration.

23. The method of any one of the preceding embodiments, wherein the method results in a reduction in the level of hair cell loss, degradation, and/or death, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to the level of hair cell loss, degradation, and/or death prior to administration.

24. The method of any one of the preceding embodiments, wherein the method results in a reduction in the spiral ganglion neuron loss, degeneration, and/or death level, optionally by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to the spiral ganglion neuron loss, degeneration, and/or death level prior to administration.

25. The method of any one of the preceding embodiments, wherein the method results in an optional decrease in Auditory Brainstem Response (ABR) threshold at any frequency of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to the ABR threshold level prior to administration.

26. The method according to any of the preceding embodiments, wherein the method results in an improved distortion product otoacoustic emission (DPOAE) profile.

27. The method according to any one of the preceding embodiments, wherein the method results in preventing, delaying or slowing the deterioration of DPOAE distribution.

28. The method of any of the preceding embodiments, wherein the method results in improved speech understanding and/or speech clarity.

29. The method of any one of the preceding embodiments, wherein the method results in preventing, delaying or slowing deterioration of speech understanding and/or speech intelligibility.

30. The method of any one of embodiments 16-29, wherein the control level is based on:

a level obtained from the subject prior to administration of the rAAV, optionally a sample from the subject.

31. The method of any one of embodiments 16-29, wherein the control level is based on:

levels resulting from administration of a rAAV that is free of the variant AAV capsid polypeptide, optionally wherein the rAAV that is free of the variant AAV capsid polypeptide comprises a rAAV capsid polypeptide selected from AAV2 and Anc80L 65.

32. The method of any one of the preceding embodiments, wherein the method results in delivery of a nucleic acid sequence encoding GJB2 to and expression in cells of the lateral wall or spiral ligament, support cells of the organ of coti, fibroblasts of the spiral ligament, claus cells, burt's cells, cells of the spiral lobe, vestibular support cells, hansen cells, dai Tesi cells, column cells, inner finger cells, outer finger cells, and/or limbic cells.

33. The method of any one of the preceding embodiments, wherein the method results in delivery of a nucleic acid sequence encoding GJB2 to and expression in at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of cells of the lateral wall or spiral ligament, support cells of the coti organ, fibroblasts of the spiral ligament, claus cells, bertschel cells, spiral convex cells, vestibular support cells, hansen cells, dai Tesi cells, columnar cells, inner finger cells, outer finger cells, limbic cells, intracochlear hair cells, extra-cochlear hair cells, spiral ganglion neurons, vestibular hair cells, vestibular ganglion support cells and/or vestibular ganglion neurons.

34. The method according to any one of the preceding embodiments, wherein the gene is GJB2.

35. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 is a non-naturally occurring sequence.

36. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 encodes mammalian GJB2.

37. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 encodes human, mouse, non-human primate, or rat GJB2.

38. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 comprises SEQ ID NO:10.

39. the method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 is codon optimized for mammalian expression.

40. The method of embodiment 39, wherein the nucleic acid sequence encoding GJB2 comprises SEQ ID NO: 11. SEQ ID NO:12 or SEQ ID NO:13.

41. the method of embodiment 40, wherein the nucleic acid sequence encoding GJB2 is codon optimized for expression in a human, rat, non-human primate, guinea pig, minipig, pig, cat, sheep, or mouse cell.

42. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 is a cDNA sequence.

43. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked C-terminal tag or N-terminal tag.

44. The method of embodiment 43, wherein the tag is a FLAG tag or an HA tag.

45. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 is operably linked to a promoter.

46. The method of embodiment 45, wherein the promoter is a ubiquitously active CBA, small CBA (smCBA), EF1a, CASI promoter, cochlear support cell promoter, GJB2 expression specific GFAP promoter, small GJB2 promoter, medium GJB2 promoter, large GJB2 promoter, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoter.

47. The method of embodiment 45 or 46, wherein the promoter is optimized to drive sufficient GJB2 expression to treat or prevent hearing loss.

48. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region.

49. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

50. The method of any one of the preceding embodiments, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked polyadenylation signal.

51. The method of embodiment 50, wherein the polyadenylation signal is an SV40 polyadenylation signal.

52. The method of embodiment 50, wherein the polyadenylation signal is a human growth hormone (hGH) polyadenylation signal.

53. The method according to any one of the preceding embodiments, wherein the polynucleotide further comprises a 27 nucleotide hemagglutinin C-terminal tag or a 24 nucleotide Flag tag; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) Ubiquitously active CBA, small CBA (smCBA), EF1a or CASI promoters; (b) Cochlear support cells or GJB2 expression specific 1.68kb GFAP, small/medium/large GJB2 promoters, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoters; operably linked to a 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal.

54. The method of any one of the preceding embodiments, wherein the polynucleotide further comprises an AAV genome cassette, optionally wherein:

(i) The AAV genome cassette is flanked by two sequence-regulating inverted terminal repeats, preferably about 143 bases in length; or (b)

(ii) The AAV genome cassette is flanked by self-complementary AAV (scAAV) genome cassettes consisting of two inverted identical repeat sequences, preferably no longer than 2.4kb, separated by an ITR (itrΔtrs) of about 113 bases that is scAAV-enabled and flanked on both ends by a sequence of about 143 bases that modulates the ITR.

55. The method of any one of the preceding embodiments, wherein the polynucleotide comprises a codon/sequence optimized human GJB2 cDNA with or without a hemagglutinin C-terminal tag or Flag tag, preferably about 27 nucleotides in length, optionally about 0.68 kilobases (kb) in size; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) A ubiquitously active CBA, preferably about 1.7kb in size, a small CBA (smCBA), preferably about 0.96kb in size, EF1a, preferably about 0.81kb in size, or a CASI promoter, preferably about 1.06kb in size; (b) A cochlear support cell or GJB2 expression specific GFAP promoter preferably about 1.68kb in size, a small GJB2 promoter preferably about 0.13kb in size, a medium GJB2 promoter preferably about 0.54kb in size, a large GJB2 promoter preferably about 1.0kb in size, or a sequential combination of 2-3 individual GJB2 expression specific promoters; operably linked to a 0.9kb 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal and further comprising two about 143 base sequence regulatory Inverted Terminal Repeats (ITRs) flanking an AAV genome cassette, or a self-complementary AAV (scAAV) genome cassette consisting of two inverted identical repeats, preferably no longer than 2.4kb, separated by about 113 base scAAV-enabled ITRs (ITR Δtrs) and flanked on both ends by about 143 base sequence regulatory ITRs.

56. The method of any one of the preceding embodiments, wherein the hearing loss is hereditary hearing loss.

57. The method of any one of the preceding embodiments, wherein the hearing loss is DFNB1 hearing loss.

58. The method of any one of the preceding embodiments, wherein the hearing loss is caused by a GJB2 mutation, optionally wherein the mutation is a homozygous mutation or a heterozygous mutation.

59. The method of any one of the preceding embodiments, wherein the hearing loss is caused by an autosomal recessive GJB2 mutant (DFNB 1).

60. The method of any one of the preceding embodiments, wherein the hearing loss is caused by an autosomal dominant GJB2 mutant (DFNA 3A).

61. The method according to any one of the preceding embodiments, wherein the administering is to the cochlea or vestibular system, optionally wherein the delivering comprises directly administering into the cochlea or vestibular system via a Round Window Membrane (RWM), oval window, or semicircular canal.

62. The method of embodiment 61, wherein the direct administration is injection.

63. The method of any one of embodiments 1-60, wherein the administration is intravenous, intraventricular, intracochlear, intrathecal, intramuscular, subcutaneous, or a combination thereof.

64. A composition for treating or preventing hearing loss associated with a deficiency in a gene in a subject in need thereof, wherein the composition comprises a recombinant adeno-associated virus (rAAV) virion comprising:

(ii) A polynucleotide comprising a nucleic acid sequence encoding said gene.

65. A composition for delivering a nucleic acid sequence encoding a gene associated with hearing loss to inner ear tissue or cells of a subject in need thereof, wherein the composition comprises an effective amount of a recombinant adeno-associated virus (rAAV) virion comprising:

(ii) A polynucleotide comprising a nucleic acid sequence encoding said gene.

66. The composition according to embodiment 64 or 65, wherein the inner ear tissue or cell is cochlear tissue or cell, or vestibular tissue or cell.

67. The composition according to embodiment 64 or 65, wherein the inner ear tissue or cell is cochlear tissue or cell.

68. The composition of any one of embodiments 64-67, wherein the variant AAV capsid polypeptide is selected from the group consisting of: mutating AAV1 capsid polypeptides;

mutating AAV2 capsid polypeptides; variant AAV3 capsid polypeptides; variant AAV4 capsid polypeptides; variant AAV5 capsid polypeptides; variant AAV6 capsid polypeptides; variant AAV7 capsid polypeptides; variant AAV8 capsid polypeptides; variant AAV9 capsid polypeptides; variant rh-AAV10 capsid polypeptides; variant AAV10 capsid polypeptides; variant AAV11 capsid polypeptides; and variant AAV12 capsid polypeptides.

69. The composition of any one of embodiments 64-68, wherein the variant AAV capsid polypeptide is a variant AAV2 capsid polypeptide.

70. The composition of any one of embodiments 64-69, wherein the variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto, optionally wherein the AAV capsid is selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides.

71. The composition of any one of embodiments 64-70, wherein the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions, insertions, and/or deletions relative to a wild type AAV2 capsid polypeptide (SEQ ID NO: 1), optionally wherein the one or more amino acid substitutions, insertions, and/or deletions occur at an amino acid residue selected from the group consisting of: q263, S264, Y272, Y444, R487, P451, T454, T455, R459, K490, T491, S492, A493, D494, E499, Y500, T503, K527, E530, E531, Q545, G546, S547, E548, K549, T550, N551, V552, D553, E555, K556, R585, R588, and Y730.

72. The composition of embodiment 71, wherein the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions with respect to a wild type AAV2 capsid polypeptide (SEQ ID NO: 1), the one or more amino acid substitutions selected from the group consisting of: q263 263 264 272 444 487 451 454 455 459 490 491 492 493 494 499 500 527, 545 546 546 547 548 548 549 550 550 551 552 555 556 585 588T, and Y730F.

73. The composition of any one of embodiments 64-72, wherein the variant AAV capsid polypeptide comprises:

74. The composition of any one of embodiments 64-73, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 27.

75. The composition of any one of embodiments 64-73, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 29.

76. The composition of any one of embodiments 64-73, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 31.

77. The composition of any one of embodiments 64-73, wherein the variant AAV capsid polypeptide comprises SEQ ID NO:33, an amino acid sequence of seq id no.

78. The composition of any one of embodiments 64-73, wherein the variant AAV capsid polypeptide comprises SEQ ID NO:35, and a sequence of amino acids.

79. The composition of any one of embodiments 64-78, wherein the gene is GJB2.

80. The composition of any one of embodiments 64-79, wherein the nucleic acid sequence encoding GJB2 is a non-naturally occurring sequence.

81. The composition of any one of embodiments 64-80, wherein the nucleic acid sequence encoding GJB2 encodes mammalian GJB2.

82. The composition of any one of embodiments 64-81, wherein the nucleic acid sequence encoding GJB2 encodes human, mouse, non-human primate, mini-pig or rat GJB2.

83. The composition of any one of embodiments 64-82, wherein the nucleic acid sequence encoding GJB2 comprises SEQ ID NO:10.

84. the composition of any one of embodiments 64-83, wherein the nucleic acid sequence encoding GJB2 is codon optimized for mammalian expression.

85. The composition of embodiment 84, wherein the nucleic acid sequence encoding GJB2 comprises SEQ ID NO: 11. SEQ ID NO:12 or SEQ ID NO:13.

86. the composition of embodiment 84, wherein the nucleic acid sequence encoding GJB2 is codon optimized for expression in a human, rat, non-human primate, guinea pig, mini-pig, cat, sheep, or mouse cell.

87. The composition of any one of embodiments 64-86, wherein the nucleic acid sequence encoding GJB2 is a cDNA sequence.

88. The composition of any one of embodiments 64-87, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked C-terminal tag or N-terminal tag.

89. The composition of embodiment 88, wherein the tag is a FLAG tag or an HA tag.

90. The composition of any one of embodiments 64-89, wherein the nucleic acid sequence encoding GJB2 is operably linked to a promoter.

91. The composition of embodiment 90, wherein the promoter is a ubiquitously active CBA, small CBA (smCBA), EF1a, CASI promoter, cochlear support cell promoter, GJB2 expression specific GFAP promoter, small GJB2 promoter, medium GJB2 promoter, large GJB2 promoter, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoter.

92. The composition of embodiment 90 or 91, wherein the promoter is optimized to drive sufficient GJB2 expression to treat or prevent hearing loss.

93. The composition of any one of embodiments 64-92, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region.

94. The composition of any one of embodiments 64-93, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

95. The composition of any one of embodiments 64-94, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked polyadenylation signal.

96. The composition of embodiment 95, wherein the polyadenylation signal is an SV40 polyadenylation signal.

97. The composition of embodiment 95, wherein the polyadenylation signal is a human growth hormone (hGH) polyadenylation signal.

98. The composition of any one of embodiments 64-97, wherein the polynucleotide further comprises a 27 nucleotide hemagglutinin C-terminal tag or a 24 nucleotide Flag tag; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) Ubiquitously active CBA, small CBA (smCBA), EF1a or CASI promoters; (b) Cochlear support cells or GJB2 expression specific 1.68kb GFAP, small/medium/large GJB2 promoters, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoters; operably linked to a 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal.

99. The composition of any one of embodiments 64-98, wherein the polynucleotide further comprises an AAV genome cassette, optionally wherein:

100. The composition of any one of embodiments 64-99, wherein the polynucleotide comprises a codon/sequence optimized human GJB2 cDNA with or without a hemagglutinin C-terminal tag or Flag tag, preferably about 27 nucleotides in length, optionally about 0.68 kilobases (kb) in size; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) A ubiquitously active CBA, preferably about 1.7kb in size, a small CBA (smCBA), preferably about 0.96kb in size, EF1a, preferably about 0.81kb in size, or a CASI promoter, preferably about 1.06kb in size; (b) A cochlear support cell or GJB2 expression specific GFAP promoter preferably about 1.68kb in size, a small GJB2 promoter preferably about 0.13kb in size, a medium GJB2 promoter preferably about 0.54kb in size, a large GJB2 promoter preferably about 1.0kb in size, or a sequential combination of 2-3 individual GJB2 expression specific promoters; operably linked to a 0.9kb 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal and further comprising two about 143 base sequence regulatory Inverted Terminal Repeats (ITRs) flanking an AAV genome cassette, or a self-complementary AAV (scAAV) genome cassette consisting of two inverted identical repeats, preferably no longer than 2.4kb, separated by about 113 base scAAV-enabled ITRs (ITR Δtrs) and flanked on both ends by about 143 base sequence regulatory ITRs.

101. A method of treating or preventing hearing loss, the method comprising administering to a subject in need thereof an effective amount of the composition of any one of embodiments 64-100.

102. A method of delivering a nucleic acid sequence encoding a gene associated with hearing loss to inner ear tissue or cells, the method comprising administering to a subject in need thereof an effective amount of the composition of any one of embodiments 64-100.

103. A method of delivering a nucleic acid sequence encoding and GJB2 to inner ear tissue or cells, the method comprising administering to a subject in need thereof an effective amount of the composition according to any one of embodiments 64-100.

104. The method or composition of any one of the preceding embodiments, wherein the subject is a mammal.

105. The method or composition of any one of the preceding embodiments, wherein the subject is a primate.

Further embodiments of the present disclosure will now be described with reference to the following examples. The examples contained herein are provided by way of illustration and not by way of limitation.

Non-limiting examples

Example 1 characterization of novel AAV capsid variants for delivery of GJB2 gene therapy for congenital hearing loss

To identify the optimal capsid for gene therapy, the novel and previously described AAV capsid variants were evaluated for ex vivo expression in rat and mouse inner ear tissue and in vivo expression in non-human primate (NHP). Exemplary AAV capsid sequences are provided in table 1.

Table 1.

Amino acid and nucleic acid sequences of exemplary AAV capsid polypeptides

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Method

Animals:P3-P5 Sprague Dawley rat pups were used for all explant experiments. AAV neutralizing antibodies pre-screening of cynomolgus monkeys (non-human primate, "NHP") aged 3-5 years, followed by bilateral dosing by injection into the cochlea via Round Window Membrane (RWM) (10) ¹⁰ vg/ear in a volume of 30 μl). NHPs were euthanized and GFP expression of cochlear sections was assessed by immunohistochemistry 12 weeks after AAV administration.

Cochlear implant:day 0: the dissected whole cochlea was mounted onto Cell-Tak coated mesh inserts and incubated overnight in growth medium containing antibiotics. Day 1: cochlea was transferred to antibiotic-free medium supplemented with 2% fbs and continuously treated with AAV (concentration range) for 120 hours. Day 6: cochlea was fixed in 4% pfa overnight and then immunostained with phalloidin, anti GFP and DAPI.

Adeno-associated virus:all capsid variants use the CBA promoter to drive expression of Green Fluorescent Protein (GFP) reporter constructs to allow for rapid and easy quantitative trending assessment.

GFP quantification:the Z-stacks from the cochlear mid-region were imaged at 63x and stitched together using Zeiss Zen Black software. The region of interest is framed for three relevant regions (spiral ligament, organ of coti and spiral rim). Each of the three regions measured a GFP/anti-GFP pixel density above the threshold. The unit of pixel density measurement is arbitrary.

Comparison of capsid variant tendencies in rat explants

Method for cochlear explant:all ex vivo studies were performed using P3-P5 Sprague Dawley rat pups. Day 0: the dissected whole cochlea was mounted onto Cell-Tak coated mesh inserts and incubated overnight in growth medium containing antibiotics. Day 1: cochlea was transferred to antibiotic-free medium supplemented with 10% fbs and continuously treated with 2e10vg AAV for 120 hours. Day 6: cochlea was fixed in 4% pfa overnight and then immunized withAnd (3) weaving chemical treatment: phalloidin (1:500), anti-GFP (1:250) and DAPI (1:1000) were then mounted in an anti-fade mounting medium. GFP transduction was measured from three regions of interest placed over the coti organ, helical margin or helical ligament. Each region above the z-series measured an anti-GFP pixel density above the threshold and the data provided was expressed in arbitrary units.

Results: figures 19-21 show a comparison of AAV capsid variant GFP coverage normalized to OMY-906 (grey bars) in the helical margin (figure 19), the coti organ (figure 20) and the helical ligament (figure 21). All capsid variants were treated at a dose of 2e10vg. Fig. 22A-22B show representative images as a blended z-stack to highlight the spiral ligament, the organ-supporting cell layer of coti, and the spiral rim. The capsids OMY-911, OMY-912, OMY-914 and OMY-915 perform best overall.

Taken together, these data demonstrate that novel AAV capsids exhibit high levels of transduction in cochlear explants compared to AAV-Anc80 and wild type AAV 2.

Comparison of capsid variant tropism at different doses

Fig. 23-26 show a comparison of AAV capsid variant GFP coverage at different doses. Fig. 23 shows fluorescence images comparing GFP coverage of OMY-912 capsid variants at the following two doses: 2e9vg and 2e10vg. Fig. 24 shows fluorescence images comparing GFP coverage of OMY-915 capsid variants at the following two doses: 2e9vg and 2e10vg. FIG. 25 shows a bar graph comparing GFP coverage of capsid variants in helical margin OMY-912 and OMY-915. FIG. 26 shows a bar graph comparing GFP coverage of capsid variants of OMY-912 and OMY-915 in the Cotinia organ. These data indicate that OMY-915 has higher overall GFP coverage at both doses tested.

Expression of connexin 26 protein in AAV-GJB2-Flag exposed rat cochlear explants

The method comprises the following steps:cochlear explants from P2-P8 rats were treated with medium containing AAV vector carrying FLAG-tagged version of the GJB2 gene. Explants were fixed 48-96 hours after treatment and treated with the targetImmunostaining with antibodies to connexin 26 and FLAG; tissues were also stained with phalloidin and DAPI to label nuclei.

Results:figure 27 shows representative images of cochlear explants exposed to OMY-914 capsid variants expressing FLAG-tagged connexin 26, indicating that this virus correctly delivers connexin 26 protein to membranes and gap junction plaques of cochlear support cells such as in the coti organ, spiral margin and spiral ligament. The FLAG staining clearly overlaps with the expression region of connexin 26, indicating that the FLAG-tagged protein targets the normal connexin 26 expression site. These data indicate that AAV-GJB2-Flag correctly delivers Flag-tagged connexin 26 protein ex vivo to supporting cells of the cochlea and overlaps with endogenous connexin 26 expression.

Expression of the postcochlear, intra-cochlear, injected AAV-GJB2-FIag, connexin 26 protein in young or adult mice

Method: post-natal day 8 (P8) or adult C57BL/6J mice were intra-cochlear injected via round window membranes with 1.0 μl of AAV containing FLAG-tagged versions of the GJB2 gene at a titer of le12 vg/mL. Mice were euthanized 2-6 weeks after injection, cochlea was fixed and immunostained with antibodies to connexin 26 and FLAG; tissues were also stained with phalloidin and DAPI to label nuclei.

Results: figure 28 shows a representative example of intra-cochlear injection of OMY-914 capsid variants containing FLAG-labeled connexin 26 in young mice (P8), demonstrating that this gene therapy product correctly delivers connexin 26 protein to membrane and gap junction plaques of cochlear support cells such as in the coti organ, spiral margin and spiral ligament.

Fig. 29 shows a representative example of intra-cochlear injection of OMY-914 capsid variants expressing FLAG-tagged connexin 26 in young mice (2-3 months old), demonstrating that this viral construct correctly delivers connexin 26 protein to membranes and gap junction plaques of adult cochlear support cells such as in the coti organ, spiral rim and spiral ligament.

These data indicate that AAV-GJB2-Flag correctly delivers the connexin 26 protein in vivo to the supporting cells of the cochlea.

In vivo AAV delivery and tropism in non-human primates

Methods for non-human primate (NHP) trend studies: AAV neutralizing antibodies pre-screening of cynomolgus monkeys ("NHP") aged 3-5 years, followed by bilateral dosing by injection into the cochlea via Round Window Membrane (RWM) (10 ¹⁰ vg/ear in a volume of 30 μl). NHPs were euthanized and GFP expression of cochlear sections was assessed by immunohistochemistry 12 weeks after AAV administration.

Results: the non-human primate (NHP) cochlea was assessed by immunohistochemistry 12 weeks after intracochlear injection of AAV (fig. 30). DAB staining expressed by GFP was pseudo red in fig. 30. Fig. 30 (top panel) shows a low magnification image of the entire cochlea and demonstrates that consistent expression from OMY-913 can be observed from basal to apical throughout the entire cochlea after a single intracochlear injection of AAV administered near the basal via a Round Window Membrane (RWM) injection. FIG. 30 (bottom panel) shows OMY-913 expression observed in the areas associated with GJB2 rescue, including the side wall (LW), the organ of Koroti (OC) supporting cells and the spiral edge (SL).

Taken together, these data demonstrate that in some embodiments, AAV described herein is capable of transducing GJB 2-related cells in the entire NHP cochlea following a single intra-cochlear injection via the Round Window Membrane (RWM).

Based on these results, and without wishing to be bound by any particular theory, it is contemplated herein that AAV capsid variants with similar transduction efficiencies at high doses may exhibit different transduction efficiencies at lower doses. Further, AAV capsid variants exhibit high levels of GFP coverage in cochlear explants compared to AAV-Anc 80. Anc80 shows a different pattern of tropism in rats compared to mouse explants. Furthermore, AAV capsid variants are able to transduce GJB 2-related cells, including supporting cells of the coti organ and the helical margin, and fibroblasts of the helical ligaments, in the entire NHP cochlea following a single RWM injection.

Example 2: AAV-mediated GJB2 gene therapy rescue of hearing loss and cochlear injury in a mouse model of congenital hearing loss caused by conditional connexin 26 knockout

The results from various mouse and human studies have revealed that Cx26 mutations can ultimately lead to almost complete degeneration of cochlear hair cells. Since the constitutive homozygous Cx26 knockout is fatal to the embryo, we used conditional knockout to study the effect of loss of Cx26 protein in inner ear cells. We utilize the method of adding Cx26 ^loxp/loxp Two different conditional knockout lines (Cx 26 cKO) were generated from mice crossed with either the inducible cre mouse line or the constitutive cre mouse line. Using the inducible cre line, we knocked out Cx26 with timing control and observed varying degrees of hearing loss and developmental defects depending on the time of cre induction. When assessed at postnatal day 30, early postnatal cre induction resulted in severe to severe hearing loss in Cx26 cKO mice (fig. 31), while later cre induction resulted in mild to moderate hearing loss that was progressive in nature. Due to embryo cre expression in inner ear tissue, the constitutive creCx26 cKO animals exhibited severe to extremely severe hearing loss across 4kHz, 8kHz, 16kHz, 32kHz and 48kHz frequencies (fig. 32). The availability of these different mouse models enabled us to evaluate AAV-mediated GdB2 gene therapy over a range of severity of hearing loss mimicking a known human phenotype. An exemplary AAV-GJB2 gene therapeutic ("therapeutic a") was constructed using one of the best performing AAV capsids of example 3, a promoter selected from sequences ID 1-6, and GJB2co369 as the gene to be delivered.

In experiments designed to assess the ability of therapeutic agent a to rescue Cx26 cKO phenotype, we performed injection of therapeutic agent a or vehicle via the post-semicircular canal (PSCC) route into two models of postnatal Cx26 cKO mice. Administration of therapeutic a to the inducible cre Cx26 cKO animals significantly restored Cx26 expression compared to vehicle and provided significant hearing improvement across multiple frequencies as measured by ABR (fig. 33A). Furthermore, cx26 cKO mice injected with therapeutic a had greatly improved cochlear morphology relative to mice injected with vehicle, which corresponds to ABR data (fig. 33A-33D). Subcellular localization of CX26 protein was normal in the rescued animals and apparent in the sulcus, claus, hansen, column and Dai Tesi cells as well as spiral bulge, and spiral border and side wall fibroblasts, and these animals showed increased numbers of surviving hair cells relative to vehicle-treated controls.

Example 3 further characterization of novel AAV capsid variants for delivery of GJB2 gene therapy for congenital hearing loss

Mutations in more than 100 genes are causally related to hearing loss. Adeno-associated virus (AAV) has proven to be a safe and effective delivery vehicle for gene therapy, with a past record of positive clinical outcomes. AAV capsids represent key regulatory elements affecting tropism. The present study evaluates the ideal tropism of novel and previously described AAV capsid variants in cochlear explant and non-human primate (NHP) studies to identify the optimal capsid for GJB2 gene therapy. We designed AAV vectors with optimized capsid, promoter and human GJB2 gene elements (therapeutic a) that provide excellent expression of CX26 in cochlear support cells and fibroblasts. We also generated the same AAV vector that expressed FLAG-tagged CX26 to allow identification of virally expressed CX26 (therapeutic a-FLAG). The best performing capsid was then packaged with the GJB2 transgene (therapeutic a) and the pharmacodynamics of the connexin 26 (CX 26) protein was assessed after in vivo administration. As described herein, in a cell-based assay, both therapeutic a and therapeutic a-FLAG induce expression of CX26 that is properly transported to the cell membrane using HeLa cells that do not normally express CX 26. Furthermore, injection of therapeutic a-FLAG into the cochlea of a mouse provides near complete CX26-FLAG expression in the cells of interest throughout the cochlea (from basal to apical).

Therapeutic A enhances FRAP signaling in HeLa cells

Method for FRAP assay:HeLa cells were seeded into 96-well plates at a density of 20,000 cells/well. After 24 hours, cells were transduced with AAV vector (moi=10,000) and adenovirus serotype 5 (moi=5). FRAP assay was performed after another 48 hoursTo allow transgene expression. For the FRAP assay, cells were incubated with calcein-AM (2.5 uM) for 30 minutes, washed in HBSS, and then imaged on an operaetta high content imaging system. The center field was photobleaching using a 40-fold objective and 488nm fluorescent illumination (5000 ms x 12 replicates). Next, the wells were imaged at 10 x magnification for 30 minutes. Fluorescence recovery was measured as the difference in 488nm intensity between the photobleaching area and the surrounding unbleached area, which was then normalized to the surrounding unbleached area to account for fluctuations in light intensity from the xenon arc fluorescent light source.

Therapeutic agent a buffer solutions for intra-cochlear administration were used in the following experiments. The therapeutic agent a construct may optionally comprise a FLAG tag.

Results: heLa cells that do not naturally express CX26 were used to evaluate the functionality of therapeutic a-driven CX26 expression. HeLa cells were incubated with therapeutic a and Ad5 for 5 hours prior to photobleaching, and then restored for 48 hours to allow transgene expression. calcein-AM hydrolyzes after cellular uptake to become fluorescent and impermeable to membranes. Intracellular calcein dyes are known to migrate to adjacent cells via functional gap junctions. The contribution of non-gap junction mediated fluorescence recovery was determined using the pan-gap junction inhibitor gastro-ketone (CBX). Fig. 34 (top panel) shows the photobleaching and image capture timelines for each FRAP test. Fluorescence recovery was measured 30 minutes after photobleaching. FIG. 34 (bottom panel) shows that therapeutic A and therapeutic A-FLAG restored fluorescence faster than untransduced HeLa cells, indicating that the transgene-driven protein may form a functional gap junction. The addition of gastro-resistant ketones reduced most of the fluorescence recovery, indicating that functional gap junctions are the major contributors to cell-to-cell dye transfer.

Taken together, these data demonstrate that therapeutic agent a-mediated delivery of GJB2 or GJB2-FLAG into HeLa cells that do not naturally express CX26 enhances the calcein-AM dye FRAP signal, suggesting that the GJB2 transgene forms a functional gap junction.

Trend of therapeutic A after PSCC delivery in P6 mice pups

The cub injection method comprises the following steps:P6C 57BL/6J mice pups were anesthetized via mild hypothermia and injected with 1 μl of therapeutic agent A-FLAG via the posterior semicircular canal (PSCC). Mice were sacrificed and perfused 25 days after injection and cochlea harvested for downstream immunohistochemical treatment. Detection of virus-expressed CX26-FLAG was performed using FLAG antibody. Cochlea/phalloidin (1:500), anti-FLAG (1:250), anti-CX 26 (1:250) and DAPI (1:1000) were treated with the following antibodies or staining agents.

Results: fig. 35A-35C show that intra-cochlear injection of therapeutic agent a-FLAG (green) into P6 mice pups via the posterior semicircular canal (PSCC) exhibited high transduction relative to endogenous CX26 expression (magenta). CX26-FLAG expression was present at high levels throughout cochlear length and formed membranous plaque-like structures in the sulcus (FIG. 35A), clausius cells (FIG. 35B) and other supporting cell types (FIG. 35C). CX26-FLAG expression is also present in fibroblasts at the helical margin and side wall and is consistent with the morphology and pattern of endogenous CX26 expression.

Taken together, these data demonstrate that direct intra-cochlear delivery of therapeutic agent a-FLAG into P6 mice pups via PSCC injection results in extensive CX26-FLAG transduction in cell types that naturally express CX 26.

Safety and trend profile of therapeutic agent a following intra-cochlear delivery of rwm+pscc windowing in P30 adult mice

Method for adult injection: adult (P30) C57BL/6J mice were injected with 1. Mu.L of either therapeutic agent A or therapeutic agent A-FLAG by direct intra-cochlear injection through the Round Window Membrane (RWM). Prior to injection, a window was opened in the posterior semicircular canal (PSCC) to allow fluid flow. Mice were sacrificed 14 or 42 days post injection and heart perfused with 4% pfa to fix tissue and cochlea harvested for downstream immunohistochemical treatment. Cochlea/phalloidin (1:500), anti-FLAG (1:250), anti-CX 26 (1:250) and DAPI (1:1000) were treated with the following antibodies or staining agents.

Results: FIG. 36 shows that intra-cochlear injection of therapeutic agent A or therapeutic agent A-FLAG via round window membrane with windowing in the posterior semi-canal of the adult mouse of age P30 is safe and does not cause intrauterine cells or disease 42 days post-surgeryInjury of outer hair cells. Figures 37 and 38 show CX26-FLAG transduction (green) in sulcus, claus cells and lateral fibroblasts 14 days post-surgery. CX26-FLAG expression in the sulcus and Claus cells is membranous and forms plaque-like structures similar to endogenous CX 26.

Overall, these data indicate that in vivo intra-cochlear delivery of therapeutic agent a-FLAG by direct intra-cochlear injection via Round Window Membrane (RWM) with post-semicircular canal (PSCC) windowing results in CX26-FLAG transduction with a clean safety profile.

EXAMPLE 4 rescue of hearing loss and cochlear degeneration in clinically relevant induced mouse models of GJB2 congenital hearing loss

Mutations in the GJB2 gene lead to the most common form of congenital non-syndromic deafness in humans. GJB2 encodes gap junction protein connexin 26 (CX 26), which CX26 is necessary for the function of non-sensory cells such as support cells and fibroblasts in the inner ear. Generally, the onset of hearing loss is premalignant and moderate to severe, however, in some subjects hearing loss due to CX26 loss can be mild and progressive. Human temporal bone studies have revealed degeneration of hair cells and supporting cells in the GJB2 mutant cochlea, while spiral ganglion neurons remain largely unaffected. In this study, AAV-based gene therapy candidate therapeutic a was evaluated in an inducible mouse model of GJB2 deficiency.

The method comprises the following steps: since homozygous Cx26 knockout is fatal to embryos in mice, we utilized the method of using the Cx26 gene ^loxp ^/loxp Mouse and tamoxifen inducible cre (Rosa-cre) ^ER ) The effect of Cx26 loss in inner ear cells was investigated by conditional knockout of Cx26 (Cx 26 cKO) generated by mouse line crosses, as shown in fig. 39A and 39B. In Cx26 flox animals, the coding region in exon 2 is flanked by a loxP site in intron 1 and a floxed neo cassette inserted into exon 2. Furthermore, we developed AAV-based gene therapy drug candidates (therapeutic a) after screening for different capsids, promoters and optimized GJB2 codons. We also created a therapeutic agent a-FLAG that expressed FLAG-tagged CX26 and via the intra-cochlear (IC) pathwayIt was administered to wild-type animals to determine the tropism of AAV-derived CX26 in the inner ear by following FLAG expression. To study the efficacy of gene therapy, therapeutic a or vehicle was postnatally administered to Cx26 cKO mice via the IC route. Auditory brainstem response was measured at postnatal day 30 (P) and cochlea was histologically treated to determine morphology and CX26 expression.

Results: adjusting the timing of tamoxifen administration allows for timing control of Cx26 knockouts, resulting in varying degrees of hearing loss and cochlear defects depending on cre activation time. Early postnatal cre activation resulted in severe to extremely severe hearing loss in Cx26 cKO mice at P30, while later cre activation resulted in progressive mild to moderate types of hearing loss. Histological examination revealed little Cx26 expression in Cx26 cKO mice. As shown in fig. 39C, intra-cochlear injection of therapeutic a-FLAG into wild-type mice during postnatal period provided extensive cochlear coverage, including all cell types that naturally express CX 26. Intra-cochlear (IC) administration of therapeutic a-FLAG to untreated mice demonstrated AAV transduction in supporting cells and fibroblasts. As shown in fig. 39D and 39E, cx26 cKO animals injected with therapeutic a exhibited substantial rescue of ABR thresholds across multiple frequencies, restoration of Cx26 expression, and retention of cochlear morphology relative to Cx26 cKO injected with vehicle.

Based on those results, and without wishing to be bound by any particular theory, it is contemplated in the present disclosure that intra-cochlear administration of therapeutic agent a successfully restores hearing, expression of Cx26 in the relevant cochlear cell types, and rescues cochlear morphology in a Cx26 cKO mouse model that partially mimics hearing defects found in human GJB2 patients.

EXAMPLE 5 rescue of hearing loss and cochlear degeneration in clinically relevant mouse models of GJB2 congenital hearing loss

GJB2 mutations represent the most common cause of hereditary hearing loss in humans. GJB2 encodes connexin 26 (CX 26), CX26 being a gap junction protein naturally expressed in fibroblasts of the helical margin and helical ligament, and in supporting cells of the organ of coti. Results from both mouse and human studies indicate that GJB2 mutations lead to an increase in Auditory Brainstem Response (ABR) threshold and support cell and hair cell degeneration. To rescue this GJB2 deficient phenotype, we attempted to deliver a functional copy of GJB2 via intra-cochlear administration of AAV. We first identified a novel class of AAV capsids that efficiently transduce cochlear cell types that naturally express CX 26. We then further optimized the AAV construct by packaging the novel capsid, promoter, and human GJB2 gene elements with and without Flag tags (therapeutic a-Flag and therapeutic a, respectively). Here we used a constitutive Cre mouse model of GJB2 hearing loss to evaluate therapeutic potential of therapeutic a.

The method comprises the following steps: to assess in vivo rescue of CX26 defects, we performed by combining CX26 ^loxp/loxp Hybridization of mice with mice expressing Cre driven by inner ear specific promoter P0 (P0-Cre) generated a mouse model with deletion of inner ear GJB2 as shown in FIGS. 40A and 40B. In Cx26 flox animals, the coding region in exon 2 is flanked by a loxP site in intron 1 and a floxed neo cassette inserted into exon 2. The onset of P0-Cre occurs during embryogenesis and previous studies have reported that plaque formation is disrupted as early as E14.5 in this model. Postnatal mice were injected via the posterior semi-regular tube with 1 μl of therapeutic a, therapeutic a-FLAG or vehicle, followed by assessment of various efficacy endpoints as early as P30. Auditory sensitivity was measured by ABR, after which cochlea was collected and immunohistochemical treatment with anti-CX 26, anti-FLAG and phalloidin to assess chemotaxis and cochlea morphology. To assess chemotaxis, cochlea and lateral wall whole-sample slides were imaged on a Zeiss LSM880 confocal microscope and FLAG or CX26 coverage was quantified.

Results: as shown in fig. 40C, intra-cochlear injection of therapeutic a-FLAG into wild-type mice during postnatal period provided extensive cochlear coverage, including all cell types that naturally express CX 26. In the presence of complete loss of hair cells and supporting cells, and severe to severe hearing loss, P0-Cre mice exhibited a significant decrease in CX26 expression and the presence of a flattened epithelial phenotype. As shown in fig. 40D and 40E, intra-cochlear administration of therapeutic a to P0-Cre mice significantly restored CX26 expression and greatly reduced the occurrence of the flattened epithelial phenotype, increased the number of hair cells present, and more importantly demonstrated improved hearing function across multiple frequencies using ABR measurements.

Based on those results, and without wishing to be bound by any particular theory, it is contemplated in the present disclosure that intra-cochlear injection of therapeutic agent a is capable of rescuing CX26 deficient hearing loss and cochlear lesions.

Example 6 delivery and trending of therapeutic agent A in non-human primate

Methods for non-human primate (NHP) trend studies: AAV neutralizing antibodies pre-screening of cynomolgus monkeys ("NHP") aged 3-5 years, followed by bilateral dosing by injection into the cochlea via Round Window Membrane (RWM) (10 ¹⁰ vg/ear in a volume of 30 μl). NHP was euthanized and cochlear slices were assessed for CX26-FLAG expression by immunohistochemistry 12 weeks after therapeutic a-FLAG administration. Cochlea/phalloidin (1:500), anti-FLAG (1:250), anti-CX 26 (1:250) and DAPI (1:1000) were treated with the following antibodies or staining agents.

Results: fig. 41 shows that intra-cochlear injection of therapeutic agent a-FLAG exhibits high transduction. CX26-FLAG expression is present at high levels in regions associated with GJB2 rescue, including the side wall (LW), the organ of Kotinia (OC) supporting cells and the helical margin (SL). CX26-FLAG expression is consistent with the morphology and pattern of endogenous CX26 expression.

Based on those results, and without wishing to be bound by any particular theory, it is contemplated herein that therapeutic agent a-FLAG is capable of transducing GJB 2-related cells in the entire NHP cochlea following intra-cochlear administration.

Although the disclosure has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it will be understood that certain changes and modifications may be practiced within the scope of the appended claims. Modifications of the above-described modes for carrying out the disclosure that are obvious to those of ordinary skill in the art of gene therapy, molecular biology, otology, and/or related arts, which will be understood in view of the foregoing disclosure, or which are routine practices or implementations of the disclosure, are intended to be within the scope of the appended claims.

All publications (e.g., non-patent documents), patents, patent application publications, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All such publications (e.g., non-patent documents), patents, patent application publications, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent application publication, or patent application was specifically and individually indicated to be incorporated by reference.

While the foregoing disclosure has been described in connection with certain preferred embodiments, it is not so limited.

Claims

1. A composition for treating or preventing hearing loss associated with a deficiency in a gene, wherein the composition comprises a recombinant adeno-associated virus (rAAV) virion comprising:

(ii) A polynucleotide comprising a nucleic acid sequence encoding said gene.

2. The composition of claim 1, wherein the variant AAV capsid polypeptide is selected from the group consisting of: mutating AAV1 capsid polypeptides;

3. The composition of claim 1, wherein the variant AAV capsid polypeptide is a variant AAV2 capsid polypeptide.

4. The composition of claim 3, wherein the variant AAV capsid polypeptide comprises the amino acid sequences listed in table 1, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto, optionally wherein the AAV capsid is selected from the group consisting of: VP1, VP2 or VP3 capsid polypeptides.

5. The composition of claim 4, wherein the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions, insertions, and/or deletions relative to a wild type AAV2 capsid polypeptide (SEQ ID NO: 1), optionally wherein the one or more amino acid substitutions, insertions, and/or deletions occur at an amino acid residue selected from the group consisting of: q263, S264, Y272, Y444, R487, P451, T454, T455, R459, K490, T491, S492, A493, D494, E499, Y500, T503, K527, E530, E531, Q545, G546, S547, E548, K549, T550, N551, V552, D553, E555, K556, R585, R588, and Y730.

6. The composition of claim 4, wherein the variant AAV capsid polypeptide comprises an amino acid sequence having one or more amino acid substitutions with respect to a wild type AAV2 capsid polypeptide (SEQ ID NO: 1), the one or more amino acid substitutions selected from the group consisting of: q263 263 264 272 444 487 451 454 455 459 490 491 492 493 494 499 500 527, 545 546 546 547 548 548 549 550 550 551 552 555 556 585 588T, and Y730F.

7. The composition of claim 1, wherein the variant AAV capsid polypeptide comprises:

8. The composition of claim 1, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO:33 or SEQ ID NO:35, or a sequence of any one of seq id no.

9. The composition of claim 1, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 29.

10. The composition of claim 1, wherein the variant AAV capsid polypeptide comprises SEQ ID NO: 31.

11. The composition of claim 1, wherein the variant AAV capsid polypeptide comprises SEQ ID NO:33, an amino acid sequence of seq id no.

12. The composition of claim 1, wherein the variant AAV capsid polypeptide comprises SEQ ID NO:35, and a sequence of amino acids.

13. The composition of claim 1, wherein the gene is GJB2.

14. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 is a non-naturally occurring sequence.

15. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 encodes mammalian GJB2.

16. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 encodes human, mouse, non-human primate, mini-pig or rat GJB2.

17. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 comprises SEQ ID NO:10.

18. the composition of claim 13, wherein the nucleic acid sequence encoding GJB2 is codon optimized for mammalian expression.

19. The composition of claim 18, wherein the nucleic acid sequence encoding GJB2 comprises SEQ ID NO: 11. SEQ ID NO:12 or SEQ ID NO:13.

20. the composition of claim 18, wherein the nucleic acid sequence encoding GJB2 is codon optimized for expression in a human, rat, non-human primate, guinea pig, minipig, pig, cat, sheep, or mouse cell.

21. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 is a cDNA sequence.

22. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked C-terminal tag or N-terminal tag.

23. The composition of claim 22, wherein the tag is a FLAG tag or an HA tag.

24. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 is operably linked to a promoter.

25. The composition of claim 24, wherein the promoter is a ubiquitously active CBA, small CBA (smCBA), EF1a, CASI promoter, cochlear support cell promoter, GJB2 expression specific GFAP promoter, small GJB2 promoter, medium GJB2 promoter, large GJB2 promoter, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoter.

26. The composition of claim 24, wherein the promoter is optimized to drive sufficient GJB2 expression to treat or prevent hearing loss.

27. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region.

28. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked 3' utr regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

29. The composition of claim 13, wherein the nucleic acid sequence encoding GJB2 further comprises an operably linked polyadenylation signal.

30. The composition of claim 29, wherein the polyadenylation signal is an SV40 polyadenylation signal or a human growth hormone (hGH) polyadenylation signal.

31. The composition of claim 13, wherein the polynucleotide further comprises a 27 nucleotide C-terminal tag of hemagglutinin or a 24 nucleotide Flag tag; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) Ubiquitously active CBA, small CBA (smCBA), EF1a or CASI promoters; (b) Cochlear support cells or GJB2 expression specific 1.68kb GFAP, small/medium/large GJB2 promoters, sequential combination of 2-3 individual GJB2 expression specific promoters, or synthetic promoters; operably linked to a 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal.

32. The composition of claim 13, wherein the polynucleotide further comprises an AAV genome cassette, optionally wherein:

33. The composition of claim 13, wherein the polynucleotide comprises a codon/sequence optimized human GJB2cDNA with or without a hemagglutinin C-terminal tag or Flag tag, preferably about 27 nucleotides in length, optionally about 0.68 kilobases (kb) in size; operably linked to one of the following promoter elements optimized to drive high GJB2 expression: (a) A ubiquitously active CBA, preferably about 1.7kb in size, a small CBA (smCBA), preferably about 0.96kb in size, EF1a, preferably about 0.81kb in size, or a CASI promoter, preferably about 1.06kb in size; (b) A cochlear support cell or GJB2 expression specific GFAP promoter preferably about 1.68kb in size, a small GJB2 promoter preferably about 0.13kb in size, a medium GJB2 promoter preferably about 0.54kb in size, a large GJB2 promoter preferably about 1.0kb in size, or a sequential combination of 2-3 individual GJB2 expression specific promoters; operably linked to a 0.9kb 3' -UTR regulatory region comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by an SV40 or human growth hormone (hGH) polyadenylation signal and further comprising two about 143 base sequence regulatory Inverted Terminal Repeats (ITRs) flanking an AAV genome cassette, or a self-complementary AAV (scAAV) genome cassette consisting of two inverted identical repeats, preferably no longer than 2.4kb, separated by about 113 base scAAV-enabled ITRs (ITR Δtrs) and flanked on both ends by about 143 base sequence regulatory ITRs.