Compositions and Methods for Treating Non-Aging Hearing Impairment in Human Subjects

Abstract

a) A first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) A second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, are provided. The composition comprises a total vg of about 4.1E10 to 4.1E12 or a total vg of about 8.1E10 to 8.1E12, and the use of these compositions in the treatment of hearing loss in a subject is also provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 343,991, filed May 19, 2022, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing The content of the sequence listing (name: 4833.017PC02_Seqlisting_ST26.xml, size: 816,150 bytes, and creation date: May 17, 2023) submitted electronically together with the application is hereby incorporated by reference in its entirety.

[0003] The present disclosure generally relates to the use of nucleic acids for treating hearing loss in a human subject.

Background Art

[0004] The ear is a complex organ and is generally described as including the outer ear, middle ear, inner ear, auditory (hearing) nerve, and the auditory system (which processes sound as it travels from the ear to the brain). In addition to detecting sound, the ear also helps maintain balance. Thus, disorders of the inner ear can cause hearing loss, tinnitus, dizziness, and balance abnormalities.

[0005] Hearing loss is one of the most common sensory deficits in humans and can occur for many reasons. Some people are born with hearing loss, while others gradually lose their hearing over time. Presbycusis (also spelled presbyacusis) is age - related hearing loss. Approximately 36 million U.S. adults report some degree of hearing loss, and one in three people over 60 and half of humans over 85 experience hearing loss.

[0006] Hearing loss may be the result of environmental factors, or a combination of genetic and environmental factors. Tinnitus, that is, the illusionary noise in the auditory system (ringing, buzzing, chirping, humming, or beating), is also present in about half of all people, and they also have a reduced sensitivity / tolerance to certain sound frequencies and volume ranges, known as hyperacusis (also spelled hyperacousis). Environmental causes of hearing loss include certain drugs, certain infections before or after birth, and long-term exposure to loud noise. Hearing loss can be caused by noise, ototoxic drugs, presbycusis, diseases, infections, or cancer affecting specific parts of the ear.

[0007] Approximately 1.5 out of 1,000 children are born with severe hearing loss, and another 2 - 3 per 1,000 children are born with partial hearing loss (Smith et al., 2005, Lancet 365:879 - 890). More than half of these cases are attributable to a genetic basis (Di Domenico, et al., 2011, J.Cell.Physiol.226:2494 - 2499).

[0008] Nonsyndromic hearing loss is hearing loss not associated with other signs and symptoms. In contrast, syndromic hearing loss includes hearing loss that occurs with abnormalities in other parts of the body. Most cases (70 - 80 percent) of hereditary hearing loss are nonsyndromic, and the remaining cases are caused by specific hereditary syndromes.

[0009] Hearing loss can be conductive (arising from the external ear or middle ear), sensorineural (arising from the inner ear or auditory nerve), or mixed. Most forms of nonsyndromic hearing loss are associated with permanent hearing loss caused by damage to structures in the inner ear (sensorineural hearing loss). Some cases of human sensorineural hearing loss are caused by abnormalities in the hair cells of the organ of Corti within the cochlea. There are also sensorineural hearing disorders involving the eighth cranial nerve (vestibulocochlear nerve) or the auditory parts of the brain. Most sensorineural hearing loss is due to hair cell dysfunction. Hair cells can be abnormal at birth or can become damaged during an individual's lifetime. There are both external causes of damage such as trauma from noise and infections, and intrinsic abnormalities such as congenital mutations in genes that play important roles in cochlear anatomy or physiology.

[0010] Hearing loss resulting from changes in the middle ear is called conductive hearing loss. Some forms of nonsyndromic hearing loss are called mixed hearing loss and involve changes in both the inner ear and the middle ear. Hearing loss present before a child can speak is classified as prelingual or congenital. Hearing loss that occurs after language development is classified as postlingual. Most autosomal recessive loci cause severe to profound prelingual hearing loss.

[0011] Nonsyndromic hearing loss can have different modes of inheritance and can occur at any age. The types of nonsyndromic hearing loss are named according to their modes of inheritance. Autosomal dominant forms are designated DFNA, autosomal recessive forms are designated DFNB, and X-linked forms are designated DFN. Each type is also numbered in the order in which it was described. For example, DFNA1 is the first described autosomal dominant form of nonsyndromic hearing loss.

[0012] Auditory neuropathy spectrum disorder (ANSD) is a hearing disorder characterized by normal outer hair cell function and abnormal or absent auditory brainstem responses, and is one of the most common diseases leading to hearing and speech communication disorders in infants and young children. Approximately 10% of children with persistent hearing loss may have ANSD. The OTOF gene is the first gene identified in autosomal recessive nonsyndromic ANSD, and mutations in OTOF have been found to account for approximately 5% of all cases of autosomal recessive nonsyndromic hearing loss in some populations (Rodriguez-Ballsteros et al. 2008 Human Mut 29 (6):823-831).

[0013] The causes of asymptomatic hearing loss are complex. Researchers have identified more than 30 genes that are associated with asymptomatic hearing loss when mutated, but the characteristics of some of these genes have not been fully elucidated. Different mutations in the same gene may be associated with different types of hearing loss, and some genes are associated with both symptomatic and asymptomatic hearing loss.

[0014] For example, genes associated with nonsyndromic hearing loss include, but are not limited to, ATP2B2, ACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB3, KCNQ4, LHFPL5, MYO1A, MYO15A, MYO6,

[0015] MYO7A, OTOF, PCDH15, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIOBP, USH1C, and WFS1.

[0016] OTOF-related hearing loss (DFNB9 non-syndromic deafness) is characterized by two phenotypes, pre-linguistic non-syndromic deafness and, less frequently, temperature-sensitive non-syndromic auditory neuropathy (TS-NSAN). Another form of progressive hearing impairment is associated with mutations in the otoferlin gene (e.g., I1573T or P1987R mutations, and / or E1700Q mutations) and is not temperature-sensitive.

[0017] Treatment for hearing loss currently consists of hearing amplification for mild to profound loss and cochlear implantation for profound to severe loss (Kral and O’Donoghue, 2010, N. Engl. J. Med. 363:1438-1450). To date, most research in this area has focused on the regeneration of cochlear hair cells, which is applicable to the most common forms of hearing loss, including presbycusis, noise-induced hearing loss, infections, and ototoxicity.

[0018] There remains a long-standing need for agents and methods to prevent or reverse hearing loss.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9-1

Figure 9-2

Figure 9-3

Figure 10-1

Figure 10-2

Figure 10-3

Figure 10-4

Figure 11

[0020] Certain embodiments of the present disclosure relate to a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises from about 4.1E10 to 4.1E12 total vg or from about 8.1E10 to 8.1E12 total vg.

[0021] In some embodiments, the composition comprises from about 4.1E10 to 4.1E12 total vg / cochlea. In some embodiments, the composition comprises about 4.1E11 total vg / cochlea. In other embodiments, the composition comprises from about 8.1E10 to 8.1E12 total vg / cochlea. In some embodiments, the composition comprises about 8.1E11 total vg / cochlea.

[0022] In some embodiments, the concentration of the composition comprises a total of about 4.5E11 to 4.5E13 vg / mL or a total of about 9E11 to 9E13 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 4.5E11 to 4.5E13 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 4.5E12 vg / mL. In other embodiments, the concentration of the composition comprises a total of about 9E11 to 9E13 vg / mL. In some embodiments, the composition comprises a total of about 9E12 vg / mL. In some embodiments, the composition comprises a first rAAV vector genome and a second rAAV vector genome in a ratio of about 1:1.

[0023] In some embodiments, the composition comprises a total of about 4.1E11 vg. In some embodiments, the composition comprises a total of about 8.1E11 vg.

[0024] Certain embodiments of the present disclosure relate to a composition comprising a first rAAV vector genome comprising a) a first expression cassette comprising a promoter, a first coding sequence encoding an N-terminal portion of otoferlin protein located 3' to the promoter, and a splicing donor signal sequence located at the 3' end of the first coding sequence, and b) a second rAAV vector genome comprising a second expression cassette comprising a splicing acceptor signal sequence, a second coding sequence encoding a C-terminal portion of otoferlin protein located 3' to the splicing acceptor signal sequence, and a polyadenylation sequence at the 3' end of the second coding sequence, wherein the composition is formulated for administration into the cochlea.

[0025] In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the composition is formulated to comprise synthetic perilymph.

[0026] In some embodiments, the composition comprises one or more buffers and one or more surfactants. In some embodiments, the buffer is selected from potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS. In some embodiments, the surfactant is selected from poloxamer 188, lubrazol, tween, ethanol, pluronic F68, and polyethylene glycol. In some embodiments, the composition comprises potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, and poloxamer 188.

[0027] In some embodiments, the formulation comprises: a) about 1.35 - 1.65 mM of potassium dihydrogen phosphate, b) about 7.29 - 8.91 mM of disodium hydrogen phosphate, c) about 2.43 - 2.97 mM of potassium chloride, d) about 154.8 - 189.2 mM of sodium chloride; and e) about 0.0001% - 0.01% of poloxamer 188. In some embodiments, the formulation comprises: a) about 1.5 mM of potassium dihydrogen phosphate, b) about 8.1 mM of disodium hydrogen phosphate, c) about 2.7 mM of potassium chloride, d) about 172 mM of sodium chloride; and e) about 0.001% of poloxamer 188.

[0028] In some embodiments, the composition comprises about 4.1E10 - 8.1E12 total vg / snail. In some embodiments, the composition comprises about 4.1E10 - 4.1E12 total vg / snail or about 8.1E10 - 8.1E12 total vg / snail. In some embodiments, the composition comprises about 4.1E11 total vg / snail. In other embodiments, the composition comprises about 8.1E11 total vg / snail.

[0029] In some embodiments, the concentration of the composition comprises about 4.5E11 - 4.5E13 total vg / mL. In some embodiments, the concentration of the composition comprises about 4.5E12 total vg / mL. In other embodiments, the concentration of the composition comprises about 9E11 - 9E13 total vg / mL. In some embodiments, the concentration of the composition comprises about 9E12 total vg / mL.

[0030] In some embodiments, the composition comprises the first rAAV vector genome and the second rAAV vector genome in a ratio of about 1:1.

[0031] In some embodiments, the nucleic acid sequence comprising the 5' portion of the otoferlin gene has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 101. In some embodiments, the nucleic acid sequence comprising the 3' portion of the otoferlin gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 107.

[0032] In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is selected from the CAG, CBA, or CMV promoter. In some embodiments, the constitutive promoter is the CAG promoter.

[0033] In some embodiments, the first rAAV vector comprises a splicing donor site and a recombination-inducing sequence. In some embodiments, the second rAAV vector comprises a splicing acceptor site, a recombination-inducing sequence, and a polyadenylation sequence.

[0034] In some embodiments, the splicing donor site has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 102. In some embodiments, the splicing acceptor site has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 106.

[0035] In some embodiments, the recombination-inducing sequence has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 103.

[0036] In some embodiments, the polyadenylation sequence is selected from bovine growth hormone, human growth hormone, mouse-β-globin, mouse-α-globin, polyoma virus, SV40, or a synthetic polyadenylation sequence. In some embodiments, the polyadenylation is the bovine growth hormone polyadenylation sequence. In some embodiments, the polyadenylation sequence has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 108.

[0037] In some embodiments, the ITR is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80 ITR. In some embodiments, the ITR is AAV2 ITR.

[0038] In some embodiments, the first expression cassette has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 96.

[0039] In some embodiments, the second expression cassette has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 105.

[0040] In some embodiments, the first and second rAAV vectors are each encapsulated by an AAV capsid. In some embodiments, the AAV capsid encapsulating the first rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80. In some embodiments, the AAV capsid encapsulating the second rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80. In some embodiments, the first rAAV vector is encapsulated by an Anc80 capsid and the second rAAV vector is encapsulated by an Anc80 capsid. In some embodiments, the Anc80 capsid comprises the polypeptide sequence of SEQ ID NO: 109.

[0041] In some embodiments, the composition is formulated for intrasnail administration. In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the composition is formulated to comprise synthetic perilymph.

[0042] In some embodiments, the composition comprises one or more buffers and one or more surfactants. In some embodiments, the buffer is selected from potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS. In some embodiments, the surfactant is selected from poloxamer 188, laurabazole, tween, ethanol, pluronic F68, and polyethylene glycol. In some embodiments, the composition comprises potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, and poloxamer 188.

[0043] In some embodiments, the formulation comprises: a) about 1.35 - 1.65 mM of monopotassium phosphate, b) about 7.29 - 8.91 mM of dibasic sodium phosphate, c) about 2.43 - 2.97 mM of potassium chloride, d) about 154.8 - 189.2 mM of sodium chloride; and e) about 0.0001% - 0.01% of poloxamer 188. In some embodiments, the formulation comprises: a) about 1.5 mM of monopotassium phosphate, b) about 8.1 mM of dibasic sodium phosphate, c) about 2.7 mM of potassium chloride, d) about 172 mM of sodium chloride; and e) about 0.001% of poloxamer 188.

[0044] Certain embodiments of the present disclosure relate to a method of treating hearing loss in a subject having a defective otoferlin gene, the method comprising administering a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR), and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises about 4.1E10 - 4.1E12 total vg or about 8.1E10 - 8.1E12 total vg within the cochlea of the subject, and wherein the first and second rAAV vectors are capable of constituting a polypeptide messenger RNA encoding a full-length human otoferlin protein in the subject.

[0045] In some embodiments, the method further comprises determining, prior to the administering step, that the subject has a defective otoferlin gene. In some embodiments, the defective otoferlin gene comprises a mutation that results in a decrease in the expression and / or activity of the otoferlin protein encoded by the gene.

[0046] In some embodiments, the method functions to reduce or prevent secondary modification of one or more cochlear structures.

[0047] Certain embodiments of the present disclosure relate to a method for expressing a recombinant full-length otoferlin protein in mammalian cells, the method comprising administering a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises from about 4.1E10 to 4.1E12 total vg or from about 8.1E10 to 8.1E12 total vg in mammalian cells, and the first and second rAAV vectors are capable of constituting a polypeptide messenger RNA encoding a full-length human otoferlin protein in mammalian cells.

[0048] In some embodiments, the mammalian cells are cochlear cells. In some embodiments, the mammalian cells are inner ear hair cells. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0049] In some embodiments, the composition is administered as a single dose. In some embodiments, the composition is administered in multiple doses. In some embodiments, the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0050] In some embodiments, the single dose comprises from about 0.01 mL to 0.2 mL. In some embodiments, the single dose comprises about 0.09 mL.

[0051] In some embodiments, the composition is administered as an injection into the round window membrane. In some embodiments, the composition is administered as a single injection. In some embodiments, the composition is administered as multiple injections. In some embodiments, the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 injections.

[0052] In some embodiments, the composition is administered through the use of a medical device. In some embodiments, the device is the device shown in FIGS. 2-5. In some embodiments, the device is a microcatheter.

[0053] In some embodiments, the composition is delivered at a controlled flow rate.

[0054] In some embodiments, the subject is between 2 and 17 years of age.

[0055] In some embodiments, administration of the composition improves the subject's auditory brainstem response (ABR) threshold response, age-appropriate behavioral hearing tests, myringotomy tests, and / or word / sentence recognition tests.

[0056] Certain aspects of the present disclosure relate to a kit comprising a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5' portion of an otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition is formulated for intratympanic administration. Other aspects of the present disclosure relate to a kit comprising a composition comprising: a) a first rAAV vector genome comprising a first expression cassette comprising a promoter, a first coding sequence located 3' of the promoter encoding an N-terminal portion of an otoferlin protein, and a splicing donor signal sequence located at the 3' end of the first coding sequence; and b) a second rAAV vector genome comprising a second expression cassette comprising a splicing acceptor signal sequence, a second coding sequence located 3' of the splicing acceptor signal sequence encoding a C-terminal portion of an otoferlin protein, and a polyadenylation sequence at the 3' end of the second coding sequence, wherein the composition is formulated for intratympanic administration.

[0057] In some aspects, the kit further comprises a filled syringe containing the composition. In other aspects, the kit further comprises a vial containing the composition.

[0058] Certain aspects of the present disclosure relate to a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises a total of about 4.1E10 to 8.1E12 vg.

[0059] In some embodiments, the composition comprises a total of about 4.1E10 to 4.1E12 vg or about 8.1E10 to 8.1E12 vg. In some embodiments, the composition comprises a total of about 4.1E10 to 4.1E12 vg. In some embodiments, the composition comprises a total of about 4.1E11 vg. In other embodiments, the composition comprises a total of about 8.1E10 to 8.1E12 vg. In some embodiments, the composition comprises a total of about 8.1E11 vg.

[0060] In some embodiments, the composition comprises about 4.1E10 to 4.1E12 vg / snail or about 8.1E10 to 8.1E12 vg / snail. In some embodiments, the composition comprises about 4.1E10 to 4.1E12 vg / snail. In some embodiments, the composition comprises about 4.1E11 vg / snail. In other embodiments, the composition comprises about 8.1E10 to 8.1E12 vg / snail. In some embodiments, the composition comprises about 8.1E11 vg / snail.

[0061] In some embodiments, the concentration of the composition comprises about 4.5E11 to 4.5E13 vg / mL. In some embodiments, the concentration of the composition comprises about 4.5E12 vg / mL. In other embodiments, the concentration of the composition comprises about 9E11 to 9E13 vg / mL. In some embodiments, the composition comprises about 9E12 vg / mL.

[0062] In some embodiments, the composition comprises a first rAAV vector genome and a second rAAV vector genome in a ratio of about 1:1.

[0063] Certain embodiments of the present disclosure relate to a composition comprising a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette operably linked to a nucleic acid sequence comprising a 5' portion of an otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR), and a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to the ITR, wherein the composition is formulated for intratympanic administration.

[0064] In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the composition is formulated to comprise synthetic perilymph.

[0065] In some embodiments, the composition comprises one or more buffers and one or more surfactants. In some embodiments, the buffer is selected from potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS. In some embodiments, the surfactant is selected from poloxamer 188, lubrazol, tween, ethanol, pluronic F68, and polyethylene glycol. In some embodiments, the composition comprises potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, and poloxamer 188.

[0066] In some embodiments, the formulation comprises: a) about 1.35 - 1.65 mM potassium dihydrogen phosphate, b) about 7.29 - 8.91 mM disodium hydrogen phosphate, c) about 2.43 - 2.97 mM potassium chloride, d) about 154.8 - 189.2 mM sodium chloride; and e) about 0.0001% - 0.01% poloxamer 188. In some embodiments, the formulation comprises: a) about 1.5 mM potassium dihydrogen phosphate, b) about 8.1 mM disodium hydrogen phosphate, c) about 2.7 mM potassium chloride, d) about 172 mM sodium chloride; and e) about 0.001% poloxamer 188.

[0067] In some embodiments, the composition comprises about 4.1E10 - 8.1E12 total vg / snail. In some embodiments, the composition comprises about 4.1E10 - 4.1E12 total vg / snail, or about 8.1E10 - 8.1E12 total vg / snail. In some embodiments, the composition comprises about 4.1E11 total vg / snail. In other embodiments, the composition comprises about 8.1E11 total vg / snail.

[0068] In some embodiments, the concentration of the composition comprises about 4.5E11 - 4.5E13 total vg / mL. In some embodiments, the concentration of the composition comprises about 4.5E12 total vg / mL. In other embodiments, the concentration of the composition comprises about 9E11 - 9E13 total vg / mL. In some embodiments, the concentration of the composition comprises about 9E12 total vg / mL.

[0069] In some embodiments, the composition comprises the first rAAV vector genome and the second rAAV vector genome in a ratio of about 1:1.

[0070] In some embodiments, the nucleic acid sequence comprising the 5' portion of the otoferlin gene has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 101. In some embodiments, the nucleic acid sequence comprising the 3' portion of the otoferlin gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 107.

[0071] In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is selected from the CAG, CBA, or CMV promoter. In some embodiments, the constitutive promoter is the CAG promoter.

[0072] In some embodiments, the first rAAV vector comprises a splicing donor site and a recombination-inducing sequence. In some embodiments, the second rAAV vector comprises a splicing acceptor site, a recombination-inducing sequence, and a polyadenylation sequence.

[0073] In some embodiments, the splicing donor site has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 102. In some embodiments, the splicing acceptor site has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 106.

[0074] In some embodiments, the recombination-inducing sequence has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 103.

[0075] In some embodiments, the polyadenylation sequence is selected from bovine growth hormone, human growth hormone, mouse-β-globin, mouse-α-globin, polyoma virus, SV40, or a synthetic polyadenylation sequence. In some embodiments, the polyadenylation is the bovine growth hormone polyadenylation sequence. In some embodiments, the polyadenylation sequence has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 108.

[0076] In some embodiments, the ITR is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80 ITR. In some embodiments, the ITR is AAV2 ITR.

[0077] In some embodiments, the first expression cassette has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 96.

[0078] In some embodiments, the second expression cassette has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 105.

[0079] In some embodiments, the first and second rAAV vectors are each encapsulated by an AAV capsid. In some embodiments, the AAV capsid encapsulating the first rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80. In some embodiments, the AAV capsid encapsulating the second rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80. In some embodiments, the first rAAV vector is encapsulated by an Anc80 capsid and the second rAAV vector is encapsulated by an Anc80 capsid. In some embodiments, the Anc80 capsid comprises the polypeptide sequence of SEQ ID NO: 109.

[0080] In some embodiments, the composition is formulated for intrasnail administration. In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the composition is formulated to include synthetic perilymph.

[0081] In some embodiments, the composition comprises one or more buffers and one or more surfactants. In some embodiments, the buffer is selected from potassium monophosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS. In some embodiments, the surfactant is selected from poloxamer 188, lubrazol, tween, ethanol, pluronic F68, and polyethylene glycol. In some embodiments, the composition comprises potassium monophosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, and poloxamer 188.

[0082] In some embodiments, the formulation comprises a) potassium dihydrogen phosphate at about 1.35 - 1.65 mM, b) disodium hydrogen phosphate at about 7.29 - 8.91 mM, c) potassium chloride at about 2.43 - 2.97 mM, d) sodium chloride at about 154.8 - 189.2 mM, and e) poloxamer 188 at about 0.0001% - 0.01%. In some embodiments, the formulation comprises a) potassium dihydrogen phosphate at about 1.5 mM, b) disodium hydrogen phosphate at about 8.1 mM, c) potassium chloride at about 2.7 mM, d) sodium chloride at about 172 mM, and e) poloxamer 188 at about 0.001%.

[0083] In some embodiments, the formulation is a sterile suspension. In some embodiments, the formulation contains sterile water.

[0084] In some embodiments, the volume of the formulation is about 0.01 mL - 0.2 mL. In some embodiments, the volume of the formulation is about 0.09 mL.

[0085] Certain aspects of the present disclosure relate to methods of treating hearing loss in a subject having a defective otoferlin gene, the method comprising administering a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises from about 4.1E10 to 8.1E12 total vg in the subject's cochlea, and wherein the first and second rAAV vectors are capable of constructing a polypeptide messenger RNA encoding a full-length human otoferlin protein in the subject. Other certain aspects of the present disclosure relate to methods of treating hearing loss in a subject having a defective otoferlin gene, the method comprising administering a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition is formulated for administration into the subject's cochlea, and wherein the first and second rAAV vectors are capable of constructing a polypeptide messenger RNA encoding a full-length human otoferlin protein in the subject.

[0086] In some embodiments, the defective otoferlin gene comprises a mutation that results in a decrease in the expression and / or activity of the otoferlin protein encoded by the gene. In some embodiments, the method further comprises determining, prior to the administration step, that the subject has a defective otoferlin gene.

[0087] Certain aspects of the present disclosure relate to methods of treating hearing loss in a subject having a biallelic otoferlin gene mutation, the method comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, the expression cassette being adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, the expression cassette being adjacent to an ITR, wherein the composition comprises from about 4.1E10 to 8.1E12 total vg within the subject's cochlea, and the first and second rAAV vectors are capable of constituting a polypeptide messenger RNA encoding a full-length human otoferlin protein in the subject. Other aspects of the present disclosure relate to methods of treating hearing loss in a subject having a biallelic otoferlin gene mutation, the method comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, the expression cassette being adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, the expression cassette being adjacent to an ITR, wherein the composition is formulated for administration into the subject's cochlea, and the first and second rAAV vectors are capable of constituting a polypeptide messenger RNA encoding a full-length human otoferlin protein in the subject.

[0088] In some aspects, the method further comprises determining that the subject has a biallelic otoferlin gene mutation prior to the administration step.

[0089] In some embodiments, the subject has clinical symptoms of bilateral severe sensorineural hearing loss. In some embodiments, the subject has clinical symptoms of bilateral severe sensorineural hearing loss without fever.

[0090] In some embodiments, the subject preserves distortion product otoacoustic emissions (DPOAE).

[0091] Other specific embodiments of the present disclosure relate to a method of expressing recombinant full-length otoferlin protein in mammalian cells, the method comprising administering a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition is formulated for intracochlear administration to mammalian cells, and the first and second rAAV vectors can constitute a polypeptide messenger RNA encoding full-length human otoferlin protein in mammalian cells.

[0092] In some embodiments, the mammalian cells are cochlear cells. In some embodiments, the mammalian cells are inner ear hair cells. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0093] In some embodiments, the composition is administered as a single dose. In some embodiments, the composition is administered in multiple doses. In some embodiments, the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0094] In some embodiments, the single dose comprises from about 0.01 mL to 0.2 mL. In some embodiments, the single dose comprises about 0.09 mL.

[0095] In some embodiments, the composition is administered as an injection into the round window membrane. In some embodiments, the composition is administered as a single injection. In some embodiments, the composition is administered as multiple injections. In some embodiments, the composition is administered as 2, 3, 4, 5, 6, 7, 8, 9, or 10 injections.

[0096] In some embodiments, the composition is administered through the use of a medical device. In some embodiments, the composition is pre-filled in the device. In some embodiments, the device is the device shown in FIGS. 2-5. In some embodiments, the device is a microcatheter. In some embodiments, the microcatheter is shaped to enter the middle ear cavity through the external auditory canal and to be able to contact the end of the microcatheter with the round window membrane (RWM). In some embodiments, the distal end of the microcatheter includes at least one microneedle having a diameter of 10-1,000 microns. In some embodiments, the at least one microneedle includes a bent portion and an inclined tip.

[0097] In some embodiments, the composition is delivered at a controlled flow rate.

[0098] In some embodiments, the subject is 2-17 years old.

[0099] In some embodiments, administration of the composition improves the subject's auditory brainstem response (ABR) threshold response, age-appropriate behavioral audiometry, tympanotomy examination, and / or word / sentence recognition test.

[0100] Certain aspects of the present disclosure relate to a kit comprising a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5' portion of an otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3' portion of an otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises a total of about 4.1E10 to 8.1E12 vg. Other specific aspects of the present disclosure relate to a kit comprising a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5' portion of an otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3' portion of an otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition is formulated for administration into the cochlea.

[0101] In some aspects, the composition is pre-filled into a device. In some aspects, the device is a microcatheter. In some aspects, the microcatheter is shaped to enter the middle ear cavity through the ear canal and contact the end of the microcatheter with the round window membrane (RWM). In some aspects, the distal end of the microcatheter comprises at least one micro-needle having a diameter of 10 to 1,000 microns.

[0102] In some aspects, the kit further comprises a device. In some aspects, the device is a device as described in any one of Figures 2 to 5. In some aspects, the device comprises a needle comprising a bent portion and a beveled tip.

[0103] In other embodiments, the kit further includes a vial containing the composition. In some embodiments, the vial is a single-use vial. In some embodiments, the kit further includes a second vial containing a diluent.

DETAILED DESCRIPTION OF THE INVENTION

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0105] The articles “a” and “an” mean one or more (i.e., at least one) of the grammatical object of the article. By way of example, “an element” encompasses one element and plural elements.

[0106] The term “about” is used herein to mean about, approximately, roughly, or within that range. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending its boundaries above and below the recited numerical values. In general, the term “about” is used herein to modify a numerical value so as to be above and below (higher or lower) the stated value by 10%.

[0107] The term “recombinant polypeptide” or “recombinant protein” refers to a polypeptide or protein produced using recombinant DNA technology, e.g., a polypeptide or protein expressed by a viral vector expression system. This term is also to be construed to mean a polypeptide or protein produced by the synthesis of a DNA molecule encoding the polypeptide or protein, where the DNA molecule expresses an amino acid sequence that identifies the protein, or polypeptide, and where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence techniques that are available and well known in the art.

[0108] The term "mutation in the otoferlin gene" refers to a modification in the wild-type otoferlin gene that results in the production of an otoferlin protein having one or more of a deletion of one or more amino acids, a substitution of one or more amino acids, and an insertion of one or more amino acids, compared to the wild-type otoferlin protein, and / or that results in a decrease in the expressed level of the encoded otoferlin protein in mammalian cells compared to the expressed level of the encoded otoferlin protein in mammalian cells that do not have the mutation. In some embodiments, the mutation can result in the production of an otoferlin protein having a deletion of one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids). In some embodiments, the mutation can result in a frameshift of the otoferlin gene. The term "frameshift" is known in the art to encompass any mutation in a coding sequence that results in a shift of the reading frame of the coding sequence. In some embodiments, the frameshift can result in a non-functional protein. In some embodiments, the point mutation can be a nonsense mutation (i.e., one that results in a premature stop codon in an exon of the gene). A nonsense mutation can result in the production of a truncated protein (compared to the corresponding wild-type protein) that may or may not be functional. In some embodiments, the mutation can result in a loss (or decrease in level) of expression of otoferlin mRNA or otoferlin protein, or both mRNA and protein. In some embodiments, the mutation can result in the production of an altered otoferlin protein having a loss or decrease in one or more biological activities (functions) compared to the wild-type otoferlin protein.

[0109] In some embodiments, the mutation is an insertion into the otoferlin gene of one or more nucleotides. In some embodiments, the mutation is in a regulatory sequence of the otoferlin gene, i.e., a portion of the gene that is not the coding sequence. In some embodiments, the mutation in the regulatory sequence can be in a promoter or enhancer region and can prevent or reduce proper transcription of the otoferlin gene.

[0110] Modifications can be introduced into the nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0111] The term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or substantially change the binding properties of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. Such families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0112] The term "encoding" refers to the unique property of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA that functions as a template for synthesizing a specified amino acid sequence. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to that gene, cDNA, or RNA produces the protein. The nucleotide sequence that is identical to the mRNA sequence and can be regarded as encoding the protein product includes both the coding strand, which is usually shown in the sequence listing, and the non-coding strand used as a template for transcription.

[0113] The term "sequence identity" is used herein to mean the relatedness between two or more amino acid (polypeptide or protein) sequences or between two or more nucleic acid (polynucleotide) sequences, which is determined by comparing the sequences. In certain embodiments, sequence identity is calculated based on the full length or a portion of two given sequence numbers. The portion can mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of both sequence numbers, or any other specific percentage. The term "identity" can also mean the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, which in some cases is determined by the matches between the strings of such sequences.

[0114] Unless otherwise specified, the "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence. The nucleotide sequence encoding a protein can also include introns.

[0115] The term "isolated" means modified or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form or, for example, in a non-native environment such as a host cell.

[0116] The terms "transfected" or "transformed" or "transduced" refer to the process by which an exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0117] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0118] As used herein, "transient" refers to the expression of an introduced gene that is not integrated for several hours, days, or weeks, and this period of expression is shorter than the period of gene expression when integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0119] The term "subject" is intended to include a living body (e.g., a mammal, a human) in which an immune response can be induced. In some embodiments, the subject is a rodent (e.g., a rat or a mouse), a rabbit, a sheep, a dog, a cat, a horse, a non-human primate or a human. In some embodiments, the subject has or is at risk of developing asymptomatic hearing loss. In some embodiments, the subject has previously been identified as having a mutation in the otoferlin gene. In some embodiments, the subject has been identified as having a mutation in the otoferlin gene and has been diagnosed with asymptomatic sensorineural hearing loss. In some embodiments, the subject has been identified as having asymptomatic sensorineural hearing loss.

[0120] As used herein, the term "therapeutic" means treatment. A therapeutic effect is obtained by a reduction, suppression, remission, or eradication of a pathological condition.

[0121] As used herein, the term "prophylaxis" means the prevention or protective treatment of a disease or a pathological condition. "Prevention" in this context includes reducing the likelihood that a subject will experience the disease.

[0122] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result. In some embodiments, a therapeutically effective amount of a composition can result in an increase in the expression level of an active otoferlin protein (e.g., wild-type full-length otoferlin protein, or a variant of an otoferlin protein having the desired activity) (e.g., as compared to the expression level prior to treatment with the composition). In some embodiments, a therapeutically effective amount of a composition can result in an increase in the expression level of an active otoferlin protein (e.g., wild-type full-length otoferlin protein, or an active variant) in a target cell (e.g., inner hair cells within the cochlea). In some embodiments, a therapeutically effective amount of a composition can result in a different cellular localization of an active otoferlin protein (e.g., wild-type full-length otoferlin protein, or an active variant) in a target cell (e.g., inner hair cells within the cochlea). In some embodiments, a therapeutically effective amount of a composition can result in an increase in the expression level of an active otoferlin protein (e.g., wild-type full-length otoferlin protein, or an active variant) and / or an increase in one or more activities of the otoferlin protein (e.g., as compared to a reference level such as the level(s) in a subject prior to treatment, the level(s) in a subject having a mutation in the otoferlin gene, or the level(s) in a subject or population of subjects having non-syndromic hearing loss) in a target cell.

[0123] The term "parenteral" administration of a composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0124] The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form, and polymers thereof. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the explicitly recited sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0125] In some embodiments of any nucleic acid described herein, the nucleic acid is DNA. In some embodiments of any nucleic acid described herein, the nucleic acid is RNA.

[0126] In the context of the present disclosure, the following abbreviations are generally used with respect to commonly occurring nucleobases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0127] As used herein, "RNA transcribed in vitro" refers to RNA synthesized in vitro, preferably mRNA. Generally, RNA transcribed in vitro is produced from an in vitro transcription vector. An in vitro transcription vector contains a template used to produce RNA transcribed in vitro.

[0128] The terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up the sequence of a protein or peptide. A polypeptide includes a peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are generally also referred to in the art as, for example, peptides, oligopeptides, and oligomers, and the many types of long chains, which are generally referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0129] The term "active otoferlin protein" means a protein encoded by DNA that, when substituted for both wild-type alleles encoding full-length otoferlin protein in the auditory hair cells (e.g., inner auditory hair cells) of a mammal that would otherwise be a wild-type mammal, and when expressed in the auditory hair cells of that mammal, results in that mammal having a hearing level approximating the normal hearing level of a similar, fully wild-type mammal. Non-limiting examples of active otoferlin proteins are full-length otoferlin proteins (e.g., any of the full-length otoferlin proteins described herein).

[0130] The term "vector" includes any genetic element such as plasmids, phages, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, etc. that can be replicated when associated with appropriate control elements and can transfer gene sequences between cells. Thus, this term encompasses cloning and expression vehicles, as well as viral vectors. In some embodiments, a useful vector is considered to be a vector in which the nucleic acid segment to be transcribed is under the transcriptional control of a promoter.

[0131] Vectors include all vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), which incorporate recombinant polynucleotides. One of ordinary skill in the art will be able to select a suitable vector and mammalian cell for making any of the nucleic acids described herein. The vector may, for example, contain sufficient cis-acting elements for expression, and other elements for expression may be supplied by the host mammalian cell or in an in vitro expression system.

[0132] "Promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or an introduced synthetic machinery that is required to initiate specific transcription of a polynucleotide sequence (e.g., a gene).

[0133] The terms "expression vector", "construct", or "expression cassette" mean any type of genetic construct that contains a nucleic acid in which some or all of the nucleic acid coding sequences can be transcribed. In some embodiments, expression includes, for example, transcription of a nucleic acid to produce a biologically active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitor) from the transcribed gene.

[0134] The terms "operably linked," "operatively disposed," "operatively linked," "under the control of," "under transcriptional control," or "transcriptional control" refer to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, such that expression of the latter is effected. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, where necessary to join two protein coding regions, are in the reading frame.

[0135] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a nucleic acid encoding a protein (e.g., an otoferlin protein), causes transcription of RNA from the nucleic acid in mammalian cells under most or all physiological conditions.

[0136] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced substantially intracellularly only when an inducer corresponding to the promoter is present intracellularly.

[0137] The term "tissue-specific" promoter refers to a promoter that is active only within a particular cell type and / or tissue (e.g., transcription of a particular gene occurs only within cells that express a transcriptional control protein that binds to the tissue-specific promoter).

[0138] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety, or a modified variant thereof, to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3' poly(A) tail is a long sequence of adenine nucleotides (often hundreds) that is added to the pre-mRNA via enzymatic action, by polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts that contain a polyadenylation signal, which is a specific sequence. The poly(A) tail and the proteins bound to it help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, nuclear export of the mRNA, and translation. Polyadenylation occurs in the nucleus immediately after DNA is transcribed into RNA, but can also occur later in the cytoplasm. After transcription has terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end of the cleavage site.

[0139] As used herein, the terms "poly(A)", "poly(A) signal sequence", "poly A sequence", or "polyadenylation sequence" refer to a sequence that induces endonuclease cleavage of an mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0140] As used herein, the term "pharmaceutically acceptable carrier" includes buffers, surfactants, salts, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, etc. that are compatible with drug administration. Additionally, auxiliary active compounds can also be incorporated into any of the compositions described herein.

[0141] As used herein, "carrier" includes every buffer, surfactant, salt, solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffer solutions, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Auxiliary active ingredients can also be incorporated into the composition.

[0142] "Viral genome" or "vector genome" or "viral vector" refers to a sequence that includes one or more polynucleotide regions encoding or containing a molecule of interest, such as, for example, proteins, peptides, and polynucleotides or multiples thereof. Viral vectors are used to deliver genetic material into cells. Viral vectors can be modified for specific applications. In some embodiments, the delivery vector includes a viral vector selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, or retrovirus vectors.

[0143] As used herein, the terms "adeno-associated virus vector" or "AAV vector" refer to any vector that contains or is derived from components of an adeno-associated vector and is suitable for infecting mammalian cells, preferably human cells. The term "AAV vector" typically refers to an AAV viral particle or virion containing a payload. AAV vectors can be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV), or can be derived from various genomes (e.g., single-stranded or self-complementary). Furthermore, AAV vectors are replication-deficient and / or can be targeted. As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh8, AAVrh10, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, AAV serotypes and clades disclosed by Ggao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), and any other AAV. See, for example, FIELDS et al. VIROLOGY, volumes 2, Chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV vector" includes derivatives of known AAV vectors. In some embodiments, "AAV vector" includes modified or artificial AAV vectors. The terms "AAV genome" and "AAV vector" may be used interchangeably.

[0144] As used herein, "recombinant AAV particle" or "rAAV particle" is an AAV virus comprising a capsid protein and an AAV vector having at least one payload region and at least one inverted terminal repeat (ITR) region.

[0145] "Serotype" with respect to a vector or viral capsid is defined by distinct immunological profiles based on capsid protein sequences and capsid structure.

[0146] The term "ratio" refers to a comparison of two or more numbers indicating their quantitative relationship to one another. In some embodiments, a ratio can be used to compare two parts within an overall amount or total quantity.

[0147] As used herein, the term "vector ratio" refers to a comparison of the amount within the vector genome (vg) of one AAV vector to the amount within the vector genome of another AAV vector.

[0148] Otogelin The human OTOF gene encodes otoferlin, which, in some aspects, is a protein that plays a crucial role in the priming, fusion, and / or replenishment of synaptic vesicles at the synapses of inner hair cells during vocal coding. To date, hundreds of mutations in the human OTOF gene have been confirmed to cause severe prelingual deafness DFNB9. Such mutations cause hearing loss in 2 - 8% of those born with autosomal recessive hereditary non-syndromic deafness in different populations (Rodriguez-Ballesteros et al. (2008) Hum. Mutat. 29 823 - 831; Choi et al. (2009) Clinical Genetics 75 237 - 243; Duman et al. (2011) Genet Test Mol Biomarkers 15 29 - 33; Varga et al. (2006) J Med Genet 43 576 - 581; Iwasa et al. (2013) BMC Med. Genet. 14 95). Mutations in both alleles of the otoferlin gene result in a localized synaptic transmission disorder between hair cells and the auditory nerve. Otoferlin enables sensory cells to release neurotransmitters in response to sound stimuli to activate auditory neurons, and those neurons transmit electronically encoded acoustic information to the brain to produce "hearing". When both alleles of OTOF are mutated, that transmission is impaired, and as a result, most subjects have hereditary bilateral severe to profound sensorineural hearing loss. For example, two substitutions in exon 15 at positions 490 and 515 within the conserved C2C domain of otoferlin cause DFNB9 (Mirqhomizadeh et al. (2002) Neurobiol. Dis. 10(2):157 - 164). Migliosi et al. discovered a novel mutation Q829X within OTOF in Spanish subjects with syndromic deafness (Migliosi et al. (2002) J. Med. Genet. 39(7):502 - 506).

[0149] Further exemplary mutations within the otoferlin gene detected in subjects with deafness, and methods of sequencing nucleic acids encoding otoferlin are described, for example, in Rodriguez - Ballesteros et al. (2003) Hum Mutat. 22:451 - 456; Wang et al. (2010) BMC Med Genet. 11:79; Yildirim - Baylan et al. (2014) Int. J. Pediatr. Otorhinolaryngol 78:950 - 953; Choi et al. (2009) Clin. Genet. 75(3):237 - 243; and Marlin et al. (2010) Biochem Biophys Res Commun 394:737 - 742.

[0150] Otoacoustic emissions from DFNB9 subjects (e.g., distortion product otoacoustic emissions (DPOAE)) are normal for at least the first 10 years of life, which indicates the morphological integrity of the inner ear and the proper functioning of outer hair cells. Thus, in some embodiments, the subject maintains DPOAE. Apart from the lack of synaptic transmission and subsequent synaptic loss, the inner ear morphology and physiology in DFNB9 are maintained for at least the first 10 years of human life. Thus, in some embodiments, restoration of OTOF and / or otoferlin function may function to reduce or prevent secondary degeneration of one or more cochlear structures.

[0151] Studies in mouse models have revealed that synapses are structurally normal and that IHCs maintain normal synapse numbers in mice within the first week of life. Approximately half of the synapses are lost between P6 and P15 (Roux et al. (2006) Cell 127 277-289). Animal models allowed us to test the effect of otoferlin mutations on synaptic transmission by recording changes in plasma membrane capacitance after vesicle fusion and activity in the auditory nerve. In otoferlin knockout (Otof- / -) mice, depolarization-induced Ca2+ influx through voltage-gated Ca2+ channels hardly induced exocytosis in IHCs (Roux et al. (2006) Cell 127 277-289). In severely deafened pachanga (OtofPga / Pga) mice carrying a random point mutation within the C2F domain, brief (<10 ms) depolarization of IHCs induced vesicle fusion of similar magnitude as in wild-type mice, but sustained stimulation revealed a strong defect in vesicle recruitment to the readily releasable pool (Pangrsic et al. (2010) Nat. Neurosci. 13 869-876). The p.Ile515Thr mutation, found in human subjects with only mildly elevated hearing thresholds but severely impaired speech comprehension and temperature-dependent deafness (Varga et al. (2006) J Med Genet 43 576-581), revealed an intermediate phenotype when tested in mouse models (Strenzke et al. (2016) EMBO J. 35:2519-2535). Humans carrying the biallelic pIl515Thr mutation have been shown to have worsening hearing loss during fever and a return to pre-fever hearing function upon defervescence (Varga et al. (2006) J Med Genet 43 576-581). OtofI515T / I515T mice showed a moderate elevation of hearing thresholds when assessed by ABR with a reduction in wave I amplitude, but normal hearing thresholds in behavioral tests and single auditory neuron recordings. RRP exocytosis is again intact, but tonic exocytosis is reduced, although not as severe as in OtofPga / Pga.In wild-type mice at room temperature, 750 vesicles can fuse per active zone per second during a sustained stimulus, but this rate drops to 350 vesicles / s / active zone in OtofI515T / I515T mice and to 200 vesicles / s / active zone in OtofPga / Pga IHC (Pangrsic et al. (2010) Nat. Neurosci. 13 869-876; Strenzke et al. (2016) EMBO J. 35 2519-2535). This correlated with a decrease in otoferlin protein levels at the IHC plasma membrane. This indicates that the amount of otoferlin varies with exocytosis and hearing (Strenzke et al. (2016) EMBO J. 35 2519-2535).

[0152] Methods for detecting gene mutations are well known in the art. Non-limiting examples of such techniques include real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.

[0153] The OTOF gene encodes otoferlin, a protein involved in synaptic vesicle exocytosis in cochlear hair cells (see, for example, Johnson and Chapman (2010) J. Cell Biol. 191(1):187-198; and Heidrych et al. (2008) Hum. Mol. Genet. 17:3814-3821).

[0154] The human OTOF gene is located on chromosome 2p23.3 and contains 48 exons encompassing approximately 132 kilobases (kb) (NCBI accession number NG009937.1). The mRNA encoding the long form of otoferlin that is expressed in the brain contains 48 exons (Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000). The forward and reverse primers that can be used to amplify each of the 48 exons of the OTOF gene are listed in Table 2 of Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000. In some instances, the full-length OTOF protein is the full-length wild-type OTOF protein. The full-length wild-type OTOF protein expressed from the human OTOF gene is 1997 residues in length.

[0155] Exemplary human wild-type otoferlin proteins are any one of the sequences of SEQ ID NOs: 1-5 or include this. Isoform e (SEQ ID NO: 5) of the human otoferlin protein is encoded by an mRNA that contains exon 48 of the otoferlin gene and does not contain exon 47 (Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000). In some embodiments, the active otoferlin protein has the sequence of SEQ ID NO: 5 but lacks 20 amino acids including the RXR motif identified in Strenzke et al., EMBO J. 35(23):2499-2615, 2016. Non-limiting examples of nucleic acids encoding the wild-type otoferlin protein are any one of SEQ ID NOs: 7-11 or include this. As can be understood in the art, at least some or all of the codons within SEQ ID NOs: 7-11 can be codon-optimized to enable optimal expression in non-human mammals or humans. Orthologs of the human otoferlin protein are known in the art.

[0156] Human otoferlin protein cDNA sequence: Human canonical (long) isoform sequence (otopetrin protein) (SEQ ID NO: 1) (also called otopetrin isoform a) (NCBI accession number AAD26117.1) Human isoform 2 (short 1) (otopetrin protein) (SEQ ID NO: 2) (also called otopetrin isoform d) (NCBI accession number NP_919304.1) Human isoform 3 (short 2) (otopetrin protein) (SEQ ID NO: 3) (also called otopetrin isoform c) (NCBI accession number NP_919303.1) Human isoform 4 (short 3) (otopetrin protein) (SEQ ID NO: 4) (also called otopetrin isoform b) (NCBI accession number NP_004793.2) Human isoform 5 (short 4) (otopetrin protein) (SEQ ID NO: 5) (also called otopetrin isoform e) (NCBI accession number NP_001274418.1) Complete cds (otopetrin cDNA) (www.ncbi.nlm.nih.gov / nuccore / AF107403.1) (SEQ ID NO: 6) (encodes the protein of SEQ ID NO: 1) Human otopetrin transcript variant 1 (www.ncbi.nlm.nih.gov / nuccore / NM_194248.2) (SEQ ID NO: 7) (encodes the protein of SEQ ID NO: 1) Human otopetrin transcript variant 2 (www.ncbi.nlm.nih.gov / nuccore / NM_004802.3) (SEQ ID NO: 8) (encodes the protein of SEQ ID NO: 4) Human otopetrin transcript variant 3 (www.ncbi.nlm.nih.gov / nuccore / NM_194322.2) (SEQ ID NO: 9) (encodes the protein of SEQ ID NO: 3) Human otopetrin transcript variant 4 (www.ncbi.nlm.nih.gov / nuccore / NM_194323.2) (SEQ ID NO: 10) (encodes the protein of SEQ ID NO: 2) Human otoferlin transcript variant 5 (www.ncbi.nlm.nih.gov / nuccore / NM_001287489.1) (SEQ ID NO:11) (encoding the protein of SEQ ID NO:5)

[0157] A non-limiting example of the human wild-type otoferlin genomic DNA sequence is SEQ ID NO: 12. The exons of SEQ ID NO: 12 are nucleotides 5001-5206 (exon 1), nucleotides 25925-25983 (exon 2), nucleotides 35779-35867 (exon 3), nucleotides 44590-44689 (exon 4), nucleotides 47100-47281 (exon 5), nucleotides 59854-59927 (exon 6), nucleotides 61273-61399 (exon 7), nucleotides 61891-61945 (exon 8), nucleotides 68626-68757 (exon 9), nucleotides 73959-74021 (exon 10), nucleotides 74404-74488 (exon 11), nucleotides 79066-79225 (exon 12), nucleotides 80051-80237 (exon 13), nucleotides 81107-81293 (exon 14), nucleotides 82690-82913 (exon 15), nucleotides 83388-83496 (exon 16), nucleotides 84046-84226 (exon 17), nucleotides 84315-84435 (exon 18), nucleotides 85950-86050 (exon 19), nucleotides 86193-86283 (exon 20), nucleotides 86411-86527 (exon 21), nucleotides 86656-86808 (exon 22), nucleotides 87382-87571 (exon 23), nucleotides 87661-87785 (exon 24), nucleotides 88206-88340 (exon 25), nucleotides 89025-89186 (exon 26), nucleotides 89589-89708 (exon 27), nucleotides 90132-90293 (exon 28), nucleotides 90405-90567 (exon 29), nucleotides 91050-91180 (exon 30), nucleotides 92549-92578 (exon 31), nucleotides 92978-93106 (exon 32), nucleotides 95225-95291 (exon 33), nucleotides 96198-96334 (exon 34), nucleotides 96466-96600 (exon 35).Nucleotides 96,848 to 96,985 (exon 36), nucleotides 97,623 to 97,750 (exon 37), nucleotides 97,857 to 98,027 (exon 38), nucleotides 98,670 to 98,830 (exon 39), nucleotides 99,593 to 99,735 (exon 40), nucleotides 100,128 to 100,216 (exon 41), nucleotides 101,518 to 101,616 (exon 42), nucleotides 101,762 to 102,003 (exon 43), nucleotides 102,669 to 102,847 (exon 44), nucleotides 102,952 to 103,052 (exon 45), nucleotides 103,494 to 103,691 (exon 46), nucleotides 105,479 to 106,496 (exon 47), and exon 48 (sequence starting with CCGGCCCGAC. See also the description of this exon in Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000).

[0158] The introns are located between each consecutive pair of exons of SEQ ID NO: 12, i.e., nucleotides 100 to 5001 (intron 1), nucleotides 5207 to 25924 (intron 2), nucleotides 25984 to 35778 (intron 3), nucleotides 35868 to 44589 (intron 4), nucleotides 44690 to 47099 (intron 5), nucleotides 47282 to 59853 (intron 6), nucleotides 59928 to 61272 (intron 7), nucleotides 61400 to 61890 (intron 8), nucleotides 61946 to 68625 (intron 9), nucleotides 68758 to 73958 (intron 10), nucleotides 74022 to 74403 (intron 11), nucleotides 74489 to 79065 (intron 12), nucleotides 79226 to 80050 (intron 13), nucleotides 80238 to 81106 (intron 14), nucleotides 81294 to 82689 (intron 15), nucleotides 82914 to 83387 (intron 16), nucleotides 83497 to 84045 (intron 17), nucleotides 84227 to 84314 (intron 18), nucleotides 84436 to 85949 (intron 19), nucleotides 86051 to 86192 (intron 20), nucleotides 86284 to 86410 (intron 21), nucleotides 86528 to 86655 (intron 22), nucleotides 86809 to 87381 (intron 23), nucleotides 87572 to 87660 (intron 24), nucleotides 87786 to 88205 (intron 25), nucleotides 88341 to 89024 (intron 26), nucleotides 89187 to 89588 (intron 27), nucleotides 89709 to 90131 (intron 28), nucleotides 90294 to 90404 (intron 29), nucleotides 90568 to 91049 (intron 30), nucleotides 91181 to 92548 (intron 31), nucleotides 92579 to 92977 (intron 32), nucleotides 93107 to 95224 (intron 33), nucleotides 95292 to 96197 (intron 34).Positions 96335 to 96465 (intron 35), positions 96601 to 96847 (intron 36), positions 96986 to 97622 (intron 37), positions 97751 to 97856 (intron 38), positions 98028 to 98669 (intron 39), positions 98831 to 99592 (intron 40), positions 99736 to 100127 (intron 41), positions 100217 to 101517 (intron 42), positions 101617 to 101761 (intron 43), positions 102004 to 102668 (intron 44), positions 102848 to 102951 (intron 45), positions 103053 to 103494 (intron 46), positions 103692 to 105478 (intron 47), and positions 106497 to 108496 (intron 48).

[0159] In some embodiments, the otoferlin gene can be split into two or more segments between and / or within any suitable exons and / or introns, and each segment is contained in a different vector of the present disclosure. In some embodiments, the otoferlin gene is split at exon 21, i.e., exons 1 to 21 (inclusive) are within the first vector and exons 22 to 48 (inclusive) are split within the second vector. In some such embodiments, the otoferlin segments in the first and second vectors are derived from the otoferlin cDNA sequence and lack introns. That is, exons 1 to 21 (inclusive) are included in the first vector and exons 22 to 48 (inclusive) are included in the second vector, and each vector lacks the otoferlin intron. In some embodiments, the otoferlin gene can be split with one or more other exons and / or introns as long as, when combined with all other components of the vector, it does not exceed the packaging capacity of the vector. Human otoferlin gene sequence (ncbi.nlm.nih.gov / nuccore / 224465243) (SEQ ID NO: 12) Mouse otoferlin protein (SEQ ID NO: 13) (NCBI accession number NP_001300696.1) Mouse otoferlin cDNA (SEQ ID NO: 14) (NCBI accession number NM_001313767.1) Mouse otoferlin gene sequence (www.ncbi.nlm.nih.gov / gene / 83762) (SEQ ID NO: 15) Accession number: NC_000071, Region: complement (30367066..30462730) GPC_000000778, NCBI reference sequence: NC_000071.6 Zebrafish otoferlin A gene sequence (www.ncbi.nlm.nih.gov / gene / 557476) (SEQ ID NO: 16) Accession number: NC_007131, Region: 31173357..31310109 GPC_000001574 NCBI reference sequence: NC_007131.7 Rhesus monkey otoferlin gene sequence (www.ncbi.nlm.nih.gov / gene / 696717) (SEQ ID NO: 17) Accession number: NC_027905 Region: complement (26723411..26826586) GPC_000002105 NCBI reference sequence: NC_027905.1 Dog otoferlin gene sequence (www.ncbi.nlm.nih.gov / gene / 607961) (SEQ ID NO: 18) Accession number: NC_006599, Region: complement (20518502..20619461) GPC_000000676 NCBI reference sequence: NC_006599.3 Chimpanzee otoferlin gene sequence (www.ncvi.nlm.nih.gov / gene / 459083) (SEQ ID NO: 19) Accession number: NC_006469, Region: complement (27006052..27107747) GPC_000002338 NCBI reference sequence: NC_006469.4 Rat otoferlin protein (SEQ ID NO: 20) Zebrafish otoferlin protein (SEQ ID NO: 21) Cow otoferlin protein (SEQ ID NO: 22) Buboon otoferlin protein (SEQ ID NO: 23)

[0160] In some embodiments, the first vector comprises the 5' portion of OTOF cDNA having a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 94. In some embodiments, the first vector comprises the 5' portion of OTOF cDNA having the sequence of SEQ ID NO: 94.

[0161] In some embodiments, the second vector comprises the 3' portion of OTOF cDNA having a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 95. In some embodiments, the second vector comprises the 5' portion of OTOF cDNA having the sequence of SEQ ID NO: 95. 5'mOTOF DNA sequence (SEQ ID NO: 94)

[0162] Some embodiments of any of the compositions described herein may include a first vector comprising the coding sequence of SEQ ID NO: 94 (or comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 94). Some embodiments of any of the compositions described herein may include a second vector comprising the coding sequence of SEQ ID NO: 95 (or comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 95).

[0163] Some embodiments of any of the compositions described herein may include a first rAAV vector having a 5' OTOF coding region that includes exon 1 to exon 21 (inclusive) of OTOF cDNA. In some embodiments, the composition includes a first vector having the nucleotide sequence of SEQ ID NO: 101 (or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 101). In some embodiments, the composition includes a first rAAV vector that includes a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 101 and that encodes the same amino acid sequence as that encoded by SEQ ID NO: 101.

[0164] In some embodiments, the compositions of the disclosure include a first rAAV vector that is a codon-optimized version of SEQ ID NO: 101, i.e., a nucleotide sequence that encodes the same amino acid sequence as that encoded by SEQ ID NO: 101 but that includes codons optimized for expression in a particular cell type, such as a mammalian cell, such as a human cell. In some embodiments, the first vector does not include any other part of the OTOF gene. In some embodiments, the first vector does not include any other part of the OTOF cDNA.

[0165] In some embodiments, the composition comprises a second rAAV vector having a 3' OTOF coding region that includes exon 22 to exon 48 (inclusive) of the OTOF cDNA. In some embodiments, the composition comprises a second vector (or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 107) that includes the nucleotide sequence of SEQ ID NO: 107. In some embodiments, the composition comprises a second rAAV vector that includes a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 108 and that encodes the same amino acid sequence as that encoded by SEQ ID NO: 107.

[0166] In some embodiments, the composition comprises a second rAAV vector that is a codon-optimized version of SEQ ID NO: 107, i.e., a nucleotide sequence that encodes the same amino acid sequence as that encoded by SEQ ID NO: 107 but that includes codons optimized for expression in a particular cell type, such as a mammalian cell, such as a human cell. In some embodiments, the second rAAV vector does not include any other part of the OTOF gene. In some embodiments, the second rAAV vector does not include any other part of the OTOF cDNA.

[0167] One of ordinary skill in the art will understand that amino acids that are not conserved among wild-type otoferrin proteins from different species can be mutated without losing activity, while amino acids that are conserved among wild-type otoferrin proteins from different species should not be mutated because they are more likely (than amino acids that are not conserved among different species) to be involved in activity.

[0168] ITR In some embodiments, the AAV vector comprises 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the rAAV vector comprises 5' and 3' inverted terminal repeats. In some embodiments, the AAV sequence of the vector comprises cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., B.J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). In some embodiments, the ITR sequence is about 145 nt in length. In some embodiments, substantially complete sequences encoding the ITRs are used in the molecule, although some minimal modification of these sequences is tolerated. The ability to modify these ITR sequences is within the skill of those in the art (see, e.g., Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York, 1989; and K. Fisher et al., J Virol. 70:520-532 (1996)). An example of such a molecule used in the present disclosure is a “cis-acting” plasmid containing the transgene, wherein the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the ITR is 145 nucleotides or comprises 145 nucleotides.

[0169] In some embodiments, the ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or Anc80 ITR. In some embodiments, the ITR is AAV2 ITR. In some embodiments, the 5’ ITR has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 97. In some embodiments, the 5’ ITR has the sequence of SEQ ID NO: 97. In some embodiments, the 3’ ITR has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 104. In some embodiments, the 3’ ITR comprises the sequence of SEQ ID NO: 104.

[0170] In some embodiments, the ITR is wild-type AAV2 ITR, for example, the 5’ ITR of SEQ ID NO: 97 and the 3’ ITR of SEQ ID NO: 104. In some embodiments, as known in the art, the ITR is derived from wild-type AAV2 ITR and includes one or more modifications, such as cleavage, deletion, substitution, or insertion. In some embodiments, the ITR comprises fewer than 145 nucleotides, for example, 127, 130, 134, or 141 nucleotides. For example, in some embodiments, the ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 nucleotides.

[0171] In some embodiments, the 5’ ITR has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 59. In some embodiments, the 3’ ITR has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 60.

[0172] In some embodiments, the 5’ AAV ITR sequence has the sequence of SEQ ID NO: 59. In some embodiments, the 3’ AAV ITR sequence has the sequence of SEQ ID NO: 60. In some embodiments, the vectors and / or constructs of the present disclosure include a 5’ AAV ITR and / or a 3’ AAV ITR. In some embodiments, the 5’ AAV ITR sequence has the sequence of SEQ ID NO: 97. In some embodiments, the 3’ AAV ITR sequence has the sequence of SEQ ID NO: 104. In some embodiments, the 5’ AAV ITR sequence is SEQ ID NO: 97 and the 3’ AAV ITR sequence is SEQ ID NO: 104. In some embodiments, the 5’ and 3’ AAV ITRs (e.g., SEQ ID NOs: 97 and 104) are adjacent to a transgene and / or a portion of a construct that includes a portion of OTOF (e.g., SEQ ID NO: 101 or 107).

[0173] In some embodiments, the recombinant AAV vector includes a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 96. In some embodiments, the recombinant AAV vector includes a nucleic acid sequence having the sequence of SEQ ID NO: 96.

[0174] In some embodiments, the recombinant AAV vector comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 105. In some embodiments, the recombinant AAV vector comprises a nucleic acid sequence having the sequence of SEQ ID NO: 105.

[0175] Promoter In some embodiments, the first recombinant adeno-associated virus (rAAV) vector comprises a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otopetrin gene. In some embodiments, the first recombinant adeno-associated virus (rAAV) vector genome comprises a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otopetrin gene.

[0176] In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is selected from the CAG, CBA, or CMV promoter. In some embodiments, the constitutive promoter is the CAG promoter.

[0177] Non-limiting examples of promoters are described herein. Further examples of promoters are known in the art.

[0178] In some embodiments, a vector encoding the N-terminal portion of an otopetrin protein (e.g., a human otopetrin protein) may comprise a promoter and / or enhancer. A vector encoding the N-terminal portion of an otopetrin protein may comprise any of the promoters and / or enhancers described herein or known in the art.

[0179] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art. In some embodiments, the promoter is an RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some embodiments, the promoter is an RNA polymerase III promoter, including but not limited to the H1 promoter, the human U6 promoter, the mouse U6 promoter, or the porcine U6 promoter. Generally, the promoter is a promoter capable of promoting transcription in cochlear cells such as hair cells. In some examples, the promoter is a cochlea-specific promoter or a cochlea-directed promoter.

[0180] Various promoters that can be used in this specification are known in the art. Non-limiting examples of promoters that can be used in this specification include the promoter of human elongation factor 1α-subunit (EF1a) (Liu et al. (2007) Exp. Mol. Med. 39(2):170-175; accession number: J04617.1; Gill et al., Gene Ther. 8(20):1539-1546, 2001; Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332; Ikeda et al., Gene Ther. 9:932-938, 2002; Gilham et al., J. Gene Med. 12(2):129-136, 2010), cytomegalovirus (Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332; Gray et al., Human Gene Ther. 22:1143-1153, 2011), human immediate early cytomegalovirus (CMV) (U.S. Patent No. 5,168,062, Liu et al. (2007) Exp. Mol. Med. 39(2):170-175; accession number: X17403.1 or KY490085.1), human ubiquitin C (UBC) (Gill et al., Gene Ther. 8(20):1539-1546, 2001; Qin et al., PLoS One 5(5):e10611, 2010), mouse phosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40), β-globin, β-actin, α-fetoprotein, γ-globulin, β-interferon, γ-glutamyltransferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g., human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, mouse MX gene, glucose-regulated protein (GRP78 and GRP94), α-2-macroglobulin, vimentin, MHC class I gene H-2κ b, HSP70, prolferin, tumor necrosis factor, thyroid-stimulating hormone α gene, immunoglobulin light chain, T-cell receptor, HLA DQα and DQβ, interleukin-2 receptor, MHC class II, MHC class II HLA-DRα, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponin I (TN I), Duchenne muscular dystrophy, human immunodeficiency virus, gibbon ape leukemia virus (GALV) promoter, promoter of HNRPA2B1-CBX1 (UCOE) (Powell and Gray (2015) Discov. Med. 19(102):49-57; Antoniou et al., Human Gene Ther. 24(4):363-374, 2013), β-glucuronidase (GUSB) (Husain et al., Gene Ther. 16:927-932, 2009), chicken β-actin (CBA) (Liu et al. (2007) Exp. Mol. Med. 39(2):170-175; stone et al. (2005) Mol. Ther. 11(6):843-848; Klein et al., Exp. Neurol.176(1):66 - 74, 2002; Ohlfest et al., Blood 105:2691 - 2698, 2005; Gray et al., Human Gene Ther. 22:1143 - 1153, 2011), human β - actin promoter (HBA) (accession number: Y00474.1), mouse myosin VIIA (musMyo7) (Boeda et al. (2001) Hum. Mol. Genet. 10(15):1581 - 1589; accession number: AF384559.1), human myosin VIIA (hsMyo7) (Boeda et al. (2001) Hum. Mol. Genet. 10(15):1581 - 1589; accession number: NG_009086.1), mouse poly(ADP - ribose) polymerase 2 (musPARP2) (Ame et al. (2001) J. Biol. Chem. 276(14):11092 - 11099; accession number: AF191547.1), human poly(ADP - ribose) polymerase 2 (hsPARP2) (Ame et al. (2001) J. Biol. Chem. 276(14):11092 - 11099; accession number: X16612.1 or AF479321.1), acetylcholine receptor epsilon subunit (AChε) (Ducclert et al. (1993) PNAS 90(7):3043 - 3047; accession number: S58221.1 or CR933736.12), Rous sarcoma virus (RSV) (Liu et al. (2007) Exp. Mol. Med. 39(2):170 - 175; accession number: M77786.1), (GFAP) (Liu et al. (2007) Exp. Mol. Med. 39(2):170 - 175; Stone et al. (2005) Mol. Ther. 11(6):843 - 848; accession number: NG_008401.1 or M67446.1), hAAT (Van Linthout et al., Human Gene Ther. 13(7):829 - 840, 2002; Cunningham et al., Mol. Ther. 16(6):1081 - 1088, 2008), and CBA hybrid (CBh) (Gray et al. (2011) Hum. Gen.Therapy 22: 1143-1153; accession number: KF926476.1 or KC152483.1). Further examples of promoters are known in the art. See, for example, Lodish, Molecular Cell Biology, Freeman and Company, New York 2007. In some embodiments, the promoter is the CMV immediate early promoter.

[0181] In some embodiments, the promoter is the CAG promoter or the CAG / CBA promoter. In some embodiments, the vectors or constructs of the present disclosure include the CAG promoter.

[0182] In some embodiments, the CAG promoter includes the nucleotide sequences of SEQ ID NOs: 98, 99, and 100 in order from 5' to 3'.

[0183] In some embodiments, the CAG promoter includes a CMV early enhancer element, a chicken beta-actin (CBA) gene sequence, and a chimeric intron / 3' splice sequence derived from the rabbit beta-globin gene.

[0184] In some embodiments, the CMV early enhancer element includes a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 98. In some embodiments, the CMV early enhancer element has the sequence of SEQ ID NO: 98.

[0185] In some embodiments, the CBA gene sequence has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 99. In some embodiments, the CBA gene sequence has the sequence of SEQ ID NO: 99.

[0186] In some embodiments, the chimeric intron / 3' splice sequence derived from the rabbit beta-globin gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 100. In some embodiments, the chimeric intron / 3' splice sequence from the rabbit beta-globin gene has the sequence of SEQ ID NO: 100.

[0187] Examples of constitutive promoters include, but are not limited to, the CAG promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (e.g., Boshhart et al. Cell 41:521-530, 1985), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1-alpha promoter (Invitrogen).

[0188] Inducible promoters enable the control of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological states (e.g., only in the acute phase, a specific differentiation state of cells, or replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those skilled in the art.

[0189] Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088), the ecdysone insect promoter (No et al. Proc. Natl. Acad. Sci. USA 93:3346 - 3351, 1996), the tetracycline repressible system (Gossen et al. Proc. Natl. Acad. Sci. USA 89:5547 - 5551, 1992), the tetracycline inducible system (see also Gossen et al. Science 268:1766 - 1769, 1995, Harvey et al. Curr. Opin. Chem. Biol. 2:512 - 518, 1998), the RU486 inducible system (Wang et al, Nat. Biotech. 15:239 - 243, 1997) and Wang et al, Gene Ther. 4:432 - 441, 1997), and the rapamycin inducible system (Magari et al. J. Clin. Invest. 100:2865 - 2872, 1997).

[0190] In some embodiments, the tissue-specific promoter has activity only in the inner ear. In some embodiments, the tissue-specific promoter has activity only within the cochlea. In some embodiments, the tissue-specific promoter has activity only within hair cells. In some embodiments, the tissue-specific promoter has activity only within inner hair cells.

[0191] Exemplary tissue-specific promoters include the following tissue-specific promoters: liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter (Kugler et al., Virology 311:89-95, 2003; Hiki et al., Gene Ther. 14:872-882, 2007; Kuroda et al., J. Gene Med. 10:1163-1175, 2008), creatine kinase (MCK) promoter (Wang et al., Gene Ther. 15:1489-1499, 2008; Talbot et al., Mol. Ther. 18:601-608, 2010; Katwal et al., Gene Ther. 20(9):930-938, 2013), mammalian desmin (DES) promoter (Talbot et al., Mol. Ther. 18:601-608, 2010), C5-12 promoter (Wang et al., Gene Ther. 15:1489-1499, 2008), α-myosin heavy chain (a-MHC) promoter, PDGF promoter (Patterna, Gene Ther. 7(15):1304-1311, 2000; Hiki et al. Gene Ther. 14:872-882, 2007), MecP2 promoter (Rategar et al., PLoS One 4:e6810, 2009; Gray et al., Human Gene Ther. 22:1143-1153, 2011), CaMKII promoter (Hioki et al., Gene Ther. 14:872-882, 2007; Kuroda et al., J. Gene Med. 10:1163-1175, 2008), mGluR2 promoter (Brene et al., Eur. J. Eurosci. 12:1525-1533, 2000; Kuroda et al., J. Gene Med. 10:1163-1175, 2008), NFL promoter (Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther.8:1323 - 1332, 2001), NFH promoter (Xu et al., Human Gene Ther. 12(5):563 - 573, 2001; Xu et al., Gene Ther. 8:1323 - 1332, 2001), nθ2 promoter (Xu et al., Human Gene Ther. 12(5):563 - 573, 2001; Xu et al., Gene Ther. 8:1323 - 1332, 2001), PPE promoter (Xu et al., Human Gene Ther. 12(5):563 - 573, 2001; Xu et al., Gene Ther. 8:1323 - 1332, 2001), Enk promoter (Xu et al., Human Gene Ther. 12(5):563 - 573, 2001; Xu et al., Gene Ther. 8:1323 - 1332, 2001), EAAT2 promoter (Su et al., Proc. Natl. Acad. Sci. USA 100:1955 - 1960, 2003; Kuroda et al., J. Gene Med. 10:1163 - 1175, 2008), GFAP promoter (Brenner et al., J. Neurosci. 14:1030 - 1037, 1994; Xu et al., Human Gene Ther. 12(5):563 - 573, 2001; Xu et al., Gene Ther. 8:1323 - 1332, 2001; Lee et al., Glia 56:481 - 493, 2008; Dirren et al., Human Gene Ther. 25:109 - 120, 2014), MBP promoter (Chen et al., Gene Ther. 5(1):50 - 58, 1998), or cardiac troponin T (cTnT) promoter, but are not limited thereto. Other exemplary promoters include the beta - actin promoter, hepatitis B virus core promoter (Sandig et al., Gene Ther. 3:1002 - 9(1996)); alpha - fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther. 7:1503 - 14(1996)), bone osteocalcin promoter (Stein et al., Mol.Biol. Rep. 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res. 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998)); immunoglobulin heavy chain promoter; T cell receptor alpha chain promoter, nerve cells, such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993); Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332, 2001), the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. U.S.A. 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron 15:373-84 (1995)), etc., which are obvious to those skilled in the art.

[0192] In some embodiments, the control sequence confers tissue-specific gene expression ability. In some cases, the tissue-specific control sequence binds to a tissue-specific transcription factor that induces transcription in a tissue-specific manner.

[0193] In some embodiments, the tissue-specific promoter is a snail-specific promoter. In some embodiments, the tissue-specific promoter is a snail hair cell-specific promoter. Non-limiting examples of snail hair cell-specific promoters include, but are not limited to, the ATOH1 promoter, the POU4F3 promoter, the LHX3 promoter, the MYO7A promoter, the MYO6 promoter, the α9ACHR promoter, and the α10ACHR promoter.

[0194] In another aspect, a native promoter for the transgene is used. A native promoter may be preferred when it is desired that the expression of the transgene mimic native expression. The native promoter may be used when it is necessary to regulate the expression of the transgene transiently or developmentally, or tissue-specifically, or in response to a specific transcriptional stimulus. In a further aspect, other native expression control elements such as enhancer elements, polyadenylation sites, or Kozak consensus sequences may also be used to mimic native expression.

[0195] Enhancer In some cases, the vector may contain a promoter sequence and / or an enhancer sequence. The term "enhancer" refers to a nucleotide sequence that can increase the transcriptional level of a nucleic acid encoding a protein of interest (e.g., otoferlin protein). The enhancer sequence (50-1500 base pairs in length) generally increases the transcriptional level by providing additional binding sites for transcription-related proteins (e.g., transcription factors). In some aspects, the enhancer sequence is found within an intron sequence. Unlike promoter sequences, enhancer sequences can act much farther away from the transcription start site (e.g., compared to the promoter). Non-limiting examples of enhancers include the RSV enhancer, the CMV enhancer, and the SV40 enhancer. An example of the CMV enhancer is described, for example, in Boshart et al., Cell 41(2):521-530, 1985.

[0196] In some embodiments, the enhancer is the CMV immediate early enhancer. In some embodiments, the CMV immediate early enhancer element comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 98. In some embodiments, the CMV immediate early enhancer comprises the sequence of SEQ ID NO: 98.

[0197] Poly(A) sequence In some embodiments, any vector provided herein may contain a poly(A) sequence. Most nascent eukaryotic mRNAs have a poly(A) tail at their 3' end, which is added during a complex process that includes cleavage of the primary transcript and a coupled polyadenylation reaction (see, e.g., Proudfoot et al., Cell 108:501-512, 2002). The poly(A) tail confers mRNA stability and translatability (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994). In some embodiments, the poly(A) sequence is positioned 3' to the nucleic acid sequence encoding the C-terminus of otoferlin protein.

[0198] The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA functionality such as localization, stability, or translation efficiency.

[0199] There are several poly(A) signal sequences that can be used, including bovine growth hormone (bgh) (Woychik et al., Proc. Natl. Acad. Sci. U.S.A. 81(13):3944-3948, 1984; U.S. Patent No. 5,122,458; Yew et al., Human Gene Ther. 8(5):575-584, 1997; Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332, 2001; Wu et al., Mol. Ther. 16(2):280-289, 2008; Gray et al., Human Gene Ther. 22:1143-1153, 2011; Choi et al., Mol. Brain 7:17, 2014), mouse-β-globin, mouse-α-globin (Orkin et al., EMBO J. 4(2):453-456, 1985; Thein et al., Blood 71(2):313-319, 1988), human collagen, polyomavirus (Batt et al., Mol. Cell Biol. 15(9):4783-4790, 1995), herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy chain gene polyadenylation signal (US2006 / 0040354), human growth hormone (hGH) (Szymanski et al., Mol. Therapy 15(7):1340-1347, 2007; Ostegaard et al., Proc. Natl. Acad. Sci. U.S.A. 102(8):2952-2957, 2005), synthetic polyA (Levitt et al., Genes Dev. 3(7):1019-1025, 1989; Yew et al., Human Gene Ther. 8(5):575-584, 1997; Ostegaard et al., Proc. Natl. Acad. Sci. U.S.A. 102(8):2952-2957, 2005; Choi et al., Mol. Brain 7:17, 2014), HIV-1 upstream poly(A) enhancer (Schampch et al., Mol. Ther.Adenovirus (L3) upstream poly(A) enhancer (Schampch et al., Mol. Ther. 15(6):1167 - 1173, 2007), hTHGB upstream poly(A) enhancer (Schampch et al., Mol. Ther. 15(6):1167 - 1173, 2007), hC2 upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6):1167 - 1173, 2007), SV40 poly(A) signal sequences, for example, those from the group consisting of SV40 late and early poly(A) signal sequences (Schek et al., Mol. Cell Biol. 12(12):5386 - 5393, 1992; Choi et al., Mol. Brain 7:17, 2014; Schambach et al., Mol. Ther. 15(6):1167 - 1173, 2007) are also included. In some embodiments, the polyA sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 68. In some embodiments, the polyA sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 76. In some embodiments, the polyA sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 77. Non - limiting examples of the poly(A) signal sequence are SEQ ID NO: 68, 76, or 77.

[0200] The poly(A) signal sequence can be the sequence AATAAA. The AATAAA sequence may be replaced with other hexanucleotide sequences homologous to AATAAA that can signal polyadenylation, such sequences including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e.g., WO06 / 12414).

[0201] In some embodiments, the poly(A) signal sequence is a synthetic polyadenylation signal site (see, e.g., the Promega pCl-neo expression vector based on Levitt el al, Genes Dev. 3(7):1019-1025, 1989). In some embodiments, the poly(A) signal sequence is the polyadenylation signal of soluble neuropilin-1 (sNRP) (AAATAAAATACGAAATG) (see, e.g., WO05 / 073384).

[0202] In some embodiments, the poly(A) sequence is the bovine growth hormone poly(A) sequence. In some embodiments, the poly A sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 108. In some such embodiments, the bGH poly(A) sequence comprises or is the sequence of SEQ ID NO: 108. In some embodiments, the vectors or constructs of the present disclosure comprise the bovine growth hormone poly A sequence represented by SEQ ID NO: 108. Further examples of poly(A) signal sequences are known in the art.

[0203] Adjacent region untranslated region (UTR) In some embodiments, any vector described herein (e.g., either of at least two different vectors) may include an untranslated region. In some embodiments, the vector may include a 5’UTR or a 3’UTR.

[0204] The untranslated region (UTR) of a gene is transcribed but not translated. The 5’UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3’UTR starts immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods as described herein to enhance the stability of otoferlin protein.

[0205] Splice donor sequences and splice acceptor sequences In other embodiments, the non-UTR sequence may be incorporated into the 5’ or 3’ UTR. In some embodiments, a portion of an intron or intron sequence can be incorporated into the flanking regions of a polynucleotide in any of the vectors, compositions, kits, and methods provided herein. Incorporation of an intron sequence can increase protein production as well as mRNA levels. The intron can be an intron from the otoferlin gene or an intron from a heterologous gene, such as a hybrid adenovirus / mouse immunoglobulin intron (Yew et al., Human Gene Ter. 8(5):575-584, 1997), an SV40 intron (Ostedgaard et al., Proc. Natl. Acad. Sci. U.S.A. 102(8):2952-2957, 2005), an MVM intron (Wu et al., Mol. Ther. 16(2):280-289, 2008), a factor IX shortened intron 1 (Wu et al., Mol. Ther. 16(2):280-289, 2008, Kurachi et al., J. Biol. Chem. 270(10):5276-5281, 1995), a chimeric θ-globin splice donor / immunoglobulin heavy chain splice acceptor intron (Wu et al., Mol. Ther. 16(2):280-289, 2008, Choi et al., Mol. Brain 7:17, 2014), an SV40 late splice donor / splice acceptor intron (19S / 16S) (Yew et al., Human Gene Ther. 8(5):575-584, 1997), a hybrid adenovirus splice donor / IgG splice acceptor (Choi et al., Mol. Brain 7:17, 1991, Huang and Gorman, Mol. Cell Biol. 10(4):1805-1810, 1990).

[0206] In some embodiments, the splice donor has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 64. In some embodiments, the splice donor has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 72. In some embodiments, the splice donor has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 74. In some embodiments, the splice donor has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 102. In some embodiments, the splice donor has the sequence of SEQ ID NO: 102.

[0207] In some embodiments, the splice acceptor has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 65. In some embodiments, the splice acceptor has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 73. In some embodiments, the splice acceptor has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 75. In some embodiments, the splice acceptor has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 106. In some embodiments, the splice acceptor has the sequence of SEQ ID NO: 106.

[0208] Non-limiting examples of splice donor sequences and splice acceptor sequences include SEQ ID NOs: 64 and 65, SEQ ID NOs: 72 and 73, SEQ ID NOs: 74 and 75, and SEQ ID NOs: 102 and 106, respectively.

[0209] In some embodiments, the splice donor sequence has the sequence of SEQ ID NO: 102. In some embodiments, the vector of the construct of the present disclosure includes the splice donor sequence of SEQ ID NO: 102. In some such embodiments, a vector or construct comprising a splice donor sequence (e.g., SEQ ID NO: 102) also includes the OTOF gene or the 5' portion of the OTOF cDNA (e.g., SEQ ID NO: 101) upstream of the splice donor sequence.

[0210] In some embodiments, the first rAAV vector has a splice donor sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 102. In some embodiments, the first rAAV vector includes a first expression cassette comprising a splice donor sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 102.

[0211] In some embodiments, the first rAAV vector genome comprises a splice donor sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 102. In some embodiments, the first rAAV vector genome comprises a first expression cassette that comprises a splice donor sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 102. In some embodiments, the splice donor has the sequence of SEQ ID NO: 102.

[0212] In some embodiments, the splice acceptor sequence has the sequence of SEQ ID NO: 106. In some embodiments, the vectors or constructs of the disclosure comprise a splice acceptor sequence of SEQ ID NO: 106. In some such embodiments, a vector or construct comprising a splice acceptor sequence (e.g., SEQ ID NO: 106) also comprises the OTOF gene or the 3’ portion of the OTOF cDNA (e.g., SEQ ID NO: 107) downstream of the splice acceptor sequence.

[0213] In some embodiments, the second rAAV vector comprises a splice acceptor sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 106. In some embodiments, the second rAAV vector comprises a second expression cassette that comprises a splice acceptor sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 106.

[0214] In some embodiments, the second rAAV vector genome comprises a splice acceptor sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 106. In some embodiments, the second rAAV vector genome comprises a second expression cassette that comprises a splice acceptor sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 106. In some embodiments, the splice acceptor has the sequence of SEQ ID NO: 106.

[0215] Recombinase-inducible sequence In some embodiments, the vectors or constructs of the present disclosure comprise one or more recombination-inducible sequences (s). In some embodiments, the recombination-inducible sequence is part of or comprises a gene sequence. In some embodiments, the recombination-inducible sequence is derived from an alkaline phosphatase gene. In some embodiments, the recombination-inducible sequence is derived from phage F1. In some such embodiments, the recombination-inducible sequence is the AK sequence derived from phage F1.

[0216] In some embodiments, the AK recombination-inducible sequence has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 103. In some embodiments, the AK recombination-inducible sequence is SEQ ID NO: 103. In some embodiments of the dual vector system of the present disclosure, each of the two vectors comprises a recombination-inducible sequence.

[0217] In some embodiments, the first rAAV vector comprises an AK recombination-inducible sequence having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 103. In some embodiments, the first rAAV vector comprises an AK recombination-inducible sequence having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 103. In some embodiments, the AK recombination-inducible sequence has the sequence of SEQ ID NO: 103.

[0218] In some embodiments, the first rAAV vector comprises an AK recombinase-inducible sequence having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 103. In some embodiments, the first rAAV vector genome comprises an AK recombinase-inducible sequence having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 103. In some embodiments, the AK recombinase-inducible sequence has the sequence of SEQ ID NO: 103.

[0219] Additional sequence In addition to the major elements identified above for the recombinant AAV vector, the vector also comprises conventional control elements operably linked to the transgene in a manner that enables its transcription, translation, and / or expression in a cell transfected with a plasmid vector produced by the present disclosure or a cell infected with a virus produced by the present disclosure.

[0220] Any of the vectors provided herein may optionally include an additional nucleotide sequence ("stuffer sequence") to optimize the total number of base pairs in the vector.

[0221] In some embodiments, the stuffer sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 54. In some embodiments, the stuffer sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 55. In some embodiments, the stuffer sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 56. In some embodiments, the stuffer sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 57. In some embodiments, the stuffer sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 58.In some embodiments, the stuffer sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 90. In some embodiments, the stuffer sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 91. SEQ ID NOS: 54-58, 90, and 91 are exemplary human Factor VIII stuffer sequences that can be used in any of the vectors described herein. Additional examples of stuffer sequences are known in the art.

[0222] Capsid In some embodiments, the recombinant AAV vectors of the disclosure are packaged into the capsids of AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rh10, rh39, rh43, or Anc80 serotypes, or hybrids of one or more thereof. In some embodiments, the capsid is derived from an ancestral serotype. For example, in some embodiments, the capsid is an Anc80 capsid (e.g., Anc80L65 capsid).

[0223] In some embodiments, the capsid comprises a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 109. In some embodiments, the capsid comprises the polypeptide represented by SEQ ID NO: 109.

[0224] Any combination of ITRs and capsids can be used in the recombinant AAV vectors of the present disclosure, for example, wild-type or variant AAV2 ITRs and Anc80 capsids, wild-type or variant AAV2 ITRs and AAV6 capsids, etc. In some embodiments of the present disclosure, the rAAV particles are rAAV2 / Anc80 particles comprising a nucleic acid vector comprising wild-type AAV2 ITRs (e.g., SEQ ID NOs: 97 and 104) adjacent to a portion of a construct comprising a transgene and / or a portion of OTOF, and encapsidated by an Anc80 capsid (e.g., comprising the polypeptide of SEQ ID NO: 109).

[0225] In some embodiments, the first and second rAAV vectors are encapsulated by an AAV capsid. In some embodiments, the AAV capsid encapsulating the first rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80. In some embodiments, the AAV capsid encapsulating the second rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80. In some embodiments, the first rAAV vector is encapsulated by an Anc80 capsid and the second rAAV vector is encapsulated by an Anc80 capsid.

[0226] In some embodiments, the first and second rAAV vector genomes are encapsulated by AAV capsids. In some embodiments, the AAV capsid encapsulating the first rAAV vector genome is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80. In some embodiments, the AAV capsid encapsulating the second rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80. In some embodiments, the first rAAV vector genome is encapsulated by an Anc80 capsid and the second rAAV vector genome is encapsulated by an Anc80 capsid.

[0227] In some embodiments, the Anc80 capsid comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 109. In some embodiments, the Anc80 capsid comprises the polypeptide of SEQ ID NO: 109.

[0228] In some embodiments, the first recombinant AAV particle comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 96. In some embodiments, the first recombinant AAV particle comprises a nucleic acid sequence having the sequence of SEQ ID NO: 96.

[0229] In some embodiments, the second recombinant AAV particle comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 105. In some embodiments, the second recombinant AAV particle comprises a nucleic acid sequence having the sequence of SEQ ID NO: 105.

[0230] Vector Provided herein are compositions and methods for treating deafness using nucleic acid therapeutics such as auditory polypeptide messenger RNA (e.g., the otoferlin gene). In some embodiments, the auditory polypeptide nucleic acid (e.g., the otoferlin gene) is present in viral vectors such as adeno-associated virus vectors, adenovirus vectors, lentivirus vectors, and retrovirus vectors. In some embodiments, the vector is an adeno-associated virus vector. In some embodiments, the vector is a recombinant adeno-associated virus vector.

[0231] The foregoing methods for packaging a recombinant vector into a desired AAV capsid to generate the rAAV (or rAAV particle) of the present disclosure are not limiting, and other suitable methods will be apparent to those skilled in the art.

[0232] Recombinant AAV vector In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) vector. As used herein, a "recombinant AAV vector" or "rAAV" includes at least a transgene or a portion thereof and control sequences (e.g., a promoter), as well as 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV construct that is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequence encoding a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product of interest. The nucleic acid coding sequence is operably linked to the control components in a manner that enables transgene transcription, translation, and / or expression in the cells of the target tissue.

[0233] Such recombinant AAV vectors are packaged into a capsid to form rAAV particles and delivered to a selected target cell (e.g., inner hair cells). In some embodiments, one or more of the recombinant AAV vectors of the present disclosure are packaged into a capsid of AAV serotype 2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rh10, rh39, rh43, or Anc80, or a capsid of one or more hybrids thereof.

[0234] In some embodiments, the rAAV of the present disclosure is capsidated by an Anc80 capsid. In some embodiments, the Anc80 capsid comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 109. In some embodiments, the Anc80 capsid comprises the polypeptide of SEQ ID NO: 109.

[0235] In some embodiments of any of the compositions described herein, the first vector comprises an ITR (e.g., any of the exemplary ITR sequences described herein), a promoter and / or enhancer (e.g., any of the exemplary enhancers described herein and any of the exemplary promoters described herein), a sequence encoding the first N-terminal portion of the human otoferlin protein (e.g., any of the exemplary sequences encoding the first N-terminal portion of the human otoferlin protein described herein), a splicing donor site (e.g., any of the exemplary splicing donor sites described herein), an AK sequence (e.g., any of the exemplary AK sequences described herein), and an ITR (e.g., any of the exemplary ITR sequences described herein).

[0236] In some embodiments of any of the compositions described herein, the second vector comprises an ITR sequence (e.g., any of the exemplary ITR sequences described herein), an AK sequence (e.g., any of the exemplary AK sequences described herein), a splicing acceptor sequence (e.g., any of the splicing acceptor sequences described herein), a sequence encoding the second portion of the human otoferlin protein (e.g., any of the exemplary sequences encoding the second C-terminal portion of the human otoferlin protein described herein), a poly(A) signal sequence (e.g., any of the exemplary poly(A) signal sequences described herein), and an ITR sequence (e.g., any of the exemplary ITR sequences described herein).

[0237] The vectors provided herein may be of different sizes. The selection of a vector for use in any of the compositions, kits, and methods described herein may depend on the size of the vector. In some embodiments, the vector(s) is an adeno-associated virus (AAV vector) and may contain a total of up to 5 kb of nucleotides. In some embodiments, the AAV vector(s) may contain a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

[0238] In some embodiments of any of the compositions, kits, and methods provided herein, two different vectors may be of the same general type of vector and may differ in size. In some embodiments, two different vectors may be of different types of vectors and may be of substantially the same size or different sizes.

[0239] Any of the vectors disclosed herein can be introduced into mammalian cells (e.g., inner hair cells in the cochlea) using a variety of different methods known in the art. Non-limiting examples of methods for introducing nucleic acids into mammalian cells include lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.

[0240] One of ordinary skill in the art will understand that any vector described herein can be introduced into mammalian cells, for example, by lipofection.

[0241] A variety of molecular biology techniques that can be used to introduce mutations and / or deletions into endogenous genes are also known in the art. Non-limiting examples of such techniques include site-directed mutagenesis, CRISPR (e.g., CRISPR / Cas9-induced knock-in mutations and CRISPR / Cas9-induced knockout mutations), and TALEN. These methods can be used to correct the sequence of a defective endogenous gene present in the chromosome of a target cell.

[0242] In some embodiments, the compositions of the present disclosure include a first rAAV vector comprising a first expression cassette comprising a 5' inverted terminal repeat, a promoter operably linked to the 5' portion of the OTOF gene or OTOF cDNA, a splice donor sequence, an AK recombination-inducing sequence, and a 3' inverted terminal repeat.

[0243] In some embodiments, the compositions of the present disclosure include a second rAAV vector comprising a second expression cassette comprising a 5' inverted terminal repeat, an AK recombination-inducing sequence, a splice acceptor sequence, the 3' portion of the OTOF gene or OTOF cDNA, a polyadenylation sequence, and a 3' inverted terminal sequence.

[0244] In some embodiments, the compositions of the present disclosure include a first rAAV vector genome comprising a first expression cassette comprising a 5' inverted terminal repeat, a promoter operably linked to the 5' portion of the OTOF gene or OTOF cDNA, a splice donor sequence, an AK recombination-inducing sequence, and a 3' inverted terminal repeat.

[0245] In some embodiments, the compositions of the present disclosure include a second rAAV vector comprising a second expression cassette comprising a 5' inverted terminal repeat, an AK recombination-inducing sequence, a splice acceptor sequence, the 3' portion of the OTOF gene or OTOF cDNA, a polyadenylation sequence, and a 3' inverted terminal sequence.

[0246] In some embodiments, the compositions of the present disclosure include a first rAAV vector comprising a first expression cassette comprising, in the 5' to 3' direction, a 5' inverted terminal repeat, a promoter operably linked to the 5' portion of the OTOF gene or OTOF cDNA, a splice donor sequence, an AK recombination-inducing sequence, and a 3' inverted terminal repeat.

[0247] In some embodiments, the compositions of the present disclosure include a second rAAV vector comprising a second expression cassette comprising, in the 5' to 3' direction, a 5' inverted terminal repeat, an AK recombination-inducing sequence, a splice acceptor sequence, the 3' portion of the OTOF gene or OTOF cDNA, a polyadenylation sequence, and a 3' inverted terminal sequence.

[0248] In some embodiments, the compositions of the present disclosure include a first rAAV vector genome comprising a first expression cassette comprising, in the 5' to 3' direction, a 5' inverted terminal repeat, a promoter operably linked to the 5' portion of the OTOF gene or OTOF cDNA, a splice donor sequence, an AK recombination-inducing sequence, and a 3' inverted terminal repeat.

[0249] In some embodiments, the compositions of the present disclosure include a second rAAV vector genome comprising a second expression cassette comprising, in the 5' to 3' direction, a 5' inverted terminal repeat, an AK recombination-inducing sequence, a splice acceptor sequence, the 3' portion of the OTOF gene or OTOF cDNA, a polyadenylation sequence, and a 3' inverted terminal sequence.

[0250] In some embodiments, the composition of the present disclosure comprises a first rAAV vector comprising a first expression cassette comprising: i) a 5' inverted terminal repeat having the sequence of SEQ ID NO: 97; ii) iii) a promoter having the sequences of SEQ ID NO: 98, 99, and 100 operably linked to the 5' portion of the OTOF gene or OTOF cDNA having the sequence of SEQ ID NO: 101; iv) a splice donor sequence having the sequence of SEQ ID NO: 102; v) an AK recombination-inducing sequence having the sequence of SEQ ID NO: 103; and vi) a 3' inverted terminal repeat having the sequence of SEQ ID NO: 104.

[0251] In some embodiments, the composition of the present disclosure comprises a second rAAV vector comprising a second expression cassette comprising: i) a 5' inverted terminal repeat having the sequence of SEQ ID NO: 97; ii) an AK recombination-inducing sequence having the sequence of SEQ ID NO: 103; iii) a splice acceptor sequence having the sequence of SEQ ID NO: 106; iv) the 3' portion of the OTOF gene or OTOF cDNA having the sequence of SEQ ID NO: 107; v) a polyadenylation sequence having the sequence of SEQ ID NO: 108; and vi) a 3' inverted terminal repeat having the sequence of SEQ ID NO: 104.

[0252] In some embodiments, the composition of the present disclosure comprises a first rAAV vector genome comprising a first expression cassette comprising: i) a 5' inverted terminal repeat having the sequence of SEQ ID NO: 97; ii) iii) a promoter having the sequences of SEQ ID NO: 98, 99, and 100 operably linked to the 5' portion of the OTOF gene or OTOF cDNA having the sequence of SEQ ID NO: 101; iv) a splice donor sequence having the sequence of SEQ ID NO: 102; v) an AK recombination-inducing sequence having the sequence of SEQ ID NO: 103; and vi) a 3' inverted terminal repeat having the sequence of SEQ ID NO: 104.

[0253] In some embodiments, the compositions of the present disclosure include a second rAAV vector genome comprising a second expression cassette that includes: i) a 5' inverted terminal repeat having the sequence of SEQ ID NO: 97; ii) an AK recombination-inducing sequence having the sequence of SEQ ID NO: 103; iii) a splice acceptor sequence having the sequence of SEQ ID NO: 106; iv) a 5' portion of the OTOF gene or OTOF cDNA having the sequence of SEQ ID NO: 107; v) a polyadenylation sequence having the sequence of SEQ ID NO: 108; and vi) a 3' inverted terminal repeat having the sequence of SEQ ID NO: 104.

[0254] In some embodiments, the compositions of the present disclosure include a first rAAV vector comprising a first expression cassette that includes, in the 5' to 3' direction: i) a 5' inverted terminal repeat having the sequence of SEQ ID NO: 97; ii), iii) a promoter having the sequences of SEQ ID NOs: 98, 99, and 100 operably linked to a 5' portion of the OTOF gene or OTOF cDNA having the sequence of SEQ ID NO: 101; iv) a splice donor sequence having the sequence of SEQ ID NO: 102; v) an AK recombination-inducing sequence having the sequence of SEQ ID NO: 103; and vi) a 3' inverted terminal repeat having the sequence of SEQ ID NO: 104.

[0255] In some embodiments, the compositions of the present disclosure include a second rAAV vector comprising a second expression cassette that includes, in the 5' to 3' direction: i) a 5' inverted terminal repeat having the sequence of SEQ ID NO: 97; ii) an AK recombination-inducing sequence having the sequence of SEQ ID NO: 103; iii) a splice acceptor sequence having the sequence of SEQ ID NO: 106; iv) a 3' portion of the OTOF gene or OTOF cDNA having the sequence of SEQ ID NO: 107; v) a polyadenylation sequence having the sequence of SEQ ID NO: 108; and vi) a 3' inverted terminal repeat having the sequence of SEQ ID NO: 104.

[0256] In some embodiments, the composition of the present disclosure comprises a first rAAV vector genome comprising a first expression cassette comprising, in the 5' to 3' direction, i) a 5' inverted terminal repeat having the sequence of SEQ ID NO: 97, ii) iii) a promoter having the sequences of SEQ ID NOs: 98, 99, and 100, operably linked to the 5' portion of an OTOF gene or OTOF cDNA having the sequence of SEQ ID NO: 101, iv) a splice donor sequence having the sequence of SEQ ID NO: 102, v) an AK recombination-inducing sequence having the sequence of SEQ ID NO: 103, and vi) a 3' inverted terminal repeat having the sequence of SEQ ID NO: 104.

[0257] In some embodiments, the composition of the present disclosure comprises a second rAAV vector genome comprising a second expression cassette comprising, in the 5' to 3' direction, i) a 5' inverted terminal repeat having the sequence of SEQ ID NO: 97, ii) an AK recombination sequence having the sequence of SEQ ID NO: 103, iii) a splice acceptor sequence having the sequence of SEQ ID NO: 106, iv) the 5' portion of an OTOF gene or OTOF cDNA having the sequence of SEQ ID NO: 107, v) a polyadenylation sequence having the sequence of SEQ ID NO: 108, and vi) a 3' inverted terminal repeat having the sequence of SEQ ID NO: 104.

[0258] In some embodiments, the composition of the present disclosure comprises a first rAAV vector comprising a first expression cassette comprising the sequence of SEQ ID NO: 96. In some embodiments, the composition of the present disclosure comprises a second rAAV vector comprising a second expression cassette comprising the sequence of SEQ ID NO: 105.

[0259] In some embodiments, the composition of the present disclosure comprises a first rAAV vector genome comprising a first expression cassette comprising the sequence of SEQ ID NO: 96. In some embodiments, the composition of the present disclosure comprises a second rAAV vector genome comprising a second expression cassette comprising the sequence of SEQ ID NO: 105.

[0260] In some embodiments, the present disclosure provides a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5' portion of an otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises a total of about 4.1E10 to 8.1E12 vg.

[0261] In some embodiments, the present disclosure provides a composition comprising: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5' portion of an otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises a total of about 4.1E10 to 4.1E12 vg or about 8.1E10 to 8.1E12 vg.

[0262] In some embodiments, the composition comprises the first rAAV vector genome and the second rAAV vector genome in a ratio of about 1:1.

[0263] In some embodiments, the composition comprises a total of about 4.1E10 to 8.1E12 vg. In some embodiments, the composition comprises a total of about 4.1E10 to 4.1E12 vg or about 8.1E10 to 8.1E12 vg.

[0264] In some embodiments, the composition comprises a total vg of about 4.1E10 - 4.1E12, about 5.1E10 - 3.1E12, about 6.1E10 - 2.1E12, about 7.1E10 - 1.1E12, about 8.1E10 - 10.1E11, about 9.1E10 - 9.1E11, about 10.1E11 - 8.1E11, about 1.1E11 - 7.1E11, about 2.1E11 - 6.1E11, or about 3.1E11 - 5.1E11. In some embodiments, the composition comprises a total vg of about 3.1E11 - 5.1E11, about 3.2E11 - 5.0E11, about 3.3E11 - 4.9E11, about 3.4E11 - 4.8E11, about 3.5E11 - 4.7E11, about 3.6E11 - 4.6E11, about 3.7E11 - 4.5E11, about 3.8E11 - 4.4E11, about 3.9E11 - 4.3E11, or about 4.0E11 - 4.2E11. In some embodiments, the composition comprises a total vg of about 4.1E11.

[0265] In some embodiments, the composition comprises a total vg of about 4.1E10 - 8.1E12 per snail. In some embodiments, the composition comprises a total vg of about 4.1E10 - 4.1E12 per snail or about 8.1E10 - 8.1E12 per snail.

[0266] In some embodiments, the composition comprises a total vg of about 4.1E10 - 4.1E12, about 5.1E10 - 3.1E12, about 6.1E10 - 2.1E12, about 7.1E10 - 1.1E12, about 8.1E10 - 10.1E11, about 9.1E10 - 9.1E11, about 10.1E11 - 8.1E11, about 1.1E11 - 7.1E11, about 2.1E11 - 6.1E11, or about 3.1E11 - 5.1E11 per snail. In some embodiments, the composition comprises a total vg of about 3.1E11 - 5.1E11, about 3.2E11 - 5.0E11, about 3.3E11 - 4.9E11, about 3.4E11 - 4.8E11, about 3.5E11 - 4.7E11, about 3.6E11 - 4.6E11, about 3.7E11 - 4.5E11, about 3.8E11 - 4.4E11, about 3.9E11 - 4.3E11, or about 4.0E11 - 4.2E11 per snail. In some embodiments, the composition comprises a total vg of about 4.1E11 per snail.

[0267] In some embodiments, the composition comprises a total vg of about 3.0E11, about 3.1E11, about 3.2E11, about 3.3E11, about 3.4E11, about 3.5E11, about 3.6E11, about 3.7E11, about 3.8E11, about 3.9E11, about 4.0E11, about 4.1E11, about 4.2E11, about 4.3E11, about 4.4E11, about 4.5E11, about 4.6E11, about 4.7E11, about 4.8E11, about 4.9E11, about 5.0E11, about 5.1E11, about 5.2E11, about 5.3E11, about 5.4E11, about 5.5E11, about 5.6E11, about 5.7E11, about 5.8E11, about 5.9E11, about 6.0E11, about 6.1E11, about 6.2E11, about 6.3E11, about 6.4E11, about 6.5E11, about 6.6E11, about 6.7E11, about 6.8E11, about 6.9E11, about 7.0E11, about 7.1E11, about 7.2E11, about 7.3E11, about 7.4E11, about 7.5E11, about 7.6E11, about 7.7E11, about 7.8E11, about 7.9E11, about 8.0E11, about 8.1E11, about 8.2E11, about 8.3E11, about 8.4E11, about 8.5E11, about 8.6E11, about 8.7E11, about 8.8E11, or about 8.9E11.

[0268] In some embodiments, the composition comprises a total vg / snail of about 3.0E11, about 3.1E11, about 3.2E11, about 3.3E11, about 3.4E11, about 3.5E11, about 3.6E11, about 3.7E11, about 3.8E11, about 3.9E11, about 4.0E11, about 4.1E11, about 4.2E11, about 4.3E11, about 4.4E11, about 4.5E11, about 4.6E11, about 4.7E11, about 4.8E11, about 4.9E11, about 5.0E11, about 5.1E11, about 5.2E11, about 5.3E11, about 5.4E11, about 5.5E11, about 5.6E11, about 5.7E11, about 5.8E11, about 5.9E11, about 6.0E11, about 6.1E11, about 6.2E11, about 6.3E11, about 6.4E11, about 6.5E11, about 6.6E11, about 6.7E11, about 6.8E11, about 6.9E11, about 7.0E11, about 7.1E11, about 7.2E11, about 7.3E11, about 7.4E11, about 7.5E11, about 7.6E11, about 7.7E11, about 7.8E11, about 7.9E11, about 8.0E11, about 8.1E11, about 8.2E11, about 8.3E11, about 8.4E11, about 8.5E11, about 8.6E11, about 8.7E11, about 8.8E11, or about 8.9E11.

[0269] In some embodiments, the composition comprises a total vg of about 8.1E10 - 8.1E12, about 9.1E10 - 7.1E12, about 10.1E10 - 6.1E12, about 1.1E11 - 5.1E12, about 2.1E11 - 4.1E12, about 3.1E11 - 3.1E12, about 4.1E11 - 2.1E12, about 5.1E11 - 1.1E12, about 6.1E11 - 10.1E11, or about 7.1E11 - 9.1E11. In some embodiments, the composition comprises a total vg of about 7.1E11 - 9.1E11, about 7.2E11 - 9.0E11, about 7.3E11 - 8.9E11, about 7.4E11 - 8.8E11, about 7.5E11 - 8.7E11, about 7.6E11 - 8.6E11, about 7.7E11 - 8.5E11, about 7.8E11 - 8.4E11, about 7.9E11 - 8.3E11, or about 8.0E11 - 8.2E11. In some embodiments, the composition comprises a total vg of about 8.1E11.

[0270] In some embodiments, the composition comprises a total of about 8.1E10 to 8.1E12, about 9.1E10 to 7.1E12, about 10.1E10 to 6.1E12, about 1.1E11 to 5.1E12, about 2.1E11 to 4.1E12, about 3.1E11 to 3.1E12, about 4.1E11 to 2.1E12, about 5.1E11 to 1.1E12, about 6.1E11 to 10.1E11, or about 7.1E11 to 9.1E11 vg / snail. In some embodiments, the composition comprises a total of about 7.1E11 to 9.1E11, about 7.2E11 to 9.0E11, about 7.3E11 to 8.9E11, about 7.4E11 to 8.8E11, about 7.5E11 to 8.7E11, about 7.6E11 to 8.6E11, about 7.7E11 to 8.5E11, about 7.8E11 to 8.4E11, about 7.9E11 to 8.3E11, or about 8.0E11 to 8.2E11 vg / snail. In some embodiments, the composition comprises a total of about 8.1E11 vg / snail.

[0271] In some embodiments, the concentration of the composition comprises a total of about 4.5E11 to 9E13 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 4.5E11 to 4.5E13 vg / mL or about 9E11 to 9E13 vg / mL.

[0272] In some embodiments, the concentration of the composition comprises a total of about 4.5E11 to 4.5E13 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 5.5E11 to 3.5E13, about 6.5E11 to 2.5E13, about 7.5E11 to 1.5E13, about 8.5E11 to 10.5E12, about 9.5E11 to 9.5E12, about 10.5E11 to 8.5E12, about 1.5E12 to 7.5E12, about 2.5E12 to 6.5E12, or about 3.5E12 to 5.5E12 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 3.5E12 to 5.5E12, about 3.6E12 to 5.4E12, about 3.7E12 to 5.3E12, about 3.8E12 to 5.2E12, about 3.9E12 to 5.1E12, about 4.0E12 to 5.0E12, about 4.1E12 to 4.9E12, about 4.2E12 to 4.8E12, about 4.3E12 to 4.7E12, or about 4.4E12 to 4.6E12 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 4.5E12 vg / mL.

[0273] In some embodiments, the concentration of the composition comprises a total of about 3.5E12, about 3.6E12, about 3.7E12, about 3.8E12, about 3.9E12, about 4.0E12, about 4.1E12, about 4.2E12, about 4.3E12, about 4.4E12, about 4.5E12, about 4.6E12, about 4.7E12, about 4.8E12, about 4.9E12, about 5.0E12, about 5.1E12, about 5.2E12, about 5.3E12, about 5.4E12, about 5.5E12, about 5.6E12, about 5.7E12, about 5.8E12, about 5.9E12, about 6.0E12, about 6.1E12, about 6.2E12, about 6.3E12, about 6.4E12, about 6.5E12, about 6.6E12, about 6.7E12, about 6.8E12, about 6.9E12, about 7.0E12, about 7.1E12, about 7.2E12, about 7.3E12, about 7.4E12, about 7.5E12, about 7.6E12, about 7.7E12, about 7.8E12, about 7.9E12, about 8.0E12, about 8.1E12, about 8.2E12, about 8.3E12, about 8.4E12, about 8.5E12, about 8.6E12, about 8.7E12, about 8.8E12, about 8.9E12, about 9.0E12, about 9.1E12, about 9.2E12, about 9.3E12, about 9.4E12, about 9.5E12, about 9.6E12, about 9.7E12, about 9.8E12, or about 9.9E12 total vg / mL.

[0274] In some embodiments, the concentration of the composition comprises a total of about 9E11 to 9E13 vg / mL. In some embodiments, the concentration of the composition is about 9E11 to 9E13, about 10E11 to 8E13, about 1E12 to 7E13, about 2E12 to 6E13, about 3E12 to 5E13, about 4E12 to 4E13, about 5E12 to 3E13, about 6E12 to 2E13, about 7E12 to 1E13, or about 8E12 to 10E12 total vg / mL. In some embodiments, the concentration of the composition is about 8E12 to 10E12, about 8.1E12 to 9.9E12, about 8.2E12 to 9.8E12, about 8.3E12 to 9.7E12, about 8.4E12 to 9.6E12, about 8.5E12 to 9.5E12, about 8.6E12 to 9.4E12, about 8.7E12 to 9.3E12, about 8.8E12 to 9.2E12, or about 8.9E12 to 9.1E12. In some embodiments, the concentration of the composition comprises 9E12 total vg / mL.

[0275] In some embodiments, the first and second rAAV vectors can constitute a polypeptide messenger RNA encoding a full-length human otoferlin protein in a subject. In some embodiments, the first rAAV vector and the second rAAV vector can reconstitute an active otoferlin gene (e.g., a full-length otoferlin gene) intracellularly after intermolecular i) concatemerization, ii) recombination, iii) trans-splicing, iv) concatemerization and trans-splicing, or v) recombination and trans-splicing.

[0276] Mammalian cells This specification also provides a cell (e.g., a mammalian cell) comprising any one of the nucleic acids, vectors (e.g., at least two different vectors described herein), or compositions described herein. One of ordinary skill in the art will understand that the nucleic acids and vectors described herein can be introduced into any mammalian cell. Non-limiting examples of vectors and methods for introducing vectors into mammalian cells are described herein. In some embodiments, the cell is a human cell, a mouse cell, a pig cell, a rabbit cell, a dog cell, a cat cell, a rat cell, a sheep cell, a cat cell, a horse cell, or a non-human primate cell. In some embodiments, the cell is a specialized cell of a snail. In some embodiments, the cell is an inner hair cell within the snail or an outer hair cell outside the snail. In some embodiments, the cell is an inner hair cell within the snail. In some embodiments, the cell is an inner hair cell within the snail.

[0277] In some embodiments, the mammalian cell is in vitro. In some embodiments, the mammalian cell is present in a mammal. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is obtained from a subject. In some embodiments, the mammalian cell is an autologous cell obtained from a subject and / or is cultured ex vivo.

[0278] Method of Use Method of Introduction into the Snail This specification also provides a method of introducing a composition described herein into the cochlea of a mammal (e.g., a human).

[0279] Also provided is a method of increasing the expression of an active otoferlin protein (e.g., a full-length otoferlin protein) in inner hair cells within the cochlea of a mammal (e.g., a human), the method comprising introducing any one of the compositions described herein into the cochlea.

[0280] Also provided is a method for treating hearing loss in a subject (e.g., a human) identified as having a defective otoferlin gene (e.g., an otoferlin gene having a mutation that results in a decrease in the expression and / or activity of the otoferlin protein encoded by the gene), the method comprising administering any of the compositions described herein to the cochlea of the subject.

[0281] Also provided is a method for treating asymptomatic sensorineural hearing loss in a subject (e.g., a human) identified as having a defective otoferlin gene (e.g., an otoferlin gene having a mutation that results in a decrease in the expression and / or activity of the otoferlin protein encoded by the gene), the method comprising administering any of the compositions described herein to the cochlea of the subject.

[0282] Also provided is a method for treating hearing loss in a subject (e.g., a human) identified as having both alleles of the otoferlin gene, the method comprising administering any of the compositions described herein to the cochlea of the subject.

[0283] In some embodiments, the methods described herein can further comprise administering a neurotrophic factor to the cochlea of the subject (e.g., substantially simultaneously with, before, or after administering any of the compositions described herein to the subject). In some embodiments, the methods described herein can further comprise administering a cochlear implant to the subject (e.g., substantially simultaneously with, before, or after administering any of the compositions described herein to the subject).

[0284] In some embodiments of any of these methods, the mammal has previously been identified as having a defective otoferlin gene (e.g., an otoferlin gene having a mutation that results in a decrease in the expression and / or activity of the otoferlin protein encoded by the gene). In some embodiments, these methods further comprise determining, prior to the introducing or administering step, that the subject has a defective otoferlin gene. In some embodiments, these methods may further comprise detecting a mutation in the otoferlin gene in the subject. In some embodiments, the method may further comprise identifying or diagnosing that the subject has non-syndromic sensorineural hearing loss.

[0285] In some embodiments, a subject identified as having biallelic otoferlin gene mutations has previously been identified as having biallelic otoferlin gene mutations. In some embodiments, these methods further comprise determining, prior to the introducing or administering step, that the subject has biallelic otoferlin gene mutations. In some embodiments, the biallelic otoferlin gene mutations can include any of the mutations of the otoferlin gene described herein. In some embodiments, the biallelic otoferlin gene mutations result in a localized synaptic transmission disorder between the subject's hair cells and the auditory nerve.

[0286] In some embodiments, the mammal or subject has clinical symptoms of severe sensorineural hearing loss. In some embodiments, the severe sensorineural hearing loss is bilateral severe sensorineural hearing loss. In some embodiments, a subject identified as having a defective otoferlin gene has clinical symptoms of severe sensorineural hearing loss at afebrile times.

[0287] In some embodiments, the mammal or subject preserves distortion product otoacoustic emissions (DPOAE). In some embodiments, the outer hair cells of the mammal or subject function normally.

[0288] In some embodiments, the method may include administering a single dose of the composition described herein to a mammal or a target snail.

[0289] In some embodiments, these methods include introducing or administering a first dose of the composition to a mammal or a target snail, evaluating the hearing function of the mammal or the target snail following the introduction or administration of the first dose, and administering additional doses of the composition to a mammal or a target snail that is found to not have hearing function within the normal range (e.g., as determined using any hearing test known in the art).

[0290] In some embodiments, the composition is administered in multiple doses. In some embodiments, the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0291] In some embodiments, the single dose includes from about 0.01 mL to 0.2 mL. In some embodiments, the single dose includes from about 0.01 to 0.2, from about 0.02 to 0.18, from about 0.03 to 0.16, from about 0.04 to 0.14, from about 0.05 to 0.13, from about 0.06 to 0.12, from about 0.07 to 0.11, from about 0.08 to 0.10 mL. In some embodiments, the single dose includes about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, or about 0.2 mL. In some embodiments, the single dose includes about 0.09 mL.

[0292] In some embodiments, the composition is administered by a single injection. In some embodiments, the composition is administered by multiple injections. In some embodiments, the composition is administered by 2, 3, 4, 5, 6, 7, 8, 9, or 10 injections.

[0293] In some embodiments of any of the methods described herein, the composition can be formulated for intrasnail administration. In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the composition further comprises one or more buffers and one or more surfactants. In some embodiments, the buffer is selected from monopotassium phosphate, dibasic sodium phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS. In some embodiments, the surfactant is selected from poloxamer 188, lubrazole, tween, ethanol, pluronic F68, and polyethylene glycol. In some embodiments, the composition comprises monopotassium phosphate, dibasic sodium phosphate, potassium chloride, sodium chloride, and poloxamer 188.

[0294] In some embodiments, the composition comprises a) from about 1.35 to 1.65 mM of monopotassium phosphate, b) from about 7.29 to 8.91 mM of dibasic sodium phosphate, c) from about 2.43 to 2.97 mM of potassium chloride, d) from about 154.8 to 189.2 mM of sodium chloride, and e) from about 0.0001% to 0.01% of poloxamer 188.

[0295] In some embodiments, the composition comprises a) about 1.35, about 1.375, about 1.4, about 1.425, about 1.45, about 1.475, about 1.5, about 1.525, about 1.55, about 1.575, about 1.6, about 1.625, or about 1.65 mM of monopotassium phosphate, b) about 7.29, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, or about 8.91 mM of dibasic sodium phosphate, c) about 2.43, about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, or about 2.97 mM of containing potassium chloride, d) about 154.8, about 160, about 165, about 170, about 175, about 180, about 185, or about 189.2 mM of sodium chloride, and e) about 0.0001%, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, or about 0.01% of poloxamer 188.

[0296] In some embodiments, the composition comprises a) about 1.5 mM of monopotassium phosphate, b) about 8.1 mM of dibasic sodium phosphate, c) about 2.7 mM of potassium chloride, d) about 172 mM of sodium chloride, and e) about 0.001% of poloxamer 188.

[0297] In some embodiments, the composition is formulated to contain synthetic lymph. In some embodiments, the synthetic lymph comprises one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the synthetic lymph further comprises one or more buffers and one or more surfactants. In some embodiments, the buffer is selected from monopotassium phosphate, dibasic sodium phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS. In some embodiments, the surfactant is selected from poloxamer 188, labrazole, tween, ethanol, pluronic F68, and polyethylene glycol. In some embodiments, the synthetic lymph comprises monopotassium phosphate, dibasic sodium phosphate, potassium chloride, sodium chloride, and poloxamer 188.

[0298] In some embodiments, the synthetic lymph comprises a) about 1.35 - 1.65 mM of monopotassium phosphate, b) about 7.29 - 8.91 mM of dibasic sodium phosphate, c) about 2.43 - 2.97 mM of potassium chloride, d) about 154.8 - 189.2 mM of sodium chloride, and e) about 0.0001% - 0.01% of poloxamer 188.

[0299] In some embodiments, the synthetic lymphatic fluid comprises a) potassium dihydrogen phosphate at about 1.35, about 1.375, about 1.4, about 1.425, about 1.45, about 1.475, about 1.5, about 1.525, about 1.55, about 1.575, about 1.6, about 1.625, or about 1.65 mM; b) disodium hydrogen phosphate at about 7.29, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, or about 8.91 mM; c) potassium chloride at about 2.43, about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, or about 2.97 mM; d) sodium chloride at about 154.8, about 160, about 165, about 170, about 175, about 180, about 185, or about 189.2 mM; and e) poloxamer 188 at about 0.0001%, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, or about 0.01%.

[0300] In some embodiments, the synthetic lymphatic fluid comprises a) potassium dihydrogen phosphate at about 1.5 mM; b) disodium hydrogen phosphate at about 8.1 mM; c) potassium chloride at about 2.7 mM; d) sodium chloride at about 172 mM; and e) poloxamer 188 at about 0.001%.

[0301] In some embodiments, the compositions described herein can be administered via intrasnail or topical administration. In some embodiments, the composition is administered by use of a medical device (e.g., any of the exemplary medical devices described herein). In some embodiments, the composition is pre-filled in the device.

[0302] In some embodiments, the device is the device shown in FIGS. 2-5. In some embodiments, the device is a microcatheter. In some embodiments, the microcatheter is shaped to enter the middle ear cavity through the external auditory canal and to be able to contact the round window membrane (RWM) with the end of the microcatheter. In some embodiments, the distal end of the microcatheter includes at least one microneedle having a diameter of 10 to 1,000 microns. In some embodiments, the at least one microneedle includes a bent portion and an inclined tip.

[0303] In some embodiments, the cochlear administration can be carried out using any of the methods described herein or known in the art. For example, the composition can be administered or introduced into the cochlea using the following surgical techniques. First, the external auditory canal is cleaned using visualization with a 0-degree, 2.5 mm rigid endoscope, and a round knife is used to sharply outline the contour of the approximately 5 mm external auditory canal tympanic flap. Then, the external auditory canal tympanic flap is lifted and advanced into the middle ear from the back. The chorda tympani nerve is identified and divided, and a curette is used to remove the (scutal) bone on the lateral wall of the epitympanum to expose the round window membrane. To enhance the distribution of the administered or introduced composition to the top, a small 2 mm fenestration can be created in the oval window using a surgical laser to allow perilymph replacement during injection of the composition through the round window membrane. Next, the microinjection device is primed and brought to the surgical field. This device is maneuvered towards the round window and the tip is placed within the bony protrusion of the round window to allow penetration of the membrane by the microneedle(s). The foot pedal is linked to allow injection of the composition in a measured, constant amount. Then, the device is retracted and the round window and the basal turn of the cochlea are sealed with a self-form patch.

[0304] In some aspects, the present disclosure describes a delivery approach that utilizes a widely accepted minimally invasive surgical technique for accessing the middle ear and / or inner ear via the external ear canal. This technique involves opening one of the physical barriers between the middle ear and the inner ear at the oval window, and then using a device (or microcatheter) as disclosed herein, such as that shown in FIGS. 2-5, to deliver the compositions disclosed herein through the round window membrane at a controlled flow rate and a constant volume.

[0305] In some aspects, the surgical technique for mammals (e.g., rodents (e.g., mice, rats, hamsters, or rabbits), primates (e.g., NHPs (e.g., macaques, chimpanzees, monkeys, or apes) or humans)) may include providing ventilation holes to increase the transduction rate by AAV vectors along the entire length of the cochlea. In some aspects, the absence of ventilation holes during surgery may result in a decrease in the transduction rate of cochlear cells by AAV vectors when compared to the transduction rate of cochlear cells by AAV vectors after surgery performed with ventilation holes provided. In some aspects, the ventilation holes facilitate a transduction rate of about 75-100% of IHCs across the entire cochlea. In some aspects, the ventilation holes enable an IHC transduction rate of about 50-70%, about 60-80%, about 70-90%, or about 80-100% at the cochlear base. In some aspects, the ventilation holes enable an IHC transduction rate of about 50-70%, about 60-80%, about 70-90%, or about 80-100% at the cochlear apex.

[0306] The delivery device described herein may be placed in the sterile field of the operating room, the end of the tube may be removed from the sterile field, and the composition disclosed herein (e.g., one or more AAV vectors) may be filled and connected to a syringe attached to a pump. After appropriate priming of the system to remove any air, the needle may then be passed through the middle ear under visualization (surgical microscope, endoscope, and / or distal tip camera). The RWM may be punctured using a needle (or micro-needle). The needle may be inserted until the stopper contacts the RWM. The device may then be held in place while the composition disclosed herein is delivered to the inner ear at a controlled flow rate over a selected duration. In some embodiments, the flow rate (or infusion rate) may include rates of about 30 μL / min, or about 25 μL / min to about 35 μL / min, or about 20 μL / min to about 40 μL / min, or about 20 μL / min to about 70 μL / min, or about 20 μL / min to about 90 μL / min, or about 20 μL / min to about 100 μL / min. In some embodiments, the flow rate is about 20 μL / min, about 30 μL / min, about 40 μL / min, about 50 μL / min, about 60 μL / min, about 70 μL / min, about 80 μL / min, about 90 μL / min, or about 100 μL / min. In some embodiments, the selected duration (i.e., the time the composition disclosed herein is flowing) may be about 3 minutes, or about 2.5 minutes to about 3.5 minutes, or about 2 minutes to about 4 minutes, or about 1.5 minutes to about 4.5 minutes, or about 1 minute to about 5 minutes. In some embodiments, the total volume of the composition disclosed herein flowing into the inner ear may be about 0.09 mL, or about 0.08 mL to about 0.10 mL, or about 0.07 mL to about 0.11 mL.

[0307] In some embodiments, the single - dose volume includes from about 0.01 mL to 0.2 mL. In some embodiments, the single - dose volume includes from about 0.01 to 0.2, from about 0.02 to 0.18, from about 0.03 to 0.16, from about 0.04 to 0.14, from about 0.05 to 0.13, from about 0.06 to 0.12, from about 0.07 to 0.11, from about 0.08 to 0.10 mL. In some embodiments, the single - dose volume includes about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, or about 0.2 mL. In some embodiments, the single - dose volume includes about 0.09 mL.

[0308] In some embodiments, the total volume of the composition disclosed herein is equal to about 40% to about 50% of the volume of the inner ear.

[0309] Once delivery is complete, the device may be removed. In some embodiments, the devices described herein may be configured as single - use disposable products. In other embodiments, the devices described herein may be configured as multi - use sterilizable products, for example, having a replaceable and / or sterilizable needle sub - assembly. Single - use devices can be appropriately discarded (e.g., in a biohazard sharps container) after administration is complete.

[0310] In some embodiments, the composition disclosed herein includes one or more rAAV vectors. In some embodiments, when more than one AAV vector is included in the composition, the AAV vectors are each different. In some embodiments, the AAV vector includes, for example, the OTOF coding region as described herein. In some embodiments, the composition includes rAAV particles comprising the AAV vectors described herein. In some embodiments, the rAAV particles are capsidated by the Anc80 capsid. In some embodiments, the Anc80 capsid includes the polypeptide of SEQ ID NO: 109.

[0311] subject In some embodiments of any of the methods described herein, the subject or mammal is a rodent, non-human primate, or human. In some embodiments of any of the methods described herein, the subject, or mammal, is an adult, teenager, young individual, juvenile individual, infantile individual, neonatal individual, or neonatal individual. In some embodiments of any of the methods described herein, the subject or mammal is 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 2-5, 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-110, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-110, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 40-60, 40-70, 40-80, 40-90, 40-100, 50-70, 50-80, 50-90, 50-100, 60-80, 60-90, 60-100, 70-90, 70-100, 70-110, 80-100, 80-110, or 90-110 years old. In some embodiments of any of the methods described herein, the subject or mammal is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months old. In some embodiments, the subject is 2-17 years old.

[0312] In some embodiments of any of the methods described herein, the method provides an improvement in hearing (e.g., any of the criteria for determining improvement in hearing described herein) in a subject in need of such improvement for at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 100 days, at least 105 days, at least 110 days, at least 115 days, at least 120 days, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0313] In some embodiments, the subject or mammal has, or is at risk of developing, non-syndromic sensorineural hearing loss. In some embodiments, the subject or mammal has been identified as having a defective otoferlin gene. In some embodiments, the subject or mammal has previously been identified as having a mutation in the otoferlin gene. In some embodiments, the subject or mammal has any of the mutations in the otoferlin gene described herein or known in the art to be associated with non-syndromic sensorineural hearing loss.

[0314] In some embodiments, the subject or mammal has been identified (e.g., via genetic testing) as a carrier of a mutation in the otoferlin gene. In some embodiments, the subject or mammal has been identified as having a mutation in the otoferlin gene and diagnosed with non-syndromic sensorineural hearing loss. In some embodiments, the subject or mammal has been identified as having non-syndromic sensorineural hearing loss.

[0315] In some embodiments, the success of treating non - symptomatic sensorineural hearing loss can be determined in a subject using any of the conventional functional audiological tests known in the art. Non - limiting examples of functional audiological tests are various types of audiometric assays (e.g., pure - tone tests, speech tests, middle - ear tests, auditory brainstem responses, and otoacoustic emissions). In some embodiments, administration of the composition improves the subject's or mammal's auditory brainstem response (ABR) threshold response, age - appropriate behavioral audiological test, tympanotomy test, and / or word / sentence recognition test.

[0316] Increased expression of active otopetrin Also provided herein is a method of increasing the expression of an active otopetrin protein (e.g., full - length otopetrin protein) in a mammalian cell, the method comprising introducing any of the compositions described herein into the mammalian cell. In some embodiments, the mammalian cell is a cochlear cell. In some embodiments, the mammalian cell is an inner ear hair cell. In some embodiments, the mammalian cell is a human cell (e.g., human inner hair cells in the cochlea). In some embodiments, the mammalian cell is in vitro. In some embodiments, the mammalian cell is in a mammal. In some embodiments, the mammalian cell is originally obtained from a mammal and / or cultured ex vivo. In some embodiments, the mammalian cell has previously been determined to have a defective otopetrin gene.

[0317] Methods for introducing any of the compositions described herein into a mammalian cell are known in the art (e.g., by use of a viral vector, e.g., any of the viral vectors described herein).

[0318] In some embodiments, the increase in the expression of an active otopetrin protein (e.g., full - length otopetrin protein) as described herein is, for example, as compared to a control or as compared to the expression level of the active otopetrin protein (e.g., full - length otopetrin protein) before introduction of the vector(s).

[0319] Method for detecting otoferlin Methods for detecting the expression and / or activity of otoferlin are known in the art. In some embodiments, the expression level of the otoferlin protein can be detected directly (e.g., detection of the otoferlin protein, or detection of otoferlin mRNA). Non-limiting examples of techniques that can be used to directly detect the expression and / or activity of otoferlin include real-time PCR, Western blotting, immunoprecipitation, immunohistochemistry, or immunofluorescence. In some embodiments, the expression of the otoferlin protein can be detected indirectly (e.g., by functional hearing tests).

[0320] Dosage and administration volume In some embodiments, the compositions disclosed herein are administered as a single dose or multiple doses. In some embodiments, the composition is administered as a single dose. In some embodiments, the composition is administered as multiple doses. In some embodiments, the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0321] In some embodiments, the composition is administered by a single injection. In some embodiments, the composition is administered by multiple injections. In some embodiments, the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 injections.

[0322] In some embodiments, the compositions disclosed herein (e.g., compositions comprising one or more AAV vectors disclosed herein) are administered in a volume of about 0.01 mL, about 0.02 mL, about 0.03 mL, about 0.04 mL, 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 1.00 mL, about 1.10 mL, about 1.20 mL, about 1.30 mL, about 1.40 mL, about 1.50 mL, about 1.60 mL, about 1.70 mL, about 1.80 mL, about 1.90 mL, or about 2.00 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.01 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.02 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.03 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.04 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.05 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.06 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.07 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.08 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 0.09 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.00 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.10 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.20 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.30 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.40 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.50 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.60 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.70 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.80 mL. In some embodiments, the compositions disclosed herein are administered in a volume of about 1.90 mL.In some embodiments, the compositions disclosed herein are administered in a volume of about 2.00 mL.

[0323] In some embodiments, the compositions disclosed herein (e.g., compositions comprising one or more AAV vectors disclosed herein) are administered in a volume of about 0.01 - 2.00 mL, about 0.02 - 1.90 mL, about 0.03 - 1.8 mL, about 0.04 - 1.70 mL, about 0.05 - 1.60 mL, about 0.06 - 1.50 mL, about 0.06 - 1.40 mL, about 0.07 - 1.30 mL, about 0.08 - 1.20 mL, or about 0.09 - 1.10 mL. In some embodiments, the compositions disclosed herein (e.g., compositions comprising one or more AAV vectors disclosed herein) are administered in a volume of about 0.01 - 2.00 mL, about 0.02 - 2.00 mL, about 0.03 - 2.00 mL, about 0.04 - 2.00 mL, about 0.05 - 2.00 mL, about 0.06 - 2.00 mL, about 0.07 - 2.00 mL, about 0.08 - 2.00 mL, about 0.09 - 2.00 mL, about 0.01 - 1.90 mL, about 0.01 - 1.80 mL, about 0.01 - 1.70 mL, about 0.01 - 1.60 mL, about 0.01 - 1.50 mL, about 0.01 - 1.40 mL, about 0.01 - 1.30 mL, about 0.01 - 1.20 mL, about 0.01 - 1.10 mL, about 0.01 - 1.00 mL, about 0.01 - 0.09 mL.

[0324] In some embodiments, the single dose comprises from about 0.01 mL to 0.2 mL. In some embodiments, the single dose comprises about 0.01 - 0.2, about 0.02 - 0.18, about 0.03 - 0.16, about 0.04 - 0.14, about 0.05 - 0.13, about 0.06 - 0.12, about 0.07 - 0.11, or about 0.08 - 0.10 mL. In some embodiments, the single dose comprises about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.012, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, or about 0.2 mL. In some embodiments, the single dose comprises about 0.09 mL.

[0325] Formulation Also provided herein are formulations comprising any of the compositions herein.

[0326] In some embodiments, the composition of the disclosure comprises: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5’ portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3’ portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises a total of about 4.1E10 to 8.1E12 vg.

[0327] In some embodiments, the composition of the disclosure comprises: a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5’ portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3’ portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition comprises a total of about 4.1E10 to 4.1E12 vg or about 8.1E10 to 8.1E12 vg.

[0328] In some embodiments, the compositions of the present disclosure include a kit comprising a composition comprising: (a) a first rAAV vector genome comprising a first expression cassette comprising a promoter, a first coding sequence encoding an N-terminal portion of an otoferlin protein located 3' to the promoter, and a splicing donor signal sequence located at the 3' end of the first coding sequence; and (b) a second rAAV vector genome comprising a second expression cassette comprising a splicing acceptor signal sequence, a second coding sequence encoding a C-terminal portion of an otoferlin protein located 3' to the splicing acceptor signal sequence, and a polyadenylation sequence at the 3' end of the second coding sequence, wherein the composition is formulated for administration into the cochlea.

[0329] In some embodiments, the compositions of the present disclosure include: (a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5' portion of an otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR); and (b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3' portion of an otoferlin gene, wherein the expression cassette is adjacent to an ITR, wherein the composition is formulated for administration into the cochlea. In some embodiments, the composition comprises the first rAAV vector genome and the second rAAV vector genome in a ratio of about 1:1.

[0330] In some embodiments, the composition comprises a total vg of about 4.1E10 to 8.1E12. In some embodiments, the composition comprises a total vg of about 4.1E10 to 4.1E12 or about 8.1E10 to 8.1E12.

[0331] In some embodiments, the composition comprises a total vg of about 4.1E10 - 4.1E12, about 5.1E10 - 3.1E12, about 6.1E10 - 2.1E12, about 7.1E10 - 1.1E12, about 8.1E10 - 10.1E11, about 9.1E10 - 9.1E11, about 10.1E11 - 8.1E11, about 1.1E11 - 7.1E11, about 2.1E11 - 6.1E11, or about 3.1E11 - 5.1E11. In some embodiments, the composition comprises a total vg of about 3.1E11 - 5.1E11, about 3.2E11 - 5.0E11, about 3.3E11 - 4.9E11, about 3.4E11 - 4.8E11, about 3.5E11 - 4.7E11, about 3.6E11 - 4.6E11, about 3.7E11 - 4.5E11, about 3.8E11 - 4.4E11, about 3.9E11 - 4.3E11, or about 4.0E11 - 4.2E11. In some embodiments, the composition comprises a total vg of about 4.1E11.

[0332] In some embodiments, the composition comprises a total vg of about 4.1E10 - 8.1E12 per snail. In some embodiments, the composition comprises a total vg of about 4.1E10 - 4.1E12 per snail or about 8.1E10 - 8.1E12 per snail.

[0333] In some embodiments, the composition comprises a total vg of about 4.1E10 - 4.1E12, about 5.1E10 - 3.1E12, about 6.1E10 - 2.1E12, about 7.1E10 - 1.1E12, about 8.1E10 - 10.1E11, about 9.1E10 - 9.1E11, about 10.1E11 - 8.1E11, about 1.1E11 - 7.1E11, about 2.1E11 - 6.1E11, or about 3.1E11 - 5.1E11 per snail. In some embodiments, the composition comprises a total vg of about 3.1E11 - 5.1E11, about 3.2E11 - 5.0E11, about 3.3E11 - 4.9E11, about 3.4E11 - 4.8E11, about 3.5E11 - 4.7E11, about 3.6E11 - 4.6E11, about 3.7E11 - 4.5E11, about 3.8E11 - 4.4E11, about 3.9E11 - 4.3E11, or about 4.0E11 - 4.2E11 per snail. In some embodiments, the composition comprises a total vg of about 4.1E11 per snail.

[0334] In some embodiments, the composition comprises a total vg of about 3.0E11, about 3.1E11, about 3.2E11, about 3.3E11, about 3.4E11, about 3.5E11, about 3.6E11, about 3.7E11, about 3.8E11, about 3.9E11, about 4.0E11, about 4.1E11, about 4.2E11, about 4.3E11, about 4.4E11, about 4.5E11, about 4.6E11, about 4.7E11, about 4.8E11, about 4.9E11, about 5.0E11, about 5.1E11, about 5.2E11, about 5.3E11, about 5.4E11, about 5.5E11, about 5.6E11, about 5.7E11, about 5.8E11, about 5.9E11, about 6.0E11, about 6.1E11, about 6.2E11, about 6.3E11, about 6.4E11, about 6.5E11, about 6.6E11, about 6.7E11, about 6.8E11, about 6.9E11, about 7.0E11, about 7.1E11, about 7.2E11, about 7.3E11, about 7.4E11, about 7.5E11, about 7.6E11, about 7.7E11, about 7.8E11, about 7.9E11, about 8.0E11, about 8.1E11, about 8.2E11, about 8.3E11, about 8.4E11, about 8.5E11, about 8.6E11, about 8.7E11, about 8.8E11, or about 8.9E11.

[0335] In some embodiments, the composition comprises a total vg / snail of about 3.0E11, about 3.1E11, about 3.2E11, about 3.3E11, about 3.4E11, about 3.5E11, about 3.6E11, about 3.7E11, about 3.8E11, about 3.9E11, about 4.0E11, about 4.1E11, about 4.2E11, about 4.3E11, about 4.4E11, about 4.5E11, about 4.6E11, about 4.7E11, about 4.8E11, about 4.9E11, about 5.0E11, about 5.1E11, about 5.2E11, about 5.3E11, about 5.4E11, about 5.5E11, about 5.6E11, about 5.7E11, about 5.8E11, about 5.9E11, about 6.0E11, about 6.1E11, about 6.2E11, about 6.3E11, about 6.4E11, about 6.5E11, about 6.6E11, about 6.7E11, about 6.8E11, about 6.9E11, about 7.0E11, about 7.1E11, about 7.2E11, about 7.3E11, about 7.4E11, about 7.5E11, about 7.6E11, about 7.7E11, about 7.8E11, about 7.9E11, about 8.0E11, about 8.1E11, about 8.2E11, about 8.3E11, about 8.4E11, about 8.5E11, about 8.6E11, about 8.7E11, about 8.8E11, or about 8.9E11.

[0336] In some embodiments, the composition comprises a total vg of about 8.1E10 - 8.1E12, about 9.1E10 - 7.1E12, about 10.1E10 - 6.1E12, about 1.1E11 - 5.1E12, about 2.1E11 - 4.1E12, about 3.1E11 - 3.1E12, about 4.1E11 - 2.1E12, about 5.1E11 - 1.1E12, about 6.1E11 - 10.1E11, or about 7.1E11 - 9.1E11. In some embodiments, the composition comprises a total vg of about 7.1E11 - 9.1E11, about 7.2E11 - 9.0E11, about 7.3E11 - 8.9E11, about 7.4E11 - 8.8E11, about 7.5E11 - 8.7E11, about 7.6E11 - 8.6E11, about 7.7E11 - 8.5E11, about 7.8E11 - 8.4E11, about 7.9E11 - 8.3E11, or about 8.0E11 - 8.2E11. In some embodiments, the composition comprises a total vg of about 8.1E11.

[0337] In some embodiments, the composition comprises a total of about 8.1E10 to 8.1E12, about 9.1E10 to 7.1E12, about 10.1E10 to 6.1E12, about 1.1E11 to 5.1E12, about 2.1E11 to 4.1E12, about 3.1E11 to 3.1E12, about 4.1E11 to 2.1E12, about 5.1E11 to 1.1E12, about 6.1E11 to 10.1E11, or about 7.1E11 to 9.1E11 vg / snail. In some embodiments, the composition comprises a total of about 7.1E11 to 9.1E11, about 7.2E11 to 9.0E11, about 7.3E11 to 8.9E11, about 7.4E11 to 8.8E11, about 7.5E11 to 8.7E11, about 7.6E11 to 8.6E11, about 7.7E11 to 8.5E11, about 7.8E11 to 8.4E11, about 7.9E11 to 8.3E11, or about 8.0E11 to 8.2E11 vg / snail. In some embodiments, the composition comprises a total of about 8.1E11 vg / snail.

[0338] In some embodiments, the concentration of the composition comprises a total of about 4.5E11 to 9E13 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 4.5E11 to 4.5E13 vg / mL or about 9E11 to 9E13 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 4.5E11 to 4.5E13 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 5.5E11 to 3.5E13, about 6.5E11 to 2.5E13, about 7.5E11 to 1.5E13, about 8.5E11 to 10.5E12, about 9.5E11 to 9.5E12, about 10.5E11 to 8.5E12, about 1.5E12 to 7.5E12, about 2.5E12 to 6.5E12, or about 3.5E12 to 5.5E12 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 3.5E12 to 5.5E12, about 3.6E12 to 5.4E12, about 3.7E12 to 5.3E12, about 3.8E12 to 5.2E12, about 3.9E12 to 5.1E12, about 4.0E12 to 5.0E12, about 4.1E12 to 4.9E12, about 4.2E12 to 4.8E12, about 4.3E12 to 4.7E12, or about 4.4E12 to 4.6E12 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 4.5E12 vg / mL.

[0339] In some embodiments, the concentration of the composition comprises a total of about 3.5E12, about 3.6E12, about 3.7E12, about 3.8E12, about 3.9E12, about 4.0E12, about 4.1E12, about 4.2E12, about 4.3E12, about 4.4E12, about 4.5E12, about 4.6E12, about 4.7E12, about 4.8E12, about 4.9E12, about 5.0E12, about 5.1E12, about 5.2E12, about 5.3E12, about 5.4E12, about 5.5E12, about 5.6E12, about 5.7E12, about 5.8E12, about 5.9E12, about 6.0E12, about 6.1E12, about 6.2E12, about 6.3E12, about 6.4E12, about 6.5E12, about 6.6E12, about 6.7E12, about 6.8E12, about 6.9E12, about 7.0E12, about 7.1E12, about 7.2E12, about 7.3E12, about 7.4E12, about 7.5E12, about 7.6E12, about 7.7E12, about 7.8E12, about 7.9E12, about 8.0E12, about 8.1E12, about 8.2E12, about 8.3E12, about 8.4E12, about 8.5E12, about 8.6E12, about 8.7E12, about 8.8E12, about 8.9E12, about 9.0E12, about 9.1E12, about 9.2E12, about 9.3E12, about 9.4E12, about 9.5E12, about 9.6E12, about 9.7E12, about 9.8E12, or about 9.9E12 total vg / mL.

[0340] In some embodiments, the concentration of the composition comprises a total of about 9E11 - 9E13 vg / mL. In some embodiments, the concentration of the composition comprises a total of about 9E11 - 9E13, about 10E11 - 8E13, about 1E12 - 7E13, about 2E12 - 6E13, about 3E12 - 5E13, about 4E12 - 4E13, about 5E12 - 3E13, about 6E12 - 2E13, about 7E12 - 1E13, or about 8E12 - 10E12 total vg / mL. In some embodiments, the concentration of the composition comprises about 8E12 - 10E12, about 8.1E12 - 9.9E12, about 8.2E12 - 9.8E12, about 8.3E12 - 9.7E12, about 8.4E12 - 9.6E12, about 8.5E12 - 9.5E12, about 8.6E12 - 9.4E12, about 8.7E12 - 9.3E12, about 8.8E12 - 9.2E12, or about 8.9E12 - 9.1E12. In some embodiments, the concentration of the composition comprises 9E12 total vg / mL.

[0341] The pharmaceutical composition of the present disclosure may comprise the rAAV vector described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0342] In some embodiments, the composition may include buffers such as neutral buffered saline, phosphate buffered saline, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS; carbohydrates such as glucose, mannose, sucrose, or dextran; mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); surfactants such as poloxamer 188, laurozol, tween, ethanol, pluronic F68, and polyethylene glycol; and preservatives.

[0343] In some embodiments, the composition includes one or more buffers and one or more surfactants. In some embodiments, the buffer is selected from potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS. In some embodiments, the surfactant is selected from poloxamer 188, laurozol, tween, ethanol, pluronic F68, and polyethylene glycol.

[0344] In some embodiments, the composition includes potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, and poloxamer 188.

[0345] In some embodiments, the composition includes a) about 1.35 - 1.65 mM of potassium dihydrogen phosphate, b) about 7.29 - 8.91 mM of disodium hydrogen phosphate, c) about 2.43 - 2.97 mM of potassium chloride, d) about 154.8 - 189.2 mM of sodium chloride, and e) about 0.0001% - 0.01% of poloxamer 188.

[0346] In some embodiments, the composition comprises: a) about 1.35, about 1.375, about 1.4, about 1.425, about 1.45, about 1.475, about 1.5, about 1.525, about 1.55, about 1.575, about 1.6, about 1.625, or about 1.65 mM of monopotassium phosphate; b) about 7.29, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, or about 8.91 mM of dibasic sodium phosphate; c) about 2.43, about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, or about 2.97 mM of potassium chloride; d) about 154.8, about 160, about 165, about 170, about 175, about 180, about 185, or about 189.2 mM of sodium chloride; and e) about 0.0001%, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, or about 0.01% of poloxamer 188.

[0347] In some embodiments, the composition comprises: a) about 1.5 mM of monopotassium phosphate; b) about 8.1 mM of dibasic sodium phosphate; c) about 2.7 mM of potassium chloride; d) about 172 mM of sodium chloride; and e) about 0.001% of poloxamer 188.

[0348] In some embodiments, the composition is formulated as a sterile suspension. In some embodiments, the sterile suspension comprises a pharmaceutically acceptable carrier. In some embodiments, the suspension comprises sterile water. In some embodiments, the composition comprises a volume of about 0.01 mL to 0.2 mL. In some embodiments, the composition comprises a volume of about 0.01 - 0.2, about 0.02 - 0.18, about 0.03 - 0.16, about 0.04 - 0.14, about 0.05 - 0.13, about 0.06 - 0.12, about 0.07 - 0.11, about 0.08 - 0.10 mL. In some embodiments, the single dose comprises about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, or about 0.2 mL. In some embodiments, the composition comprises a volume of about 0.09 mL.

[0349] In some embodiments, the compositions of the present disclosure are formulated for intrasnail administration. In some embodiments, the compositions of the present disclosure are formulated for intravenous administration.

[0350] In some embodiments, the composition is formulated to comprise synthetic perilymph. In some embodiments, the synthetic perilymph comprises one or more buffers and one or more surfactants. In some embodiments, the buffer is selected from potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS. In some embodiments, the surfactant is selected from poloxamer 188, lubrazole, tween, ethanol, pluronic F68, and polyethylene glycol.

[0351] In some embodiments, the synthetic lymphatic fluid comprises potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, and poloxamer 188. In some embodiments, the synthetic lymphatic fluid comprises a) potassium dihydrogen phosphate at about 1.35 - 1.65 mM, b) disodium hydrogen phosphate at about 7.29 - 8.91 mM, c) potassium chloride at about 2.43 - 2.97 mM, d) sodium chloride at about 154.8 - 189.2 mM, and e) poloxamer 188 at about 0.0001% - 0.01%.

[0352] In some embodiments, the synthetic lymphatic fluid comprises a) potassium dihydrogen phosphate at about 1.35, about 1.375, about 1.4, about 1.425, about 1.45, about 1.475, about 1.5, about 1.525, about 1.55, about 1.575, about 1.6, about 1.625, or about 1.65 mM, b) disodium hydrogen phosphate at about 7.29, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, or about 8.91 mM, c) potassium chloride at about 2.43, about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, or about 2.97 mM, d) sodium chloride at about 154.8, about 160, about 165, about 170, about 175, about 180, about 185, or about 189.2 mM, and e) poloxamer 188 at about 0.0001%, about 0.00025%, about 0.0005%, about 0.00075%, about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, or about 0.01%.

[0353] In some embodiments, the synthetic lymphatic fluid comprises a) potassium dihydrogen phosphate at about 1.5 mM, b) disodium hydrogen phosphate at about 8.1 mM, c) potassium chloride at about 2.7 mM, d) sodium chloride at about 172 mM, and e) poloxamer 188 at about 0.001%.

[0354] In some embodiments, the compositions described herein may further comprise one or more agents (e.g., liposomes or cationic lipids) that facilitate entry of the nucleic acids or any vectors described herein into primate cells. In some embodiments, any of the vectors described herein can be formulated using natural and / or synthetic polymers. Non-limiting examples of polymers that can be included in any of the compositions described herein include formulations by DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.), Mirus Bio (Madison, Wis.), and Roche Madison (Madison, Wis.), including but not limited to, PhaseRX polymer formulations such as SMARTT POLYMER TECHNOLOGY® (PhaseRX, Seattle, Wash.), DMRI / DOPE, poloxamer, the VAXFECTIN® adjuvant by Vical (San Diego, Calif.), chitosan, cyclodextrin by Calando Pharmaceuticals (Pasadena, Calif.), dendrimers, and poly(lactic-co-glycolic acid) (PLGA) polymers, the RONDEL™ (RNAi / oligonucleotide nanoparticle delivery) polymer (Arrowhead Research Corporation, Pasadena, Calif.), and pH-responsive block copolymers such as, but not limited to, block copolymers produced by PhaseRX (Seattle, Wash.).Many of these polymers have been demonstrated to be effective in delivering oligonucleotides to mammalian cells in vivo (e.g., deFougerolles, Human Gene Ther. 19:125-132, 2008 Rozema et al., Proc. Natl. Acad. Sci. U.S.A. 104:12982-12887, 2007, Rozema et al., Proc. Natl. Acad. Sci. U.S.A. 104:12982-12887, 2007, Hu-Lieskovan et al., Cancer Res. 65:8984-8982, 2005, Heidel et al., Proc. Natl. Acad. Sci. U.S.A. 104:5715-5721, 2007). Any composition described herein can be, for example, a pharmaceutical composition.

[0355] In some embodiments, the composition comprises a pharmaceutically acceptable carrier (e.g., phosphate buffered saline, saline, or bacteriostatic water). When formulated, the solution is administered in a manner compatible with the dosage form and in an amount therapeutically effective. The formulations are readily administered in various dosage forms (e.g., injection solutions, injectable gels, drug release capsules, etc.).

[0356] The compositions provided herein can be formulated, for example, to be compatible with their intended route of administration. Non-limiting examples of intended routes of administration are local administration (e.g., intracochlear administration).

[0357] Also provided are kits containing any of the compositions described herein. In some embodiments, the kit can include a solid composition (e.g., a lyophilized composition containing at least two different vectors described herein) and a liquid for solubilizing the lyophilized composition. In some embodiments, the kit can include a filled syringe containing any of the compositions described herein.

[0358] In some embodiments, the kit may include a composition pre-filled in the device. In some embodiments, the device is a microcatheter. In some embodiments, the microcatheter is shaped to enter the middle ear cavity through the external auditory canal and to be able to contact the RWM with the end of the microcatheter. In some embodiments, the distal end of the microcatheter is composed of at least one micro-needle having a diameter of 10 to 1,000 microns.

[0359] In some embodiments, the kit may include a device. In some embodiments, the device is the device provided herein. In some embodiments, the device is the device described in any one of FIGS. 2 to 5. In some embodiments, the device includes a needle including a bent portion and a beveled tip.

[0360] In some embodiments, the kit includes a vial containing any of the compositions described herein (e.g., formulated as an aqueous composition, e.g., an aqueous pharmaceutical composition). In some embodiments, the vial is a single-use vial or a multi-use vial.

[0361] In some embodiments, the vial is a single-use vial. In some embodiments, the single-use vial contains the composition at a concentration (target concentration) appropriate for intratympanic administration. In some embodiments, the target concentration is about 4.5E11 to 9E13 vg / mL. In some embodiments, the target concentration is about 1E13 total vg / mL.

[0362] In some embodiments, the kit includes a second vial containing a diluent. In some embodiments, the second vial is a single-use vial or a multi-use vial. In some embodiments, the diluent includes one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients of the composition. In some embodiments, the concentration of one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients of the composition corresponds to the concentration of one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients contained in the composition. In some embodiments, the diluent includes a) about 1.5 mM potassium dihydrogen phosphate, b) about 8.1 mM disodium hydrogen phosphate, c) about 2.7 mM potassium chloride, d) about 172 mM sodium chloride, and e) about 0.001% poloxamer 188. In some embodiments, the diluent is utilized to prepare the concentration of the composition to be administered to a subject in need of administration of the composition.

[0363] In one aspect, the kit may include instructions for carrying out any of the methods described herein. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or sterile dispersions for immediate preparation of injectable sterile solutions or sterile dispersions and sterile powders. The dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy syringeability exists. It must be stable under manufacturing and storage conditions and protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of the injectable composition can be brought about by using in the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0364] For example, in the case of administration of an aqueous injection solution, the solution can be suitably buffered if necessary, and the liquid diluent can first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this context, the sterile aqueous media that can be used will be known to those skilled in the art. For example, a single dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous drip fluid or injected into the planned injection site (see, for example, "Remington’s Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in the dosage will necessarily occur depending on the condition of the host. In any case, it is the responsibility of the person administering the dose to determine the appropriate dosage for an individual host.

[0365] Sterile injectable solutions are prepared by incorporating the required amount of the active rAAV into a suitable solvent, optionally containing various other ingredients enumerated herein, and subsequently filtering the solution sterile. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a basic dispersion medium and a sterile vehicle containing the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying techniques and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0366] The rAAV compositions disclosed herein can also be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution is administered in a manner compatible with the dosage form and in an amount therapeutically effective. The formulations are readily administered in various dosage forms such as injection solutions, drug release capsules, etc.

[0367] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present disclosure into suitable host cells. In particular, the transgene delivered by the rAAV vector can be formulated by encapsulation in any of lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc. for delivery.

[0368] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved stability in serum and circulating half-life have been developed (U.S. Patent No. 5,741,516). Further, various methods of liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0369] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering rAAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Patent No. 5,656,016 as a device for enhancing the rate and effectiveness of drug permeation into and through the circulatory system. Other alternative drug delivery methods contemplated are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208), and feedback controlled delivery (U.S. Patent No. 5,697,899).

[0370] Administration of the subject compositions can be carried out in any convenient manner, including by aerosol inhalation, injection, oral ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to a subject by intraarterial, subcutaneous, intradermal, intranodular, intramedullary, intramuscular, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the nucleic acid compositions of the disclosure are administered to a subject by intradermal or subcutaneous injection. In one aspect, the nuclear compositions of the disclosure are administered by i.v. injection.

[0371] Devices and surgical methods In some aspects, the present specification provides techniques (e.g., systems, methods, devices, etc.) that can be used to treat hearing loss and other hearing-related diseases, disorders, and conditions. Examples of such techniques are also included, for example, in WO2017223193 and WO2019084145, each of which is incorporated herein by reference in its entirety. In one aspect, the present disclosure provides a therapeutic delivery system for treating hearing loss and other hearing-related diseases, disorders, and conditions. In one aspect, a therapeutic delivery system is provided, the therapeutic delivery system comprising: i) a medical device capable of creating one or more incisions in the round window membrane of the inner ear of a human subject in need thereof; and ii) an effective amount of a therapeutic composition comprising one or more adeno-associated virus (AAV) vectors, wherein the one or more AAV vectors are capable of encoding full-length auditory polypeptide messenger RNA in target cells of the inner ear. In some aspects of the means for performing the surgical method, the method comprises administering an effective amount of the therapeutic composition of the present disclosure into the cochlea of a human subject in need thereof, wherein the therapeutic composition can be administered using a medical device comprising: a) means for creating one or more incisions in the round window membrane; and b) an effective amount of the therapeutic composition.

[0372] The present specification provides a surgical method for the treatment of hearing loss. In one aspect, the method comprises introducing a first incision into the cochlea of a human subject at a first incision point and administering an effective amount of a therapeutic composition provided herein (e.g., any composition described herein) into the cochlea. In one aspect, the therapeutic composition (e.g., any composition described herein) is administered to the subject at the first incision point. In one aspect, the therapeutic composition is administered to the subject within or through the first incision. In one aspect, the therapeutic composition is administered to the subject within or through the oval window membrane of the cochlea. In one aspect, the therapeutic composition is administered to the subject within or through the round window membrane of the cochlea.

[0373] In some embodiments, the compositions disclosed herein can be administered to a subject by a surgical procedure. In some embodiments, administration, e.g., via a surgical procedure, involves injecting a composition disclosed herein into the inner ear via a delivery device described herein. In some embodiments, the surgical procedures disclosed herein include performing a transcanal myringotomy, performing a microscopic stapedotomy using a laser, and injecting a composition disclosed herein into the inner ear via a delivery device described herein.

[0374] In some embodiments, the surgical procedure includes performing a transcanal myringotomy, performing a microscopic stapedotomy using a laser, injecting a composition disclosed herein into the inner ear via a delivery device described herein, applying a sealant around the round window and / or oval window of the subject, and lowering the external auditory meatal tympanic flap of the subject into its anatomical position.

[0375] In some embodiments, the surgical procedure includes performing a transcanal myringotomy, preparing the round window of the subject, performing a microscopic stapedotomy using a laser, preparing both a delivery device described herein and a composition disclosed herein for delivery to the inner ear, injecting a composition disclosed herein into the inner ear via the delivery device, applying a sealant around the round window and / or oval window of the subject, and lowering the external auditory meatal tympanic flap of the subject into its anatomical position.

[0376] In some embodiments, performing a microscopic stapedotomy using a laser includes using a KTP otologic laser and / or a CO2 otologic laser.

[0377] In some embodiments, the composition comprises one or more AAV vectors. In some aspects, when more than one AAV vector is included in the composition, the AAV vectors are each different. In some aspects, the AAV vector comprises an OTOF coding region, such as described herein. In some aspects, the composition comprises rAAV particles comprising the AAV vectors described herein. In some aspects, the rAAV particles are capsidated by an Anc80 capsid. In some aspects, the Anc80 capsid comprises the polypeptide of SEQ ID NO: 109.

[0378] For example, in one aspect, the therapeutic composition is administered using a medical device capable of creating a plurality of incisions within the round window membrane. In one aspect, the medical device comprises a plurality of microneedles. In one aspect, the medical device comprises a plurality of microneedles comprising a substantially circular first face, each microneedle having a diameter of at least about 10 microns. In one aspect, the medical device comprises a base and / or reservoir capable of holding the therapeutic composition. In one aspect, the medical device comprises a plurality of hollow microneedles each individually comprising a lumen capable of transporting the therapeutic composition. In one aspect, the medical device comprises means for generating at least a partial vacuum.

[0379] As another example, the compositions disclosed herein are administered using a device and / or system specifically designed for an endolymphatic administration route. In some aspects, the design elements of the devices described herein may include maintaining the sterility of the injected fluid, minimizing air bubbles introduced into the inner ear, the ability to accurately deliver a small volume at a controlled rate, delivery via the ear canal by a surgeon, minimizing damage to the round window membrane (RWM) or to the inner ear, such as the cochlear structure beyond the RWM, and / or minimizing back-leakage of the injected fluid through the RWM.

[0380] The devices, systems, and methods provided herein also describe the potential for safely and efficiently delivering compositions to the inner ear to treat conditions and disorders for which delivery of the compositions disclosed herein to the inner ear would be beneficial, including but not limited to, for example, hearing impairments described herein. As another example, by providing pores in the oval window and injecting through the RWM, the compositions disclosed herein can be dispersed throughout the cochlea while minimizing dilution at the site of action. The development of the devices described enables surgical administration procedures to be performed through the external ear canal in humans. The devices described can be removed from the ear after injecting a quantity of fluid into the perilymph of the cochlea. In a subject, the device may be advanced through the external ear canal either under surgical microscope control or with an endoscope.

[0381] Exemplary devices for use in any of the methods disclosed herein are described in FIGS. 2-5. FIG. 2 illustrates an exemplary device 10 for delivering fluid to the inner ear. Device 10 includes a knurled handle 12 and a distal handle adhesive 14 (e.g., an epoxy such as loctite 4014) that couples to a telescoping hypodermic needle support 24. The knurled handle 12 (or handle portion) may include a knurling mechanism and / or grooves to enhance the grip. The knurled handle 12 (or handle portion) can be about 5 mm to about 15 mm thick, or about 5 mm to about 12 mm thick, or about 6 mm to about 10 mm thick, or about 6 mm to about 9 mm thick, or about 7 mm to about 8 mm thick. The knurled handle 12 (or handle portion) can be hollow so that fluid can pass through device 10 during use. Device 10 may also include a proximal handle adhesive 16 at the proximal end 18 of the knurled handle 12, a needle subassembly 26 (shown in FIG. 3) having a stopper 28 (shown in FIG. 3) at the distal end 20 of device 10, and a strain relief mechanism 22. The strain relief mechanism 22 may be composed of a Santoprene material, a Pebax material, a polyurethane material, a silicone material, a nylon material, and / or a thermoplastic elastomer. The telescoping hypodermic needle support 24 surrounds and supports a bent needle 38 (shown in FIG. 3) disposed therein.

[0382] Referring to FIG. 2, the stopper 28 may be composed of a thermoplastic material or a plastic polymer (e.g., a UV-curable polymer), as well as other suitable materials, and may be used to prevent the bending needle 38 from being inserted too deeply into the ear canal (e.g., to prevent the insertion of the bending needle 38 into the side wall or other inner ear structures). The device 10 may also include a tapered portion 23 disposed between the knurled handle 12 and the distal handle adhesive 14 coupled to the telescopic hypodermic needle support 24. The knurled handle 12 (or handle portion) may include a tapered portion 23 at the distal end of the handle portion 12. The device 10 may also include a tube 36 fluidly connected to the proximal end 16 of the device 10, which functions as a fluid inlet line connecting the device to an upstream component (e.g., in some embodiments, a pump, syringe, and / or upstream component that may be coupled to a control system and / or a power source (not shown)). In some aspects, the bending needle 38 (shown in FIG. 3) extends from the distal end 20, through the telescopic hypodermic needle support 24, through the tapered portion 23, through the knurled handle 12, and through the strain relief mechanism 22, and is in direct fluid connection with the tube 36. In other embodiments, the bending needle 38 is in fluid connection with the hollow interior of the knurled handle (e.g., via the telescopic hypodermic needle support 24), and then is in fluid connection with the tube 36 at the proximal end 16. In embodiments where the bending needle 38 does not extend completely inside the device 10, the contact area (e.g., between the overlapping nested hypodermic tubes 42), the tolerance, and / or the sealant between the interface components must be sufficient to prevent the therapeutic fluid from leaking out of the device 10 (which operates at a relatively low pressure (e.g., about 1 Pascal to about 50 Pa, or about 2 Pa to about 20 Pa, or about 3 Pa to about 10 Pa)).

[0383] FIG. 3 illustrates a side view of a bent needle subassembly 26 according to an aspect of the present disclosure. The bent needle subassembly 26 includes a needle 38 having a bent portion 32. The bent needle subassembly 26 may also include a stopper 28 coupled to the bent portion 32. The bent portion 32 includes an angled tip 34 at the distal end 20 of the device 10 for piercing the eardrum (e.g., RWM). The needle 38, the bent portion 32, and the angled tip 34 are hollow so that fluid can flow therethrough. The angle 46 (as shown in FIG. 5) of the bent portion 32 may vary. The geometry of the stopper 28 may be cylindrical, disc-shaped, annular, domed, and / or other suitable shapes. The stopper 28 may be formed at a predetermined position on the bent portion 32. For example, the stopper 28 may be disposed concentrically around the bent portion 32 using an adhesive or compression fitting. Examples of adhesives include UV curable adhesives (such as Dymax 203A-CTH-F-T), elastomeric adhesives, thermosetting adhesives (such as epoxy or polyurethane), or emulsion adhesives (such as polyvinyl acetate). The stopper 28 conforms concentrically around the bent portion 32 such that the angled tip 34 is inserted into the ear at a desired insertion depth. The bent needle 38 may be formed from a straight needle using incremental forming and other suitable techniques.

[0384] FIG. 4 illustrates a perspective view of an exemplary device 10 for delivering fluid to the inner ear. The tube 36 can have a length of about 1300 mm (dimension 11 in FIG. 4) to about 1600 mm, or about 1400 mm to about 1500 mm, or about 1430 mm to about 1450 mm. The strain relief mechanism 22 can have a length of about 25 mm to about 30 mm (dimension 15 in FIG. 4), or a length of about 20 mm to about 35 mm. The handle 12 can have a length of about 155.4 mm (dimension 13 in FIG. 4), or about 150 mm to about 160 mm, or about 140 mm to about 170 mm. The telescoping hypodermic needle support 24 can have two or more nested hypodermic tubes, for example, three nested hypodermic tubes 42A, 42B, and 42C, or four nested hypodermic tubes 42A, 42B, 42C, and 42D. The total length of the hypodermic tubes 42A, 42B, 42C and the tip assembly 26 (dimension 17 in FIG. 4) can be about 25 mm to about 45 mm, or about 30 mm to about 40 mm, or about 35 mm. Further, the telescoping hypodermic needle support 24 can have a length of about 36 mm, or about 25 mm to about 45 mm, or can form about 30 mm to about 40 mm. The three nested hypodermic tubes 42A, 42B, and 42C can each have a length of 3.5 mm, 8.0 mm, and 19.8 mm, plus or minus about 20%. The innermost nested hypodermic tube (or the narrowest portion) of the telescoping hypodermic needle support 24 can be arranged concentrically around the needle 38.

[0385] FIG. 5 illustrates a perspective view of a bent needle subassembly 26 coupled to the distal end 20 of the device 10, according to an aspect of the present disclosure. As shown in FIG. 5, the bent needle subassembly 26 may include a needle 38 coupled to a bent portion 32. In other aspects, the bent needle 38 may be a single needle (e.g., a straight needle that is subsequently bent to include a desired angle 46). The needle 38 may be a 33-gauge needle, or may include a gauge of about 32 to about 34, or about 31 to 35. With finer gauges, care must be taken so that the tube 36 does not twist or become damaged. The needle 38 can be attached to the handle 12 to safely and accurately position the needle 38 in the inner ear. As shown in FIG. 5, the bent needle subassembly 26 may also include a stopper 28 disposed around the bent portion 32. FIG. 5 also shows that the bent portion 32 may include an angled tip 34 for piercing the eardrum (e.g., RWM). The stopper 28 may have a height 48 of about 0.5 mm, or about 0.4 mm to about 0.6 mm, or about 0.3 mm to about 0.7 mm. The bent portion 32 may have a length 52 of about 1.45 mm, or about 1.35 mm to about 1.55 mm, or about 1.2 mm to about 1.7 mm. In other aspects, the bent portion 32 may have a length greater than 2.0 mm such that the distance between the distal end of the stopper 28 and the distal end of the angled tip 34 is about 0.5 mm to about 1.7 mm, or about 0.6 mm to about 1.5 mm, or about 0.7 mm to about 1.3 mm, or about 0.8 mm to about 1.2 mm. FIG. 5 shows that the stopper 28 may have a geometry that is cylindrical, disc-shaped, and / or dome-shaped. Those skilled in the art will understand that other geometries may be used.

[0386] The present disclosure will be described in further detail by reference to the following examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein.

[0387] Other assays, including those described in the Examples section of this specification and those known in the art, can also be used to evaluate the auditory polypeptides and nucleic acid constructs of the present disclosure.

[0388] One of ordinary skill in the art will appreciate that the compounds of the present disclosure can be made and utilized, and the claimed methods can be practiced, using the foregoing description and the examples that follow as illustrative descriptions without further elaboration. The following examples specifically point out various aspects of the present disclosure and should in no way be construed as limiting the remainder of the present disclosure.

Examples

[0389] Example 1: Adeno-Associated Virus (AAV) Trans-Splicing Strategy Two different rAAV vectors can be used to reconstitute an active otoferlin gene (e.g., the full-length otoferlin gene) intracellularly after intermolecular concatenation and trans-splicing. See, for example, Yan et al., Proc. Natl. Acad. Sci. U.S.A. 97:12; 6716-6721, 2000, which is hereby incorporated by reference in its entirety.

[0390] In some aspects, two different rAAV vectors are used. In some aspects, the first rAAV vector comprises a first expression cassette comprising a promoter (e.g., any of the promoters described herein), a first coding sequence encoding an N-terminal portion of the otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the N-terminal portions of the otoferlin protein described herein) positioned 3' to the promoter, and a splicing donor signal sequence positioned at the 3' end of the first coding sequence.

[0391] In some embodiments, the second rAAV vector comprises a second expression cassette comprising a splicing acceptor signal sequence, a second coding sequence encoding a C-terminal portion of the otoferlin protein (i.e., the entire portion not included in the N-terminal portion of the otoferlin protein) (e.g., any of the sizes of portions of the otoferlin protein described herein and / or any of the C-terminal portions of the otoferlin protein described herein) positioned at the 3' end of the splicing acceptor signal sequence, and a polyadenylation sequence (e.g., any of the polyadenylation sequences described herein) at the 3' end of the second coding sequence.

[0392] In some embodiments, the first rAAV vector genome comprises a first expression cassette comprising a promoter (e.g., any of the promoters described herein), a first coding sequence encoding an N-terminal portion of the otoferlin protein (e.g., any of the sizes of portions of the otoferlin protein described herein and / or any of the N-terminal portions of the otoferlin protein described herein) positioned 3' of the promoter, and a splicing donor signal sequence positioned at the 3' end of the first coding sequence.

[0393] In some embodiments, the second rAAV vector genome comprises a second expression cassette comprising a splicing acceptor signal sequence, a second coding sequence encoding a C-terminal portion of the otoferlin protein (i.e., the entire portion not included in the N-terminal portion of the otoferlin protein) (e.g., any of the sizes of portions of the otoferlin protein described herein and / or any of the C-terminal portions of the otoferlin protein described herein) positioned at the 3' end of the splicing acceptor signal sequence, and a polyadenylation sequence (e.g., any of the polyadenylation sequences described herein) at the 3' end of the second coding sequence.

[0394] In some embodiments, each of the encoded portions is at least 30 amino acid residues in length (e.g., at least 50 amino acids, at least 75 amino acids, or at least 100 amino acids in length), the amino acid sequence of each of the encoded portions does not overlap with the sequences of other encoded portions, and neither of the two different rAAV vectors encodes an active otopetrin protein (e.g., a full-length otopetrin protein). When the two coding sequences of the two rAAV vectors are expressed in mammalian cells (e.g., any of the mammalian cells described herein), splicing occurs between the splicing donor signal sequence and the splicing acceptor signal sequence, thereby forming a recombinant mRNA encoding an active otopetrin protein (e.g., a full-length otopetrin protein).

[0395] In any of these examples of methods, the amino acid sequence of any of the encoded portions does not overlap with the sequences of any other encoded portions, and neither of the single vectors encodes an active otopetrin protein (e.g., a full-length otopetrin protein).

[0396] Each of the two different rAAV vectors contains a coding sequence encoding a different portion of the otopetrin protein, and each of the encoded portions can be at least 30 amino acids (e.g., from about 30 amino acids to about 800 amino acids, or any other sub-range within this range described herein).

[0397] In some embodiments, each of the two different vectors comprises a coding sequence encoding a different portion of the otopetrin protein, and each of the encoded portions can encode up to 80% (e.g., up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, or up to 70%) of the amino acid sequence of SEQ ID NO:5 such that each of the encoded portions does not overlap. In some embodiments, each of the two different vectors comprises a coding sequence encoding a different portion of the otopetrin protein, and each of the encoded portions encodes up to 80% (e.g., up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, or up to 70%) of the amino acid sequence of SEQ ID NO:5 such that each of the encoded portions does not overlap.

[0398] Each of the two rAAV vectors may further comprise inverted terminal repeat sequences (ITRs) that allow for head-to-tail recombination. The ITRs are then removed via splicing. For example, the ITRs can be palindromic double D ITRs as described in Yan et al., Proc. Natl. Acad. Sci. U.S.A. 97(12):6716-6721, 2000, which is hereby incorporated by reference in its entirety. For example, the ITRs can be AAV serotype 2 ITRs as described in Gosh et al., Mol. Ther. 16:124-130, 2008, and Gosh et al., Human Gene Ther. 22:77-83, 2011. Non-limiting examples of splicing acceptor and / or donor signal sequences are known in the art. See, for example, Reich et al., Human Gene Ther. 14(1):37-44, 2003, and Lai et al. (2005) Nat. Biotechnol. 23(11):1435-1439, 2005, 2005. The splicing donor and acceptor signal sequences can be any endogenous intron splicing signal of a gene (e.g., the otopetrin gene).

[0399] For example, the splicing donor signal sequence can be 5’-GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCT-3’ (SEQ ID NO: 64), and the splicing acceptor signal can be 5’-ATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG-3’ (SEQ ID NO: 110) (see, for example, Trapani et al., EMBO Mol. Med. 6(2):194-211, 2014).

[0400] In some embodiments, the splicing donor sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 102. In some embodiments, the splice donor sequence has the sequence of SEQ ID NO: 102.

[0401] In some embodiments, the splicing acceptor sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 106. In some embodiments, the splicing acceptor sequence has the sequence of SEQ ID NO: 106.

[0402] Methods for assessing splicing and splicing efficiency are known in the art (see, for example, Lai et al., Nat. Biotechnol. 23(11):1435-1439, 2005).

[0403] Example 2: Hybrid vector trans-splicing strategy using a highly recombinogenic exogenous gene region of alkaline phosphatase (AP) Two different nucleic acid vectors (e.g., AAV vectors) can also be used by any of the methods described herein to reconstitute an active otoferlin gene (e.g., the full-length otoferlin gene) intracellularly after intermolecular i) concatemerization, ii) recombination, iii) trans-splicing, iv) concatemerization and trans-splicing, or v) recombination and trans-splicing. This strategy is a hybrid strategy because it involves concatemerization, homologous recombination, and / or trans-splicing. See, for example, Gosh et al., Mol. Ther. 16:124-130, 2008, Gosh et al., Human Gene Ther. 22:77-83, 2011, and Duan et al., Mol. Ther. 4:383-391, 2001, each of which is incorporated herein by reference in its entirety.

[0404] Recombination can occur at highly recombinogenic DNA sequences that allow for sequence-independent recombination. Non-limiting examples of recombinogenic sequences are the alkaline phosphatase (AP) gene. For example, the recombinogenic sequence can be the central third of human placental AP complementary DNA, which is 872 bp in length (see, e.g., Gosh et al., 2008). The two different nucleic acid vectors contain a recombinogenic sequence (e.g., any of the recombinogenic sequences described herein).

[0405] The hybrid vector is constructed based on a trans-splicing vector as described in Example 7. Thus, an active otoferlin gene (e.g., a full-length otoferlin gene) can be reconstituted using either ITR-mediated recombination and trans-splicing or recombination-inducing sequence-mediated (e.g., AP gene-mediated) recombination and trans-splicing. After trans-splicing, the active otoferlin gene (e.g., a full-length otoferlin gene) is reconstituted in the genomic DNA of mammalian cells (e.g., any mammalian cell described herein).

[0406] In some embodiments, the first rAAV vector comprises a first expression cassette including a promoter (e.g., any of the promoters described herein), a first coding sequence encoding an N-terminal portion of an otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the N-terminal portions of the otoferlin protein described herein) positioned 3' to the promoter, a splicing donor signal sequence positioned at the 3' end of the first coding sequence, and a first recombination-inducing sequence (e.g., an alkaline phosphatase recombination-inducing sequence) positioned 3' to the splicing donor signal sequence.

[0407] In some embodiments, the second rAAV vector comprises a second expression cassette including a second recombination-inducing sequence (e.g., an alkaline phosphatase recombination-inducing sequence), a splicing acceptor signal sequence positioned 3' to the second recombination-inducing sequence, a second coding sequence encoding a C-terminal portion of an otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the C-terminal portions of the otoferlin protein described herein) positioned at the 3' end of the splicing acceptor signal sequence, and a polyadenylation sequence (e.g., any of the polyadenylation sequences described herein) at the 3' end of the second coding sequence.

[0408] In some embodiments, the first rAAV vector genome comprises a first expression cassette including a promoter (e.g., any of the promoters described herein), a first coding sequence encoding an N-terminal portion of otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the N-terminal portions of the otoferlin protein described herein) positioned 3′ to the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a first recombinase-inducible sequence (e.g., an alkaline phosphatase recombinase-inducible sequence) positioned 3′ to the splicing donor signal sequence.

[0409] In some embodiments, the second rAAV vector genome comprises a second expression cassette including a second recombinase-inducible sequence (e.g., an alkaline phosphatase recombinase-inducible sequence), a splicing acceptor signal sequence positioned 3′ to the second recombinase-inducible sequence, a second coding sequence encoding a C-terminal portion of otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the C-terminal portions of the otoferlin protein described herein) positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence (e.g., any of the polyadenylation sequences described herein) at the 3′ end of the second coding sequence.

[0410] In some embodiments, each of the coded portions is at least 30 amino acid residues in length (e.g., at least 50 amino acids, at least 75 amino acids, or at least 100 amino acids in length), the amino acid sequences of each of the coded portions do not overlap, and neither single vector of the two different vectors encodes an active otoferlin protein (e.g., full-length otoferlin protein). When introduced into a mammalian cell (e.g., any of the mammalian cells described herein), splicing occurs between the splicing donor signal sequence and the splicing acceptor signal sequence, thereby forming an RNA that encodes an active otoferlin protein (e.g., full-length otoferlin protein).

[0411] Based on the strategies provided above, one of ordinary skill in the art will understand how to develop strategies using four, five, or six vectors.

[0412] The coding sequences provided in the two nucleic acid vectors (e.g., two) do not overlap. Each of the two different vectors may contain a coding sequence encoding a different portion of the otoferlin protein, and each of the coded portions is, for example, at least 30 amino acids (e.g., from about 30 amino acids to about 1600 amino acids, or any other sub-range within this range described herein).

[0413] In some embodiments, each of the two different rAAV vectors comprises a coding sequence encoding a different portion of the otoferlin protein, each of the encoded portions encoding up to 80% (e.g., up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%) of SEQ ID NO: 5 such that each of the encoded portions does not overlap. In some embodiments, each of the two different rAAV vectors comprises a coding sequence encoding a different portion of the otoferlin protein, each of the encoded portions encoding up to 80% (e.g., up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, or up to 70%) of SEQ ID NO: 5 such that each of the encoded portions does not overlap.

[0414] As described in Example 7, each of the two rAAV vectors may further comprise inverted terminal repeat sequences (ITRs) that allow for head-to-tail recombination. The ITRs are then removed via splicing. Examples of ITRs as well as splicing acceptor and / or splicing donor signal sequences are known in the art and are described in Example 7.

[0415] Example 3: Hybrid vector trans-splicing strategy using the highly recombinogenic exogenous gene region (AK) of F1 phage Two different rAAV vectors can also be used in any of the methods described herein to reconstitute an active otoferlin gene (e.g., a full-length otoferlin gene) intracellularly after intermolecular i) concatemerization, ii) recombination, iii) trans-splicing, iv) concatemerization and trans-splicing, or v) recombination and trans-splicing. This strategy is a hybrid strategy because it involves concatemerization, homologous recombination and / or trans-splicing. See, e.g., Trapani et al., EMBO Mol. Med. 6(2):194-211, 2014, which is incorporated herein by reference in its entirety.

[0416] As used herein, the F1 phage recombination induction region (AK) is used to enable recombination independent of the coding sequence. The F1 phage recombination induction region may be a 77 bp recombination induction region derived from the F1 phage genome as described in Trapani et al. (2014) EMBO Mol. Med. 6(2):194 - 211, 2014. The two different rAAV vectors contain the F1 phage recombination induction region. Since the hybrid vector is constructed based on the trans - splicing vector as described in Example 7, the rAAV vector encoding the active otoferlin protein (e.g., full - length stereocilin protein) can be generated using either ITR - mediated recombination and trans - splicing, or recombination and trans - splicing induced by the F1 phage recombination induction region. After trans - splicing, a nucleic acid encoding the active otoferlin protein (e.g., full - length otoferlin protein) is generated in mammalian cells (e.g., any of the mammalian cells described herein).

[0417] In some embodiments, two rAAV vectors are used. In some embodiments, the first rAAV vector comprises a first expression cassette including a promoter (e.g., any of the promoters described herein), a first coding sequence encoding an N - terminal portion of the otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the N - terminal portions of the otoferlin protein described herein) positioned 3' to the promoter, a splicing donor signal sequence positioned 3' to the first coding sequence, and an F1 phage recombination - inducible sequence positioned 3' to the splicing donor signal sequence.

[0418] In some embodiments, the second rAAV vector comprises a second expression cassette comprising an F1 phage recombination-inducing region, a splicing acceptor signal sequence located 3' to the F1 phage recombination-inducing region, a second coding sequence encoding a C-terminal portion of otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the C-terminal portions of the otoferlin protein described herein) positioned at the 3' end of the splicing acceptor signal sequence, and a polyadenylation sequence (e.g., any of the polyadenylation sequences described herein) at the 3' end of the second coding sequence.

[0419] In some embodiments, the first rAAV vector genome comprises a first expression cassette comprising a promoter (e.g., any of the promoters described herein), a first coding sequence encoding an N-terminal portion of otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the N-terminal portions of the otoferlin protein described herein) positioned 3' to the promoter, a splicing donor signal sequence positioned at the 3' end of the first coding sequence, and an F1 phage-derived sequence located 3' to the splicing donor signal sequence.

[0420] In some embodiments, the second rAAV vector genome comprises a second expression cassette comprising an F1 phage recombination-inducing region, a splicing acceptor signal sequence located 3' to the F1 phage recombination-inducing region, a second coding sequence encoding a C-terminal portion of otoferlin protein (e.g., any of the sizes of a portion of the otoferlin protein described herein and / or any of the C-terminal portions of the otoferlin protein described herein) positioned at the 3' end of the splicing acceptor signal sequence, and a polyadenylation sequence (e.g., any of the polyadenylation sequences described herein) at the 3' end of the second coding sequence.

[0421] In some embodiments, each of the encoded portions is at least 30 amino acid residues in length (e.g., at least 50 amino acids, at least 75 amino acids, or at least 100 amino acids in length), the amino acid sequences of each of the encoded portions do not overlap, and neither of the two different rAAV vectors encodes an active otopetrin protein (e.g., a full-length otopetrin protein). When an rAAV vector is introduced into a mammalian cell (e.g., any of the mammalian cells described herein), splicing occurs between the splicing donor signal sequence and the splicing acceptor signal sequence, thereby forming a recombinant nucleic acid that encodes an active otopetrin protein (e.g., a full-length otopetrin protein).

[0422] The coding sequences provided in each of the two rAAV vectors do not overlap. Each of the two different rAAV vectors contains a coding sequence encoding a different portion of the otopetrin protein, and each of the encoded portions is at least 30 amino acids (e.g., from about 30 amino acids to about 1600 amino acids, or any of the partial ranges within this range described herein).

[0423] In some embodiments, each of the two different rAAV vectors comprises a coding sequence encoding a different portion of the otoferlin protein, and each of the encoded portions encodes at least one exon and at least one intron of SEQ ID NO: 12 (e.g., at least two exons and at least one intron, at least two exons and at least two introns, at least three exons, at least one intron, at least three exons and at least two introns, or at least three exons and at least three introns). In some embodiments, each of the two different rAAV vectors comprises a coding sequence encoding a different portion of the otoferlin protein, and each of the encoded portions encodes up to 80% of SEQ ID NO: 5 (e.g., up to 10%, 20%, 30%, 40%, 50%, 60%, or 70% of SEQ ID NO: 5) such that the encoded portions do not overlap. In some embodiments, each of the two different rAAV vectors comprises a coding sequence encoding a different portion of the otoferlin protein, and each of the encoded portions encodes up to 80% of SEQ ID NO: 5 (e.g., up to 10%, 20%, 30%, 40%, 50%, 60%, or 70% of SEQ ID NO: 5) such that the encoded portions do not overlap.

[0424] As described in Example 7, each of the at least two nucleic acid vectors may further comprise inverted terminal repeats (ITRs) that allow head-to-tail recombination. The ITRs are then removed via splicing. Examples of ITRs as well as splicing acceptor and / or splicing donor signals are known in the art and are described in Example 7.

[0425] Example 4: In Vitro Expression of Full-Length Human Otoferlin Using Two Vectors Similar to other dual vector approaches, two transgenes, each containing a part of the full-length transcript, are packaged into separate vectors and provided together to contact a target, such as a target cell population within a subject in need thereof. This example provides a set of vectors generated to each contain a nucleic acid sequence comprising a part of the coding sequence of the human otoferlin (OTOF) gene or OTOF cDNA.

[0426] AAVAnc80-hOTOF is composed of two recombinant vectors (AAVAnc80-5’hOTOF and AAVAnc80-3’hOTOF) each containing a single-stranded DNA genome of 4452 and 3905 nucleotides (excluding ITRs) (Figure 1). The single-stranded DNA genomes are encapsidated by the AAVAnc80 (also called Anc80L65; Zinn 2015) capsid.

[0427] The upstream DNA genome (AAVAnc80-5’hOTOF) contains a eukaryotic expression cassette encoding the following promoter and regulatory sequences: the cytomegalovirus (CMV) immediate early enhancer element (SEQ ID NO: 98); the chicken beta-actin (CBA) gene sequence (SEQ ID NO: 99) located between the 5’ flanking region and the proximal region of the second exon; and the 3’ splice sequence (SEQ ID NO: 100) derived from the rabbit beta-globin (RBG) gene. This is generally referred to as the CAG promoter (Miyasaki 1989, Nwa 1991, Orban 2009). Following this hybrid regulatory element are the human OTOF (hOTOF) coding sequence, exons 1-21 (including both ends) (SEQ ID NO: 101), a synthetic splice donor (SD) (Trapani 2014) (SEQ ID NO: 102) to facilitate trans-splicing, and a 77 base pair (bp) AK recombination-inducing sequence (Trapani 2014, Trapan 2015) (SEQ ID NO: 103). The full-length AAVAnc80-5’hOTOF sequence has the sequence of SEQ ID NO: 96.

[0428] The downstream DNA genome (AAVAnc80-3’hOTOF) contains a eukaryotic expression cassette encoding the following: the same 77bp AK recombination-inducing sequence (Trapani 2014, Trapani 2015) (SEQ ID NO: 103); a synthetic splice acceptor (SA) to facilitate trans-splicing (Trapani 2014) (SEQ ID NO: 106); the human OTOF (hOTOF) coding sequence, exons 22-45 (including both ends) and exon 47 (excluding non-coding exon 46) (SEQ ID NO: 107); and the bovine growth hormone (bGH) polyadenylation (pA) signal (SEQ ID NO: 108). Each expression cassette is adjacent to AAV2 inverted terminal repeats (ITRs) (SEQ ID NOs: 97 and 104, respectively). The full-length AAVAnc80-3’hOTOF sequence has the sequence of SEQ ID NO: 105.

[0429] Target cells need to receive copies of both the upstream and downstream transgenes. Based on a dual-vector design, these transgenes recombine at the DNA level, leading to the production of full-length mRNA transcripts (McClements 2017), specifically the production of human OTOF based on isoform 5 according to NCBI accession number NM_001287489.1 (128…6121).

[0430] In some embodiments, when each of the upstream and downstream vectors is administered to a subject in need thereof, the constructs concatenate within a given cell. In some embodiments, the concatenated full-length OTOF is expressed, generating a functional otoferrin protein.

[0431] In some embodiments, when each of the upstream and downstream vectors is administered to a subject in need thereof, the constructs recombine within a given cell. In some embodiments, the recombined full-length OTOF is expressed, generating a functional otoferrin protein.

[0432] Pairs of these vectors are used to treat human subjects suffering from or prone to hearing loss. A composition comprising both vectors of the dual AAV vector system, AAVAnc80.AKhOTOF5 and AAVAnc80.AKhOTOF3, is introduced into at least one cochlea of a human subject. Auditory function is tested in the human subject on days 15, 30, 45, 60, and 90 after administration and compared to the auditory function of the human subject before treatment or a human subject not receiving treatment.

[0433] The AAVAnc80-5’hOTOF construct comprises a CAG promoter (specified by SEQ ID NOs: 98, 99, and 100) comprising two ITRs (SEQ ID NOs: 97 and 104), a CMV immediate enhancer element (SEQ ID NO: 98), the CMV immediate enhancer element (SEQ ID NO: 98), a chicken beta-actin gene sequence (SEQ ID NO: 99), and a chimeric intron comprising a 3’ splice sequence from a rabbit beta-globin gene (SEQ ID NO: 100), a 5’ OTOF coding region (SEQ ID NO: 101), an SD intron sequence (SEQ ID NO: 102), and an AK recombination-inducing sequence (SEQ ID NO: 103). Full-length AAVAnc80-5’hOTOF is represented by SEQ ID NO: 96.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

[0434] The AAVAnc80-3’hOTOF construct contains two ITRs (SEQ ID NOs: 97 and 104), an AK recombination-inducing sequence (SEQ ID NO: 103), an SA intron sequence (SEQ ID NO: 106), a 3’OTOF coding region (SEQ ID NO: 107), and a bgH polyA sequence (SEQ ID NO: 108). The full-length AAVAnc80-3’hOTOF is represented by SEQ ID NO: 105.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 3

[0435] Example 5: In Vivo Expression of Full-Length Human Otoferlin Using Two Vectors Wild-type or Otof - / - Mice (p23 ± 2 days) were administered either vehicle or the AAVAnc80-hOTOF dual hybrid vector described in Example 4 via intracochlear injection. Otoferlin expression in inner hair cells was examined 1 month after administration (Figures 6A - 6C). Robust expression of full-length human otoferlin was observed only in inner hair cells and not in other cochlear cells in Otof mice administered the AAVAnc80-hOTOF dual hybrid vector (Figure 6C). - / - In mice, it was observed only in inner hair cells and not in other cochlear cells (Figure 6C).

[0436] Either a vehicle or the flag-tagged AAVAnc80-hOTOF dual hybrid vector described in Example 4 was administered to non-human primates (NHP) via intracochlear injection. One month after administration, otoferlin expression in inner hair cells was examined (Figs. 7A-7B). Otoferlin-flag was detected only in inner hair cells and not in other cochlear nerve regions or supporting cell regions (Fig. 7B).

[0437] Next, the auditory function of Otof mice administered either a vehicle or the AAVAnc80-hOTOF dual hybrid vector described in Example 4 was evaluated. Auditory brainstem responses (ABRs) were evaluated 15, 30, 45, and 60 days after administration (Fig. 8A), or 1, 2, 3, 4.5, or 6 months after administration (Fig. 8B). Otof mice administered a vehicle - / - had no measurable auditory brainstem responses (Figs. 8A-8B). Approximately 70% of Otof mice administered the AAVAnc80-hOTOF dual hybrid vector showed recovery of auditory brainstem responses by day 15 (Fig. 8A). At least 80% of Otof- / - mice administered the AAVAnc80-otoferlin dual hybrid vector at 5-fold the dose had recovered auditory brainstem responses for at least 6 months (Fig. 8B). The degree of recovery of auditory function was dependent on the dose administered. - / - mice administered a vehicle - / - had no measurable auditory brainstem responses (Figs. 8A-8B). Approximately 70% of Otof mice administered the AAVAnc80-hOTOF dual hybrid vector showed recovery of auditory brainstem responses by day 15 (Fig. 8A). At least 80% of Otof- / - mice administered the AAVAnc80-otoferlin dual hybrid vector at 5-fold the dose had recovered auditory brainstem responses for at least 6 months (Fig. 8B). The degree of recovery of auditory function was dependent on the dose administered.

[0438] Example 6: In Vivo Biodistribution of Full-Length Human Otoferlin Using Two Vectors Non-human primates received bilateral cochlear implantation with either a vehicle, the flag-tagged AAVAnc80-hOTOF dual hybrid vector described in Example 4, or a spike-in positive control (OTOF-transduced HEK293FT cell lysate added to NHP tissue lysate), and the biodistribution of otoferlin in the submandibular lymph nodes, liver, and spleen was evaluated 1 month after administration (Figures 9A-9C). Human otoferlin-flag mRNA expression by RT-qPCR was positive only in the liver and spleen and was positive in only some of the animals (Figures 9A-9C). Human otoferlin-flag protein was not detected in the liver or spleen by Western blotting (Figures 9D-9E).

[0439] Example 7: In Vivo Survival and Function of Cochlear Hair Cells after Administration of Full-Length Human Otoferlin Using Two Vectors The survival of cochlear hair cells was quantified, and the local tolerability of cochlear implantation with either a vehicle or the flag-tagged AAVAnc80-hOTOF dual hybrid vector described in Example 4 was evaluated in non-human primates (NHP) (Figures 10A-10C) or Otof - / - mice (Figures 10D-10F). AAVAnc80-Otof was well tolerated both systemically and locally, and no adverse effects were observed in clinical pathology, ear pathology, systemic histopathology, and / or auditory function (Figures 10A-10F).

[0440] Auditory function and cochlear function were evaluated before and 6 months after cochlear implantation with either a vehicle or the flag-tagged AAVAnc80-hOTOF dual hybrid vector described in Example 4 in non-human primates (NHP) (Figures 11A-11B). Auditory function was measured using auditory brainstem response (ABR), and cochlear function was measured using distortion product otoacoustic emissions (DPOAE). No effect of otoferlin expression or dose on the shift in ABR or DPOAE thresholds (before vs. after administration) was observed.

[0441] Other aspects It should be understood that the words used are words of illustration rather than limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

[0442] Although the disclosure has been described in some degree of scope and in some degree of detail with respect to several described aspects, it is intended that no such detail or aspect or any particular aspect should be limiting, but reference should be made to the appended claims to provide the broadest interpretation of such claims from the perspective of the prior art and thus be construed so as to effectively encompass the intended scope of the disclosure.

[0443] Although the disclosure has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate rather than limit the scope of the disclosure as defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0444] All publications, patent applications, patents, and other references mentioned in this specification are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, will control. In addition, section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. a) A first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein said expression cassette is adjacent to an inverted terminal repeat (ITR), said first recombinant adeno-associated virus (rAAV) vector genome (vg), and b) A second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein said expression cassette is adjacent to an ITR, said second rAAV vector genome, comprising A composition comprising about 4.1E10 to 4.1E12 total vg or about 8.1E10 to 8.1E12 total vg.

2. The composition according to claim 1, comprising about 4.1E10 to 4.1E12 total vg / snail or about 8.1E10 to 8.1E12 total vg / snail.

3. The composition according to claim 1 or 2, comprising about 4.1E10 to 4.1E12 total vg / snail.

4. The composition according to any one of the preceding claims, comprising about 4.1E11 total vg / snail.

5. The composition according to claim 1 or 2, comprising about 8.1E10 to 8.1E12 total vg / snail.

6. The composition according to any one of claims 1, 2, or 5, comprising about 8.1E11 total vg / snail.

7. The composition according to any one of the preceding claims, wherein the concentration of said composition comprises about 4.5E11 to 4.5E13 total vg / mL or about 9E11 to 9E13 total vg / mL.

8. The composition according to any one of claims 1 to 4, wherein the concentration of said composition comprises about 4.5E11 to 4.5E13 total vg / mL.

9. The composition according to any one of claims 1 to 4 or 8, wherein the concentration of said composition comprises about 4.5E12 total vg / mL.

10. The composition according to any one of claims 1 to 2 or 5 to 7, wherein the concentration of said composition comprises about 9E11 to 9E13 total vg / mL.

11. The composition according to any one of claims 1 to 2, 5 to 7, or 10, wherein the concentration of said composition comprises about 9E12 total vg / mL.

12. The composition according to any one of the preceding claims, comprising the first rAAV vector genome and the second rAAV vector genome in a ratio of about 1:

1.

13. The composition according to any one of claims 1 to 4 or 7 to 9, comprising a total of about 4.1E11 vg.

14. The composition according to any one of claims 1, 2, 5 to 7, or 10 to 12, comprising a total of about 8.1E11 vg.

15. (a) A first rAAV vector genome comprising a first expression cassette including a promoter, a first coding sequence encoding an N-terminal portion of an otoferlin protein located 3' to the promoter, and a splicing donor signal sequence located at the 3' end of the first coding sequence, and (b) A second rAAV vector genome comprising a second expression cassette including a splicing acceptor signal sequence, a second coding sequence encoding a C-terminal portion of an otoferlin protein located 3' to the splicing acceptor signal sequence, and a polyadenylation sequence at the 3' end of the second coding sequence, A composition formulated for intratympanic administration.

16. The composition according to claim 15, comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.

17. The composition according to claim 15 or 16, formulated to contain synthetic perilymph.

18. The composition according to any one of claims 15 to 17, further comprising one or more buffers and one or more surfactants.

19. The composition according to claim 18, wherein the buffer is selected from potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS.

20. The composition according to claim 18 or 19, wherein the surfactant is selected from poloxamer 188, labrasol, tween, ethanol, pluronic F68, and polyethylene glycol.

21. The composition according to any one of claims 15 to 20, further comprising potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, and poloxamer 188.

22. The formulation is a) about 1.35 to 1.65 mM of potassium dihydrogen phosphate, b) about 7.29 to 8.91 mM of disodium hydrogen phosphate, c) about 2.43 to 2.97 mM of potassium chloride, d) about 154.8 to 189.2 mM of sodium chloride, and e) about 0.0001% to 0.01% of poloxamer 188. The composition according to any one of claims 15 to 21, comprising

23. wherein the formulation is a) about 1.5 mM of monopotassium phosphate, b) about 8.1 mM of disodium phosphate, c) about 2.7 mM of potassium chloride, d) about 172 mM of sodium chloride, and e) about 0.001% of poloxamer 188, The composition according to any one of claims 15 to 22, comprising

24. The composition according to any one of claims 15 to 23, comprising about 4.1E10 to 4.1E12 total vg / snail or about 8.1E10 to 8.1E12 total vg / snail.

25. The composition according to any one of claims 15 to 24, comprising about 4.1E11 total vg / snail.

26. The composition according to any one of claims 15 to 24, comprising about 8.1E11 total vg / snail.

27. The composition according to any one of claims 15 to 25, wherein the concentration of the composition comprises about 4.5E12 total vg / mL.

28. The composition according to any one of claims 15 to 24 or 26, wherein the concentration of the composition comprises about 9E12 total vg / mL.

29. The composition according to any one of claims 15 to 28, comprising the first rAAV vector genome and the second rAAV vector genome in a ratio of about 1:

1.

30. The composition according to any one of the preceding claims, wherein the nucleic acid sequence comprising the 5' portion of the otoferlin gene has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

101.

31. The composition according to any one of the preceding claims, wherein the nucleic acid sequence comprising the 3' portion of the otoferlin gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

107.

32. The composition according to any one of the preceding claims, wherein the promoter is selected from a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

33. The composition according to claim 32, wherein the promoter is a constitutive promoter.

34. The composition according to claim 33, wherein the constitutive promoter is selected from a CAG, CBA, or CMV promoter.

35. The composition according to claim 34, wherein the constitutive promoter is a CAG promoter.

36. The composition according to any one of the preceding claims, wherein the first rAAV vector contains a splicing donor site and a recombination-inducible sequence.

37. The composition according to any one of the preceding claims, wherein the second rAAV vector contains a splicing acceptor site, a recombination-inducible sequence, and a polyadenylation sequence.

38. The composition according to claim 36, wherein the splicing donor site has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

102.

39. The composition according to claim 37, wherein the splicing acceptor site has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

106.

40. The composition according to any one of claims 36 to 39, wherein the recombination-inducible sequence has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

103.

41. The composition according to any one of claims 36 to 40, wherein the polyadenylation sequence is selected from bovine growth hormone, human growth hormone, mouse-β-globin, mouse-α-globin, polyomavirus, SV40, or a synthetic polyadenylation sequence.

42. The composition according to claim 41, wherein the polyadenylation is a bovine growth hormone polyadenylation sequence.

43. The composition according to any one of claims 36 to 42, wherein the polyadenylation sequence has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

108.

44. The composition according to any one of the preceding claims, wherein the ITR is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80 ITR.

45. The composition according to claim 44, wherein the ITR is AAV2 ITR.

46. The composition according to any one of the preceding claims, wherein the first expression cassette has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

96.

47. The composition according to any one of the preceding claims, wherein the second expression cassette has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

105.

48. The composition according to any one of the preceding claims, wherein the first rAAV vector and the second rAAV vector are each encapsulated by an AAV capsid.

49. The composition according to claim 48, wherein the AAV capsid encapsulating the first rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80.

50. The composition according to claim 48, wherein the AAV capsid encapsulating the second rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80.

51. The composition according to claim 49 or 50, wherein the first rAAV vector is encapsulated by an Anc80 capsid and the second rAAV vector is encapsulated by an Anc80 capsid.

52. The composition according to any one of claims 49 to 51, wherein the Anc80 capsid contains the polypeptide sequence of SEQ ID NO:

109.

53. The composition according to any one of claims 1 to 14 or 30 to 52, formulated for intrasnail administration.

54. The composition according to any one of claims 1 to 14 or 30 to 53, comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.

55. The composition according to any one of claims 1 to 14 or 30 to 54, formulated to contain synthetic perilymph.

56. The composition according to any one of claims 1 to 14 or 30 to 55, further comprising one or more buffering agents and one or more surfactants.

57. The composition according to claim 56, wherein the buffering agent is selected from potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS.

58. The composition according to claim 56, wherein the surfactant is selected from poloxamer 188, labrasol, tween, ethanol, pluronic F68, and polyethylene glycol.

59. The composition according to any one of claims 1 to 14 or 30 to 58, further comprising monopotassium phosphate, dibasic sodium phosphate, potassium chloride, sodium chloride, and poloxamer 188.

60. The preparation is a) about 1.35 to 1.65 mM of monopotassium phosphate, b) about 7.29 to 8.91 mM of dibasic sodium phosphate, c) about 2.43 to 2.97 mM of potassium chloride, d) about 154.8 to 189.2 mM of sodium chloride, and e) about 0.0001% to 0.01% of poloxamer 188, the composition according to any one of claims 53 to 59.

61. The preparation is a) about 1.5 mM of monopotassium phosphate, b) about 8.1 mM of dibasic sodium phosphate, c) about 2.7 mM of potassium chloride, d) about 172 mM of sodium chloride, and e) about 0.001% of poloxamer 188, the composition according to any one of claims 53 to 60.

62. A method for treating hearing loss in a subject having a defective otoferlin gene, comprising administering to the subject snail the composition according to any one of the preceding claims, wherein the first rAAV vector and the second rAAV vector can constitute a polypeptide messenger RNA encoding a full-length human otoferlin protein in the subject.

63. The method according to claim 62, further comprising determining before the administering step that the subject has a defective otoferlin gene.

64. The method according to claim 63, wherein the defective otoferlin gene comprises a mutation that results in a decrease in the expression and / or activity of the otoferlin protein encoded by the gene.

65. The method according to any one of claims 62 to 64, wherein the method functions to reduce or prevent secondary degeneration of one or more snail structures.

66. A method for expressing recombinant full-length otoferlin protein in a mammalian cell, comprising administering to the mammalian cell the composition according to any one of claims 1 to 61, wherein the first rAAV vector and the second rAAV vector can constitute a polypeptide messenger RNA encoding a full-length human otoferlin protein in the mammalian cell.

67. The method according to claim 66, wherein the mammalian cell is a snail cell.

68. The method according to claim 67, wherein the mammalian cell is an inner ear hair cell.

69. The method according to claim 62, wherein the subject is a mammal.

70. The method according to claim 62, wherein the subject is a human.

71. The method according to any one of claims 62 to 70, wherein the composition is administered in a single dose.

72. The method according to any one of claims 62 to 70, wherein the composition is administered in multiple doses.

73. The method according to claim 72, wherein the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

74. The method according to any one of claims 62 to 71, wherein the single dose comprises about 0.01 mL to 0.2 mL.

75. The method according to claim 74, wherein the single dose comprises about 0.09 mL.

76. The method according to any one of claims 62 to 75, wherein the composition is administered as an injection into the round window membrane.

77. The method according to claim 76, wherein the composition is administered in a single injection.

78. The method according to claim 76, wherein the composition is administered in multiple injections.

79. The method according to claim 78, wherein the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 injections.

80. The method according to any one of claims 62 to 79, wherein the composition is administered through the use of a medical device.

81. The method according to claim 80, wherein the device is the device shown in FIGS. 2 to 5.

82. The method according to claim 80, wherein the device is a microcatheter.

83. The method according to any one of claims 62 to 82, wherein the composition is delivered at a controlled flow rate.

84. The method according to any one of claims 62 to 83, wherein the subject is 2 to 17 years old.

85. The method according to any one of claims 62 to 84, wherein the administration of the composition improves the subject's auditory brainstem response (ABR) threshold response, age-appropriate behavioral audiometry, myringotomy examination, and / or word / sentence recognition test.

86. A kit comprising the composition according to any one of claims 1 to 61.

87. The kit according to claim 86, further comprising a pre-filled syringe containing the composition.

88. The kit according to claim 86, further comprising a syringe containing the composition.

89. a) A first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR), the first recombinant adeno-associated virus (rAAV) vector genome (vg), and b) A second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, the second rAAV vector genome, A composition comprising a total of about 4.1E10 to 8.1E12 vg.

90. The composition according to claim 89, comprising a total of about 4.1E10 to 4.1E12 vg.

91. The composition according to claim 89 or 90, comprising a total of about 4.1E11 vg.

92. The composition according to claim 89 or 90, comprising about 4.1E10 to 4.1E12 vg / snail.

93. The composition according to any one of claims 89 to 92, comprising about 4.1E11 vg / snail.

94. The composition according to any one of claims 89 to 93, wherein the concentration of the composition comprises about 4.5E11 to 4.5E13 vg / mL.

95. The composition according to any one of claims 89 to 94, wherein the concentration of the composition comprises about 4.5E12 vg / mL.

96. The composition according to claim 89, comprising a total of about 8.1E10 to 8.1E12 vg.

97. The composition according to claim 89 or 96, comprising a total of about 8.1E11 vg.

98. The composition according to claim 89 or 96, comprising about 8.1E10 to 8.1E12 vg / snail.

99. The composition according to claim 89 or any one of claims 96 to 98, comprising about 8.1E11 vg / snail.

100. The composition according to claim 89 or any one of claims 96 to 99, wherein the concentration of the composition comprises about 9E11 to 9E13 vg / mL.

101. The composition according to claim 89 or any one of claims 96 to 100, wherein the concentration of the composition comprises about 9E12 vg / mL.

102. The composition according to any one of claims 89 to 101, comprising the first rAAV vector genome and the second rAAV vector genome in a ratio of about 1:

1.

103. a) A first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising the 5' portion of the otoferlin gene, wherein the expression cassette is adjacent to an inverted terminal repeat (ITR), the first recombinant adeno-associated virus (rAAV) vector genome (vg), and b) A second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising the 3' portion of the otoferlin gene, wherein the expression cassette is adjacent to an ITR, the second rAAV vector genome, A composition formulated for administration into the cochlea.

104. The composition according to claim 103, comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.

105. The composition according to claim 103 or 104, formulated to contain synthetic perilymph.

106. The composition according to any one of claims 103 to 105, further comprising one or more buffering agents and one or more surfactants.

107. The composition according to claim 106, wherein the buffering agent is selected from potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS.

108. The composition according to claim 106 or 107, wherein the surfactant is selected from poloxamer 188, laurabazole, tween, ethanol, pluronic F68, and polyethylene glycol.

109. The composition according to any one of claims 103 to 108, further comprising potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride, and poloxamer 188.

110. The formulation is a) about 1.35 to 1.65 mM of potassium dihydrogen phosphate, b) about 7.29 to 8.91 mM of disodium hydrogen phosphate, c) about 2.43 to 2.97 mM of potassium chloride, d) about 154.8 to 189.2 mM of sodium chloride, and e) about 0.0001% to 0.01% of poloxamer 188, the composition according to any one of claims 103 to 109.

111. The formulation is a) about 1.5 mM of monopotassium phosphate, b) about 8.1 mM of dibasic sodium phosphate, c) about 2.7 mM of potassium chloride, d) about 172 mM of sodium chloride, and e) about 0.001% of poloxamer 188, the composition according to any one of claims 103 to 110.

112. The composition according to any one of claims 103 to 111, comprising about 4.1E10 to 8.1E12 total vg / snail.

113. The composition according to any one of claims 103 to 112, comprising about 4.1E10 to 4.1E12 total vg / snail or about 8.1E10 to 8.1E12 total vg / snail.

114. The composition according to any one of claims 103 to 113, comprising about 4.1E11 total vg / snail.

115. The composition according to any one of claims 103 to 113, comprising about 8.1E11 total vg / snail.

116. The composition according to any one of claims 103 to 115, wherein the concentration of the composition comprises about 4.5E11 to 9E13 total vg / mL.

117. The composition according to any one of claims 103 to 114 or 116, wherein the concentration of the composition comprises about 4.5E12 total vg / mL.

118. The composition according to any one of claims 103 to 113, 115, or 116, wherein the concentration of the composition comprises about 9E12 total vg / mL.

119. The composition according to any one of claims 103 to 118, comprising the first rAAV vector genome and the second rAAV vector genome in a ratio of about 1:

1.

120. The nucleic acid sequence comprising the 5' portion of the otoferlin gene has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 101, the composition according to any one of claims 89 to 119.

121. The composition according to any one of claims 89 to 120, wherein the nucleic acid sequence containing the 3' portion of the otoferlin gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

107.

122. The composition according to any one of claims 89 to 121, wherein the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

123. The composition according to claim 122, wherein the promoter is a constitutive promoter.

124. The composition according to claim 123, wherein the constitutive promoter is selected from a CAG, CBA, or CMV promoter.

125. The composition according to claim 124, wherein the constitutive promoter is a CAG promoter.

126. The composition according to any one of claims 89 to 125, wherein the first rAAV vector contains a splicing donor site and a recombination-inducing sequence.

127. The composition according to any one of claims 89 to 126, wherein the second rAAV vector contains a splicing acceptor site, a recombination-inducing sequence, and a polyadenylation sequence.

128. The composition according to claim 126, wherein the splicing donor site has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

102.

129. The composition according to claim 127, wherein the splicing acceptor site has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

106.

130. The composition according to any one of claims 126 to 129, wherein the recombinant-inducible array has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

103.

131. The composition according to any one of claims 126 to 130, wherein the polyadenylation array is selected from bovine growth hormone, human growth hormone, mouse-β-globin, mouse-α-globin, polyomavirus, SV40, or a synthetic polyadenylation array.

132. The composition according to claim 131, wherein the polyadenylation is a bovine growth hormone polyadenylation array.

133. The composition according to any one of claims 126 to 132, wherein the polyadenylation array has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

108.

134. The composition according to any one of claims 89 to 133, wherein the ITR is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80 ITR.

135. The composition according to claim 134, wherein the ITR is AAV2 ITR.

136. The composition according to any one of claims 89 to 135, wherein the first expression cassette has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

96.

137. The composition according to any one of claims 89 to 136, wherein the second expression cassette has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

105.

138. The composition according to any one of claims 89 to 137, wherein the first rAAV vector and the second rAAV vector are each encapsulated by an AAV capsid.

139. The composition according to claim 138, wherein the AAV capsid encapsulating the first rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80.

140. The composition according to claim 138, wherein the AAV capsid encapsulating the second rAAV vector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80.

141. The composition according to claim 139 or 140, wherein the first rAAV vector is encapsulated by an Anc80 capsid and the second rAAV vector is encapsulated by an Anc80 capsid.

142. The composition according to any one of claims 139 to 141, wherein the Anc80 capsid contains the polypeptide sequence of SEQ ID NO:

109.

143. The composition according to any one of claims 89 to 102 or 120 to 142, formulated for intrasnail administration.

144. The composition according to any one of claims 89 to 102 or 120 to 143, comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.

145. The composition according to any one of claims 89 to 102 or 120 to 144, formulated to contain synthetic perilymph.

146. The composition according to any one of claims 89 to 102 or 120 to 145, further comprising one or more buffering agents and one or more surfactants.

147. The composition according to claim 146, wherein the buffering agent is selected from monopotassium phosphate, dibasic sodium phosphate, potassium chloride, sodium chloride, Tris HCl, Tris base, histidine, boric acid, citric acid, glycine, HEPES, and MOPS.

148. The composition according to claim 146, wherein the surfactant is selected from poloxamer 188, labrasol, tween, ethanol, pluronic F68, and polyethylene glycol.

149. The composition according to any one of claims 89 to 102 or 143 to 148, further comprising monopotassium phosphate, dibasic sodium phosphate, potassium chloride, sodium chloride, and poloxamer 188.

150. The formulation is a) about 1.35 to 1.65 mM of monopotassium phosphate, b) about 7.29 to 8.91 mM of dibasic sodium phosphate, c) about 2.43 to 2.97 mM of potassium chloride, d) about 154.8 to 189.2 mM of sodium chloride, and e) about 0.0001% to 0.01% of poloxamer 188, and the composition according to any one of claims 143 to 149.

151. The formulation is a) about 1.5 mM of monopotassium phosphate, b) about 8.1 mM of dibasic sodium phosphate, c) about 2.7 mM of potassium chloride, d) about 172 mM of sodium chloride, and e) about 0.001% of poloxamer 188, and the composition according to any one of claims 143 to 150.

152. The composition according to any one of claims 103 to 119 or 143 to 151, wherein the formulation is a sterile suspension.

153. The composition according to any one of claims 103 to 119 or 143 to 152, wherein the formulation contains sterile water.

154. The composition according to any one of claims 103 to 119 or 143 to 153, wherein the volume of the formulation is about 0.01 mL to 0.2 mL.

155. The composition according to any one of claims 103 to 119 or 143 to 154, wherein the volume of the formulation is about 0.09 mL.

156. A method for treating hearing loss in a subject having a defective otoferlin gene, the method comprising administering to the subject snail a composition according to any one of claims 89 to 155, wherein the first rAAV vector and the second rAAV vector are capable of constituting a polypeptide messenger RNA encoding a full-length human otoferlin protein in the subject.

157. The method according to claim 156, wherein the defective otoferlin gene comprises a mutation that results in a decrease in the expression and / or activity of the otoferlin protein encoded by the gene.

158. The method according to claim 156 or 157, further comprising determining before the administering step that the subject has a defective otoferlin gene.

159. A method for treating hearing loss in a subject identified as having otoferlin gene mutations in both alleles, the method comprising administering to the subject snail a composition according to any one of claims 89 to 155, wherein the first rAAV vector and the second rAAV vector are capable of constituting a polypeptide messenger RNA encoding a full-length human otoferlin protein in the subject.

160. The method according to claim 159, further comprising determining before the administering step that the subject has otoferlin gene mutations in both alleles.

161. The method according to any one of claims 156 to 160, wherein the subject has clinical symptoms of bilateral severe sensorineural hearing loss.

162. The method according to claim 161, wherein the subject has clinical symptoms of bilateral severe sensorineural hearing loss without fever.

163. The method according to any one of claims 156 to 162, wherein the method functions to reduce or prevent secondary degeneration of one or more snail structures.

164. The method according to any one of claims 156 to 163, wherein the subject preserves distortion product otoacoustic emissions (DPOAE).

165. A method for expressing recombinant full-length otoferlin protein in mammalian cells, comprising administering to the mammalian cells the composition according to any one of claims 89 to 155, wherein the first rAAV vector and the second rAAV vector can constitute a polypeptide messenger RNA encoding the full-length human otoferlin protein in the mammalian cells.

166. The method according to claim 165, wherein the mammalian cell is a cochlear cell.

167. The method according to claim 166, wherein the mammalian cell is an inner ear hair cell.

168. The method according to claim 156 or 159, wherein the subject is a mammal.

169. The method according to claim 156 or 159, wherein the subject is a human.

170. The method according to any one of claims 156 to 169, wherein the composition is administered in a single dose.

171. The method according to any one of claims 156 to 169, wherein the composition is administered in multiple doses.

172. The method according to claim 171, wherein the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

173. The method according to any one of claims 156 to 172, wherein the single dose comprises about 0.01 mL to 0.2 mL.

174. The method according to claim 173, wherein the single dose comprises about 0.09 mL.

175. The method according to any one of claims 156 to 174, wherein the composition is administered as an injection into the round window membrane.

176. The method according to claim 175, wherein the composition is administered as a single injection.

177. The method according to claim 175, wherein the composition is administered as multiple injections.

178. The method according to claim 177, wherein the composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, or 10 injections.

179. The method according to any one of claims 156 to 178, wherein the composition is administered through the use of a medical device.

180. The method according to claim 179, wherein the composition is pre-filled in the device.

181. The method according to claim 179 or 180, wherein the device is the device shown in FIGS. 2 to 5.

182. The method according to claim 179 or 180, wherein the device is a microcatheter.

183. The method according to claim 182, wherein the microcatheter enters the middle ear cavity through the external auditory canal and is shaped such that the end of the microcatheter can be brought into contact with the round window membrane (RWM).

184. The method according to claim 182 or 183, wherein the distal end of the microcatheter includes at least one micro-needle having a diameter of 10 to 1,000 microns.

185. The method according to claim 184, wherein the at least one micro-needle includes a bent portion and an inclined tip.

186. The method according to any one of claims 156 to 185, wherein the composition is delivered at a controlled flow rate.

187. The method according to any one of claims 156 to 185, wherein the subject is 2 to 17 years old.

188. The method according to any one of claims 156 to 187, wherein the administration of the composition improves the auditory brainstem response (ABR) threshold response, age-appropriate behavioral audiometry, myringotomy examination, and / or word / sentence recognition examination of the subject.

189. A kit comprising the composition according to any one of claims 89 to 155.

190. The kit according to claim 189, wherein the composition is pre-filled in a device.

191. The kit according to claim 190, wherein the device is a microcatheter.

192. The kit according to claim 191, wherein the microcatheter enters the middle ear cavity through the external auditory canal and is shaped such that the end of the microcatheter can be brought into contact with the round window membrane (RWM).

193. The kit according to claim 191 or 192, wherein the distal end of the microcatheter is composed of at least one micro-needle having a diameter of 10 to 1,000 microns.

194. The kit according to claim 189, further comprising a device.

195. The kit according to claim 194, wherein the device is the device described in any one of FIGS. 2 to 5.

196. The kit according to claim 194 or 195, wherein the device includes a needle having a bent portion and an inclined tip.

197. The kit according to any one of claims 194 to 196, further comprising a syringe containing the composition.

198. The kit according to claim 197, wherein the vial is a single-use vial.

199. The kit according to claim 197 or 198, further comprising a second vial containing a diluent.

Images