Compositions and methods for treating non-age-related hearing impairment in human subjects

Abstract

Provided herein are compositions comprising at least two different nucleic acid vectors, each of which comprises a coding sequence encoding a different portion of an otoferlin protein, and uses of these compositions for treating hearing loss in a subject. The present disclosure is based on the discovery that a composition comprising at least two different nucleic acid vectors, each of which comprises a coding sequence encoding a different portion of an otoferlin protein, can be used to generate a sequence encoding an active otoferlin protein (e.g., a full-length otoferlin protein) in mammalian cells, thereby treating non-syndromic sensorineural hearing loss in a subject in need thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 979,792, filed February 21, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on February 21, 2020, is named 2013615-0002_SL.txt and is 1,298,432 bytes in size.

[0003] Technical Field The present disclosure relates generally to the use of nucleic acids to treat hearing loss in a human subject. [Background technology]

[0004] Background of the Disclosure The ear is a complex organ, classically described as including the outer ear, middle ear, inner ear, hearing (acoustic) nerve, and 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. Therefore, disorders of the inner ear can cause hearing loss, tinnitus, spatial disorientation, and imbalance. Spatial disorientation is a hallucination of movement and is a primary symptom of vestibular system disorders. Spatial disorientation can be caused by problems in the inner ear or central nervous system. Common inner ear causes of spatial disorientation include vestibular neuritis (sudden unilateral vestibular loss), Meniere's disease (episodic spatial disorientation), benign paroxysmal positional vertigo (BPPV), and bilateral vestibular loss. Common central nervous system causes of spatial disorientation include postconcussion syndrome, cervical vertigo, vestibular migraine, cerebrovascular disease, and acoustic neuroma.

[0005] Hearing loss is one of the most common human sensory losses and can occur for many reasons. Some people are born with hearing loss, while others lose it slowly over time. Presbycusis (also spelled presbyacusis) is age-related hearing loss. Approximately 36 million adult Americans report some degree of hearing loss, with one in three over the age of 60 and half of those over the age of 85 experiencing hearing loss.

[0006] Hearing loss can be the result of environmental factors or a combination of genetic and environmental factors. Approximately half of all people with tinnitus—a hallucination in the auditory system (ringing, buzzing, ringing, humming, or beating sounds)—also have hypersensitivity / reduced tolerance to certain audible frequencies and volume ranges, known as hyperacusis (also written as hyperacusis). Williams syndrome (also known as Williams-Beuren syndrome) is a multisystem disorder caused by a hemizygous deletion of a 1.6 Mb region in 7q11.23 encompassing approximately 26 genes, including the gene encoding LIM kinase 1 (LIMK1). Individuals with Williams syndrome exhibit hyperacusis and progressive hearing loss, and the early onset of hyperacusis suggests that it may be related to one of the deleted genes.

[0007] Environmental causes of hearing loss include certain medications, certain infections before or after birth, and prolonged exposure to loud noise. Hearing loss can be caused by noise, ototoxic agents, presbycusis, disease, infection, or cancer affecting specific parts of the ear. Ischemic injury can cause hearing loss through pathophysiological mechanisms initiated by the immune system. As another example, autoimmune inner ear disease (AIED) is characterized by rapidly progressive bilateral sensorineural hearing loss, which occurs when the body's immune system mistakes cells in the inner ear for viruses or bacteria and attacks them.

[0008] Approximately 1.5 children in 1,000 are born with profound hearing loss, and an additional 2-3 children in 1,000 are born with partial hearing loss (Smith et al., 2005, Lancet 365:879-890). More than half of these cases are attributable to genetic causes (Di Domenico, et al., 2011, J. Cell. Physiol. 226:2494-2499).

[0009] Non-syndromic hearing loss is hearing loss that is not associated with other signs and symptoms. In contrast, syndromic hearing loss involves hearing loss that occurs in conjunction with abnormalities in other parts of the body.

[0010] Most cases of genetic hearing loss (70 percent to 80 percent) are nonsyndromic; the remaining cases are caused by specific genetic syndromes.

[0011] Hearing loss can be conductive (originating from the ear canal or middle ear), sensorineural (originating from the inner ear or auditory nerve), or mixed. Many forms of nonsyndromic hearing loss are associated with permanent hearing loss caused by damage to structures within the inner ear (sensorineural hearing loss). The majority of human sensorineural hearing loss is caused by abnormalities in the hair cells of the organ of Corti within the cochlea. There are also very rare sensorineural hearing disorders involving the eighth cranial nerve (vestibulocochlear nerve) or the auditory portion of the brain. In the rarest of these types of hearing loss, only the auditory center of the brain is affected. In this situation, cortical hearing loss can occur, in which sounds can be heard at normal thresholds, but the perceived quality of the sound is so poor that speech cannot be understood. However, most sensorineural hearing loss results from defective hair cell function. Hair cells may be abnormal at birth or damaged during an individual's lifetime. There are both exogenous insults, such as noise trauma and infection, and intrinsic abnormalities, such as congenital mutations to genes that play important roles in the anatomy or physiology of the cochlea.

[0012] Hearing loss resulting from changes in the middle ear is called conductive hearing loss. Some forms of nonsyndromic hearing loss involve changes in both the inner and middle ear and are called mixed hearing loss. Hearing loss that is present before a child learns to speak is classified as prelingual or congenital. Hearing loss that occurs after speech development is classified as postlingual. Most autosomal recessive loci cause severe to profound prelingual hearing loss.

[0013] Non-syndromic hearing loss can have different inheritance patterns and can occur at any age. Types of non-syndromic hearing loss are named according to their inheritance patterns. The autosomal dominant form is designated DFNA, the autosomal recessive form is designated DFNB, and the X-linked form is designated DFN. Each type is also numbered in the order in which it was described. For example, DFNA1 was the first autosomal dominant type of non-syndromic hearing loss described.

[0014] In 75 to 80 percent of cases, the condition is inherited in an autosomal recessive pattern, meaning that both copies of the gene in each cell carry the mutation. Usually, each parent of an individual with autosomal recessive hearing loss carries one copy of the mutated gene but is not affected by this form of hearing loss.

[0015] Another 20 to 25 percent of nonsyndromic hearing loss cases are autosomal dominant, meaning that one copy of the altered gene in each cell is sufficient to cause hearing loss. People with autosomal dominant hearing loss most often inherit an altered copy of the gene from one parent who has hearing loss.

[0016] One to two percent of cases show an X-linked pattern of inheritance, meaning that the mutated gene responsible for the condition is located on the X chromosome (one of the two sex chromosomes). Males with X-linked nonsyndromic hearing loss tend to develop hearing loss earlier in life and more severely than females who inherit a mutated copy of the same gene. A hallmark of X-linked inheritance is that fathers cannot pass the X-linked trait to their sons.

[0017] Mitochondrial non-syndromic hearing loss, which is caused by changes to mitochondrial DNA, occurs in less than 1 percent of cases in the United States. The altered mitochondrial DNA is passed down from the mother to all of her sons and daughters. This type of hearing loss is not inherited from the father.

[0018] Auditory neuropathy spectrum disorder (ANSD), a hearing disorder characterized by normal outer hair cell function and abnormal or absent auditory brainstem response, is one of the most common disorders that cause hearing and speech communication barriers in infants and young children. Approximately 10% of children with permanent hearing loss may have ANSD. The OTOF gene was the first gene identified for autosomal recessive nonsyndromic ANSD, and mutations in OTOF account for approximately 5% of all cases of autosomal recessive nonsydromic hearing loss in some populations (Rodriguez-Ballesteros et al. 2008 Human Mut 29(6):823-831).

[0019] The causes of nonsyndromic hearing loss are complex. Researchers have identified more than 30 genes that, when altered, are associated with nonsyndromic hearing loss, although some of these genes have not been fully characterized. Different mutations in the same gene may be associated with different types of hearing loss, and some genes are associated with both syndromic and nonsyndromic hearing loss.

[0020] For example, genes associated with non-syndromic 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, MYO7A, OTOF, PCDH15, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIOBP, USH1C, and WFS1.

[0021] The most common cause of hearing loss is nonsyndromic hearing loss and deafness, DFNB1 (also known as GJB2-related DFNB1 nonsyndromic hearing loss and deafness, autosomal recessive deafness 1, and sensorineural nonsyndromic recessive deafness 1). Nonsyndromic hearing loss and deafness (DFNB1) is characterized by congenital, nonprogressive, mild to severe sensorineural hearing impairment. It is caused by mutations in GJB2 (encoding the protein connexin 26) and GJB6 (encoding connexin 30). Diagnosis of DFNB1 relies on molecular genetic testing to identify deafness-causing mutations in GJB2 and upstream cis-regulatory elements that modify the gap junction beta-2 protein (connexin 26). Molecular genetic testing for GJB2 detects deafness-causing mutations in these genes in >99% of cases. Unlike some other forms of hearing loss, DFNB1 nonsyndromic hearing loss and deafness does not affect balance or movement. The degree of hearing loss is difficult to predict based on which gene mutation is present. Even when members of the same family have DFNB1 nonsyndromic hearing loss and deafness, the degree of hearing loss can vary between them.

[0022] Mutations in the genes encoding connexin 26 (Cx26) and / or Cx30 are associated with approximately half of all cases of human autosomal nonsyndromic prelingual hearing loss. Cx26 and Cx30 are the two major Cx isoforms found in the cochlea, which coassemble to form hybrid (heteromeric and heterotypic) gap junctions (GJs) (Ahmad, et al., Proc. Natl. Acad. Sci., 2007, 104(4):1337-1341). Nonsyndromic hearing loss and hearing impairment, DFNA3, are caused by dominant-negative pathogenic variants in the GJB2 or GJB6 genes, which alter either the connexin 26 (Cx26) or connexin 30 (Cx30) proteins, respectively, and are characterized by mild to severe, progressive high-frequency sensorineural hearing loss that occurs before or after language acquisition.

[0023] OTOF-associated hearing loss (DFNB9 nonsyndromic hearing loss) is characterized by two phenotypes: prelingual nonsyndromic hearing loss and, less frequently, temperature-sensitive nonsyndromic auditory neuropathy (TS-NSAN).

[0024] Another form of progressive hearing loss is associated with mutations in the otoferlin gene (eg, the I1573T or P1987R mutation, and / or the E1700Q mutation) or is not temperature sensitive.

[0025] Pendred syndrome / DFNB4 (deafness with goiter) is an autosomal recessive disorder that accounts for 7.5% of all cases of congenital hearing loss. Pendred syndrome is associated with mutations in the PDS gene (also known as DFNB4, EVA, PDS, TDH2B, and solute transporter family 26, member 4, SLC26A4) on the long arm of chromosome 7 (7q31), which encodes the pendrin protein. Mutations in this gene also cause enlarged vestibular aqueduct syndrome (EVA or EVAS), another congenital cause of hearing loss; certain mutations are more likely to cause EVAS, while others are more associated with Pendred syndrome. (Azaiez, et al. (December 2007), Hum. Genet. 122 (5): 451-7)

[0026] Transmembrane protease, serine 3, is an enzyme encoded by the TMPRSS3 gene (also known as DFNB10, DFNB8, ECHOS1, and TADG12). This gene was identified through its association with both congenital and childhood-onset autosomal recessive hearing loss. Mutations in TMPRSS3 are associated with postlingual and rapidly progressive hearing loss. The protein encoded by the TMPRSS3 gene contains a serine protease domain, a transmembrane domain, an LDL receptor-like domain, and a scavenger receptor cysteine-rich domain. Serine proteases are known to be involved in various biological processes, and their dysfunction often causes human diseases and disorders. This gene is expressed in the fetal cochlea and many other tissues and is thought to be involved in the development and maintenance of the inner ear or perilymphatic and endolymphatic contents. This gene has also been identified as a tumor-associated gene overexpressed in ovarian tumors. Four alternative splicing variants have been described, two of which encode identical products.

[0027] DFN3 hearing loss is caused by a mutation in the POU3F4 gene, located on the X chromosome. In people with this condition, one of the small bones in the middle ear (the stapes) cannot move normally, which interferes with hearing. This characteristic symptom of DFN3 is called stapes fixation. At least four other regions of the X chromosome have been implicated in hearing loss, but the gene involved has not been identified. DFNB59 (deafness, autosomal recessive 59), also known as Pejvakin or PJVK, is a 352-amino acid protein that belongs to the gasdermin family in vertebrates. DFNB59 is encoded by a gene mapping to human chromosome 2q31.2, which is essential for proper function of auditory pathway neurons and outer hair cell function. Mutations in DFNB59 are thought to cause nonsyndromic sensorineural hearing loss autosomal recessive type 59, a form of sensorineural hearing loss characterized by absent or severely abnormal auditory brainstem responses but normal otoacoustic emissions (auditory neuropathy or auditory dyssynchrony). DFNB59 shares significant similarity with DFNA5, indicating that these genes share a common origin.

[0028] Alport syndrome is caused by mutations in the COL4A3, COL4A4, and COL4A5 genes, which are involved in collagen biosynthesis. Mutations in any of these genes prevent the proper production or assembly of type IV collagen network, a critical structural component of the basement membrane in the kidney, inner ear, and eye. One of the criteria used in diagnosing Alport syndrome is bilateral sensorineural hearing loss in the 2000-8000 Hz range. Hearing loss develops gradually, is not present in early childhood, and generally appears before the age of 30.

[0029] Defects in ion channels are associated with hearing loss, and DFNA2 nonsyndromic hearing loss is inherited as an autosomal dominant mutation in the KCNQ4 gene, which encodes the potassium voltage-gated channel subfamily KQT member 4, also known as the voltage-gated potassium channel subunit Kv7.4. DFNA2 nonsyndromic hearing loss is characterized by progressive, symmetric, primarily high-frequency sensorineural hearing loss (SNHL) across all frequencies. In younger individuals, hearing loss is mild at low frequencies and moderate at high frequencies; in older individuals, hearing loss is moderate at low frequencies and severe to profound at high frequencies. While hearing impairment is often detected during routine hearing evaluations in school-age children, hearing is likely damaged from birth, particularly at high frequencies. Most affected individuals first require hearing aids to aid in sound amplification between the ages of 10 and 40. By age 70, all people with DFNA2 hearing loss have severe to profound hearing loss.

[0030] Mutations in the KCNE1 and KCNQ1 genes cause Jerber-Lange-Nielsen syndrome (JLNS), a form of long QT syndrome associated with severe bilateral hearing loss. This condition is an autosomal recessive disorder affecting an estimated 1.6–6 per million children, accounting for less than 10 percent of all cases of long QT syndrome. Norway and Sweden have significantly higher case numbers, up to 1:200,000. Proteins produced by the KCNE1 and KCNQ1 genes function together to form potassium channels that transport positively charged potassium ions out of cells. The movement of potassium ions through these channels is important for maintaining normal function of the inner ear and heart muscle.

[0031] EAST / SeSAME syndrome, characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt wasting, is thought to be caused by mutations in the KCNJ10 inwardly rectifying potassium channel.

[0032] Subjects with Bartter syndrome with sensorineural hearing loss type 4 (also known as Bartter syndrome IV or BSND) have a defect in the Cl − channel accessory subunit.

[0033] Mutations in the ATP6V1B1 gene, which is expressed in both the kidney and the cochlea, are associated with distal renal tubular acidosis (DRTA). A significant percentage of children with autosomal recessive DRTA have also been found to experience progressive bilateral sensorineural hearing loss.

[0034] Usher syndrome (also known as Hallgren syndrome, Usher-Hallgren syndrome, retinitis pigmentosa-deafness syndrome, and retinal dystrophic deafness syndrome) is a rare disorder caused by mutations in any one of at least 10 genes. It causes a combination of hearing loss and gradual vision impairment and is the leading cause of deafblindness. Hearing loss is caused by a defective inner ear, while vision loss results from retinitis pigmentosa (RP), a degeneration of retinal cells. Usher syndrome has three clinical subtypes, designated Type I, Type II, and Type III. Subjects with Type I Usher syndrome are born profoundly deaf, begin to lose vision within the first 10 years of life, and children walk late due to problems with the vestibular system and exhibit balance difficulties. Subjects with Type II Usher syndrome are not born deaf, but have hearing loss but do not appear to have significant balance problems. They begin to lose vision later in life (by age 20), but may retain some vision into middle age. Subjects with Usher Syndrome Type III are not born deaf, but experience a gradual loss of hearing and vision and may or may not have balance difficulties.

[0035] Mouse models of congenital hearing loss have been generated by creating a null mutation in the gene encoding vesicular glutamate transporter-3 (VGLUT3). Recently, hearing was restored in VGLUT3 knockout mice using viral-mediated gene therapy (Akil, et al., 2012, Neuron 75:283-293).

[0036] Math1 (the mouse homolog of ATH1), also known as the atonal Drosophila homolog of HATH1 or (ATOH1), is essential for hair cell development in the inner ear, and Math1 has therefore been proposed to function as a "hair cell-promoting gene" in the developing sensory epithelium (Bermingham et al., 1999, Science 284:1837-1841). Several studies have now shown that viral-mediated delivery of Math1 results in hair cell regeneration in the traumatized mouse cochlea and improvement of both hearing and balance (Baker et al., 2009, Adv. Otorhinolaryngol. 66:52-63; Husseman and Raphael, 2009, Adv. Otorhinolaryngol. 66:37-51; Izumikawa et al., 2008, Hear. Res. 240:52-56; Kawamoto et al., 2003, J. Neurosci. 23:4395-4400; Praetorius et al., 2010, Acta Otolaryngol. 130:215-222; Staecker et al., 2007, Otol. Neurotol. 28:223-231).

[0037] Mutations in the WFS1 gene cause more than 90 percent of cases of Wolfram syndrome type 1, a condition affecting many body systems that is most often characterized by high blood sugar levels (diabetes mellitus) caused by a lack of the hormone insulin and progressive vision loss due to degeneration of the nerves that carry information from the eye to the brain (optic atrophy). However, people with Wolfram syndrome often also have excessive urine production (diabetes insipidus), hearing loss caused by changes in the inner ear (sensorineural hearing loss), urinary tract problems, reduced levels of the sex hormone testosterone in men (hypogonadism), or pituitary gland dysfunction, which can result in neurological or psychiatric disorders. Approximately 65 percent of people with Wolfram syndrome have sensorineural hearing loss, the severity of which can range from hearing loss that begins at birth to mild hearing loss that begins in adolescence and worsens over time. Additionally, about 60 percent of people with Wolfram syndrome develop neurological or psychiatric disorders, most commonly problems with balance and coordination (ataxia), which typically begin in early adulthood.

[0038] The WFS1 gene encodes a protein called wolframin, which is thought to regulate the amount of calcium within cells. When Wolfram syndrome is caused by a mutation in the WFS1 gene, it is inherited in an autosomal recessive pattern, and the wolframin protein has reduced or absent function. As a result, intracellular calcium levels are unregulated and the endoplasmic reticulum does not function properly. If the endoplasmic reticulum does not have enough functional wolframin, the cell triggers its own cell death (apoptosis). The death of cells in the pancreas, particularly the cells that produce insulin (beta cells), leads to diabetes mellitus in people with Wolfram syndrome. The gradual loss of cells along the optic nerve ultimately leads to blindness in affected individuals. Cell death in other body systems likely causes the various signs and symptoms of Wolfram syndrome type 1.

[0039] Mutations in the mitochondrial genes MT-TS1 and MT-RNR1 have been found to increase the risk of developing nonsyndromic hearing loss. Nonsyndromic mitochondrial hearing loss and hearing loss are characterized by moderate to severe hearing loss. Pathogenic variants in MT-TS1 are usually associated with childhood-onset sensorineural hearing loss. Pathogenic variants in MT-RNR1 are associated with hearing loss when exposed to certain antibiotic drugs called aminoglycosides (ototoxicity) and / or a predisposition to late-onset sensorineural hearing loss, although some people with mutations in the MT-RNR1 gene develop hearing loss even without exposure to these antibiotics. Hearing loss associated with aminoglycoside ototoxicity is bilateral and severe to profound and occurs within days to weeks after administration of any amount (even a single dose) of an aminoglycoside antibiotic, such as gentamicin, tobramycin, amikacin, kanamycin, or streptomycin.

[0040] Treatment of hearing loss currently consists of hearing amplification for mild to severe loss and cochlear implantation for severe to profound loss (Kral and O'Donoghue, 2010, N. Engl. J. Med. 363:1438-1450). To date, the majority of research in this field has focused on cochlear hair cell regeneration, which is applicable to the most common forms of hearing loss, including presbycusis, noise-induced injury, infection, and ototoxicity.

[0041] In animal models of cochlear ischemia, ischemic damage can be prevented by compounds such as insulin-like growth factor-1 (IGF-1), AM-111 (an apoptosis inhibitor), edaravone (a free radical scavenger), ginsenoside RB1 (Kappo), glial cell line-derived neurotrophic factor (GDNF), BDNF, CNTF, SOD1, SOD2, necrostatin-1, DFNA5, and MSRB3. However, it appears that combinations of substances may be more effective than single compounds (e.g., complementary therapies modulating oxidative stress, exotoxicity, blood flow, calcium and stimuli overload, apoptotic pathways, neurotrophic, or hormonal regulatory mechanisms).

[0042] The inhibition of apoptosis (mitochondrial-induced) induced by JNK-1 can be prevented by compounds such as dominant-negative JNK-1 and d-stereoisomer JNK-1 (Mol. Pharmacol. 2007 March; 71(3):654-66, the contents of which are incorporated herein by reference in their entirety).

[0043] There remains a long-felt need for agents and methods for preventing or reversing hearing loss. [Prior art documents] [Non-patent literature]

[0044] [Non-Patent Document 1] Smith et al., 2005, Lancet 365:879-890 [Non-patent document 2] Di Domenico, et al., 2011, J. Cell. Physiol. 226:2494-2499 [Non-patent document 3] Rodriguez-Ballesteros et al. 2008 Human Mut 29(6):823-831 [Brief explanation of the drawings]

[0045] [Figure 1] Figure 1 shows an exemplary schematic diagram of the genetic maps of the 5' and 3' vectors for dual AAV transduction in inner hair cells (IHCs) using a trans-splicing approach. In the 5' vector, the CMV enhancer (CMVe) and human β-actin promoter (hbA) drive transcription of mRNA encoding eGFP and the P2A peptide, which is cleaved during translation. The 5' vector also contains a cDNA encoding the N-terminal portion of otoferlin and a splice donor site (SD). The SD DNA sequence was provided by Trapani et al. (2014) EMBO Mol Med 6 194-211. In the 3' vector, a splice acceptor site (SA) was subcloned after the first inverted terminal repeat (ITR) and before the 3' portion of the otoferlin coding sequence. This is followed by an mRNA stabilizing sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a polyadenylation signal (pA).

[0046] [Figure 2] Figure 2 is an exemplary schematic diagram of the genetic maps of the 5' and 3' vectors for dual AAV transduction in IHC using a hybrid approach. In addition to the elements described in Figure 1, highly recombinogenic sequences from F1 phage (AK) sequences were included at the 3' end of the 5' vector and the 5' end of the 3' vector to ensure accurate vector assembly (Trapani et al. (2014) EMBO Mol Med 6 194-211).

[0047] [Figure 3] Figure 3 is a graph showing the amplitude of the auditory brainstem response (ABR) over time in untransduced Otof− / − mice (n=25; thin black line) and Otof− / − mice after dual AAV-mediated otoferlin expression (n=14; gray lines represent individual animals, thick black line is the average response across all animals).

[0048] [Figure 4] Figure 4 is a graph showing ABR amplitude over time in untransduced Otof− / − mice (n=25; long-dashed line), Otof− / − mice after dual AAV-mediated otoferlin expression (n=14; short-dashed line), and wild-type Otofwt / wt mice after dual AAV transduction with enhanced green fluorescent protein (eGFP) (n=5; thin black lines represent individual animals, thick black line is the average amplitude across all animals).

[0049] [Figure 5] 5 is an exemplary graph showing ABR thresholds (in dB SPL) versus frequency (in kHz) in response to a pure tone or click stimulus. The minimum sound pressure level (SPL) at which waves can be detected in response to a pure tone or click stimulus is shown for individual Otof− / − animals with dual AAV-mediated otoferlin expression (open circles represent individual animals, and the hatched circle is the average threshold across all animals), untransduced Otof− / − animals (solid circles), and Otofwt / wt animals after dual AAV transduction with eGFP (checkered circles).

[0050] [Figure 6] Figure 6 is a graph showing the transduction rates of IHCs along the cochlea (e.g., whole Corti, apical turn, and midbasal / basal turn) as determined by immunohistochemistry using two different antibodies (one binding to the N-terminal otoferlin fragment and the other to the very C-terminal portion of otoferlin).

[0051] [Figure 7]Figure 7 shows a set of immunohistochemistry images of one organ of Corti from an Otof- / - mouse after dual AAV-mediated otoferlin expression. Calbindin was used as a marker for inner and outer hair cells. Cells expressing eGFP indicate viral transduction of at least the 5' virus. The left and middle panels show anti-otoferlin antibody staining (Abcam) for the N-terminal half of otoferlin. The right panel shows anti-otoferlin antibody staining (Synaptic Systems) for the C-terminus. Collectively, all three panels demonstrate that full-length otoferlin is expressed in IHC. Importantly, despite AAV transducing several cell types in the organ of Corti (as indicated by eGFP fluorescence), otoferlin expression was restricted to inner hair cells. Scale bar: 100 μm.

[0052] [Figure 8] Figure 8 is a graph showing Ca2+ currents in response to depolarizing stimuli for Otof- / - IHCs (white; n=10), Otof- / - IHCs after dual AAV-mediated otoferlin expression (hatched; n=8), and wild-type IHCs from background-matched control animals (black; n=6).

[0053] [Figure 9] Figure 9 shows plasma membrane capacitance (ΔCm) over the duration of depolarization in dual AAV-transduced IHCs from Otof- / - mice (black line, individual IHCs; filled hatched diamonds: mean ± sem; n = 8), two individual dual AAV-transduced IHCs from injected Otof- / - mice that showed only trace amounts of exocytosis (dashed lines), and background-matched wild-type controls (black diamonds; n = 6). Wild-type data from Strenzke et al. (2016) EMBO J. 35 2519-2535 are shown as black circles for comparison.

[0054] [Figure 10]Figure 10 is a representative graph showing Qreal versus depolarization duration (ms) in Otof− / − IHCs (white diamonds), dual AAV-transduced IHCs from Otof− / − mice (black circles), and background-matched wild-type controls (black diamonds).

[0055] [Figure 11] Figure 11 is a representative plasmid map of pAAV-AK-SA-3'mOTOF-EWB.

[0056] [Figure 12] Figure 12 is a representative plasmid map of pAAV-SA-3'mOTOF-EWB.

[0057] [Figure 13] Figure 13 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD.

[0058] [Figure 14] Figure 14 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD-AK.

[0059] [Figure 15] Figure 15 is a graph showing plasma membrane capacitance (ΔCm) versus duration of depolarization in IHCs of Otof− / − mice transduced with the two vectors shown in Figure 2 (medium shading; thick line), in IHCs of wild-type mice (dark shading; medium thick line), and in IHCs of Otof− / − mice.

[0060] [Figure 16] Figure 16 is a representative graph showing Qreal versus depolarization duration (ms) in IHCs of Otof- / - (white diamonds), Otof- / - mice transduced with the two vectors shown in Figure 2 (gray diamonds), and background-matched wild-type controls (black diamonds).

[0061] [Figure 17] Figure 17 is a representative plasmid map of pAAV-AK-SA-3'mOTOF-EWB.

[0062] [Figure 18] Figure 18 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD.

[0063] [Figure 19] Figure 19 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD-AK.

[0064] [Figure 20] Figure 20 is a representative plasmid map of pAAV-SA-3'mOTOF-EWB.

[0065] [Figure 21] FIG. 21 is a representative schematic diagram of a portion of pAKOS102 (SEQ ID NO: 43).

[0066] [Figure 22] FIG. 22 is a representative schematic diagram of a portion of pAKOS103 (SEQ ID NO: 44).

[0067] [Figure 23] FIG. 23 is a representative schematic diagram of a portion of pAKOS103.

[0068] [Figure 24] FIG. 24 is a representative plasmid map of pAKOS104 (SEQ ID NO: 45).

[0069] [Figure 25] FIG. 25 is a representative schematic diagram of a portion of pAKOS104-DHFR (SEQ ID NO: 46).

[0070] [Figure 26]FIG. 26 is a representative schematic diagram of a portion of pAKOS104-DHFR.

[0071] [Figure 27] FIG. 27 is a representative schematic diagram of a portion of pAKOS105 (SEQ ID NO: 47).

[0072] [Figure 28] FIG. 28 is a representative schematic diagram of a portion of pAKOS105.

[0073] [Figure 29] FIG. 29 is a representative schematic diagram of a portion of pAKOS105_GFP (SEQ ID NO: 48).

[0074] [Figure 30] FIG. 30 is a representative schematic diagram of a portion of pAKOS106 (SEQ ID NO: 49).

[0075] [Figure 31] Figure 31 is a representative schematic diagram of a portion of pAKOS106.

[0076] [Figure 32] FIG. 32 is a representative schematic diagram of a portion of pAKOS107 (SEQ ID NO: 50).

[0077] [Figure 33] Figure 33 is a representative schematic diagram of a portion of pAKOS107.

[0078] [Figure 34] FIG. 34 is a representative schematic diagram of a portion of pAKOS108 (SEQ ID NO: 51).

[0079] [Figure 35] Figure 35 is a representative schematic diagram of a portion of pAKOS108.

[0080] [Figure 36]FIG. 36 is a representative schematic diagram of a portion of pAKOS109 (SEQ ID NO: 52).

[0081] [Figure 37] FIG. 37 is a representative schematic diagram of a Factor VIII stuffer (SEQ ID NOs: 54-57).

[0082] [Figure 38-1] Figure 38 is a representative schematic diagram of p109 (SEQ ID NO: 84). [Figure 38-2] Figure 38 is a representative schematic diagram of p109 (SEQ ID NO: 84).

[0083] [Figure 39-1] Figure 39 is a representative schematic diagram of p105 (SEQ ID NO: 85). [Figure 39-2] Figure 39 is a representative schematic diagram of p105 (SEQ ID NO: 85).

[0084] [Figure 40] Figure 40 is a representative schematic diagram of the 105.WPRE.

[0085] [Figure 41] Figure 41 is a representative schematic diagram of p108.

[0086] [Figure 42] Figure 42 is a representative schematic diagram of 1OTOF18.CL1.

[0087] [Figure 43] Figure 43 is a representative schematic diagram of 19OTOF48.

[0088] [Figure 44] Figure 44 is a representative schematic diagram of 1OTOF20.CL1.

[0089] [Figure 45] Figure 45 is a representative schematic diagram of 21OTOF48.WPRE.

[0090] [Figure 46] Figure 46 is a representative schematic diagram of 1OTOF21.CL1.

[0091] [Figure 47] Figure 47 is a representative schematic diagram of 22OTOF48.WPRE.

[0092] [Figure 48] Figure 48 is a representative schematic diagram of 105.pA.NTF3.CMVd.

[0093] [Figure 49] FIG. 49 is an immunoblot showing the expression of full-length human otoferlin in HEK293FT cells transfected with various plasmid pairs as indicated.

[0094] [Figure 50] FIG. 50 is a table showing quantification of full-length human otoferlin expression from three replicates of the experiment described in FIG.

[0095] [Figure 51] Figure 51 is a graph of click ABR thresholds in wild-type, untreated Otof mice, or Otof mice that were untreated or treated with dual AAV Anc80.hOtof vectors (p105 and p109 vectors). Hearing acuity was measured in Otof mice administered DualAAV Anc80.hOtof vectors (p105 and p109 vectors) 26-28 and 91 days after treatment.

[0096] [Figure 52]Figure 52 is a graph of tone burst ABR thresholds in wild-type, untreated Otof mice, or Otof mice that were untreated or treated with dual AAV Anc80.hOtof vectors (p105 and p109). Hearing acuity was measured in Otof mice administered DualAAV Anc80.hOtof vectors (p105 and p109 vectors) 26-28 and 91 days after treatment.

[0097] [Figure 53-1] Figure 53 is a representative schematic diagram of a portion of pAAV-HBA-eGFP-P2A-5'mOTOF.SD (SEQ ID NO: 87). [Figure 53-2] Figure 53 is a representative schematic diagram of a portion of pAAV-HBA-eGFP-P2A-5'mOTOF.SD (SEQ ID NO: 87).

[0098] [Figure 54-1] Figure 54 is a representative schematic diagram of a portion of pAAV-SA-3'mOTOF.WPRE (SEQ ID NO: 88). [Figure 54-2] Figure 54 is a representative schematic diagram of a portion of pAAV-SA-3'mOTOF.WPRE (SEQ ID NO: 88).

[0099] [Figure 55] Figure 55 is a representative schematic diagram of a portion of pAAV-HBA-eGFP-P2A-5'mOTOF.SD-AK.

[0100] [Figure 56] Figure 56 is a representative schematic diagram of a portion of pAAV-AK-SA-3'mOTOF.WPRE.

[0101] [Figure 57] Figure 57 is a representative schematic diagram of a portion of pAAV-CMV-5'hOTOF-SD-AK.

[0102] [Figure 58]Figure 58 is a representative schematic diagram of a portion of pAAV-HBA-5'hOTOF-SD-AP.

[0103] [Figure 59] Figure 59 is a representative schematic diagram of a portion of pAAV-HBA-5'hOTOF-SD-AK.

[0104] [Figure 60] Figure 60 is a representative schematic diagram of a portion of pAAV-HBA-5'hOTOFcodop-SD-AK.

[0105] [Figure 61] Figure 61 is a representative schematic diagram of a portion of pAAV-HBA-5'hOTOFcodop-SD.

[0106] [Figure 62] Figure 62 is a representative schematic diagram of a portion of pAAV-CMV-5'hOTOFcodop-SD.

[0107] [Figure 63] Figure 63 is a representative schematic diagram of a portion of pAAV-CMV-5'hOTOFcodop-SD-AK.

[0108] [Figure 64] Figure 64 is a representative schematic diagram of a portion of pAAV-CBA-5'hOTOFcodop-SD-AK.

[0109] [Figure 65] Figure 65 is a representative schematic diagram of a portion of pAAV-CBA-5'hOTOF-SD.

[0110] [Figure 66] Figure 66 is a representative schematic diagram of a portion of pAAV-SA-3'OTOF.

[0111] [Figure 67] Figure 67 is a representative schematic diagram of a portion of pAAV-AP-SA-3'OTOF.

[0112] [Figure 68] Figure 68 is a representative schematic diagram of a portion of pAAV-AK-SA-3'OTOFcodop.

[0113] [Figure 69]Figure 69 is a representative immunoblot showing the expression of full-length human otoferlin in HEK293FT cells transfected using the DNA transfection reagent jetPRIME® with 600 ng pairs of the various plasmids indicated. Lane 1 contained a prestained protein ladder. Lane 2 contained protein samples from HEK293FT cells transfected with vector pAKOS104 (shown in Figures 24 and 59) and vector pAKOS105 (shown in Figures 27, 28, and 39). Lane 3 contained protein samples from HEK293FT cells transfected with vector pAKOS108 (shown in Figures 34, 35, 41, and 57) and vector pAKOS105 (shown in Figures 27, 28, and 39). Lane 4 contained a protein sample of HEK293FT cells transfected with vector pAKOS109 (shown in Figures 36 and 38) and vector pAKOS105 (shown in Figures 27, 28, and 39). Lane 5 contained a protein sample of HEK293FT cells transfected with vector pAAV-HBA-5'hOTOFcodop-SD-AK (shown in Figure 60) and vector pAAV-AK-SA-3'OTOFcodop (shown in Figure 68). Lane 6 contained a protein sample of HEK293FT cells transfected with vector pAAV-CMV-5'hOTOFcodop-SD-AK (shown in Figure 63) and vector pAAV-AK-SA-3'OTOFcodop (shown in Figure 68). Lane 7 contained a protein sample of HEK293FT cells transfected with vector pAAV_CBA-5'hOTOFcodop-SD-AK (shown in Figure 64) and vector pAAV-AK-SA-3'OTOFcodop (shown in Figure 68). Lane 8 contained a protein sample of HEK293FT cells transfected with vector pAKOS102 (shown in Figure 21) and vector pAKOS103 (shown in Figures 22, 23, and 66).Lane 9 contained a protein sample from HEK293FT cells transfected with the CBA.TS vector and vector pAKOS103 (shown in Figures 22, 23, and 66). Lane 10 contained a protein sample from HEK293FT cells transfected with vector pAKOS106 (shown in Figures 30, 31, and 58) and vector pAKOS107 (shown in Figures 32, 33, and 67). Ninety-six hours after transfection, cells were harvested, lysed using RIPA buffer, and analyzed in a 4-12% Bolt protein gel, which was then transferred to a nitrocellulose membrane. Human otoferlin was detected using an anti-OTOF polyclonal antibody (Thermo PA5-52935). Human beta-actin was used as the primary antibody for internal loading control between lanes. Experiments were repeated in triplicate. Relative quantitative measurements for each experiment are provided by immunoblot, along with the mean measurement and standard deviation (STDEV).

[0114] [Figure 70]Figure 70 is an immunoblot showing the expression of full-length human otoferlin in HEK293FT cells transfected with the indicated plasmid pairs at various multiplicities of infection. HEK293FT cells were seeded overnight at 4 x 104 cells per well on a 96-well plate. Six hours after seeding, dual vectors were added to each well. Ninety-six hours after transfection, cells were harvested, lysed using RIPA buffer, and analyzed in a 4-12% Bolt protein gel, which was then transferred to a nitrocellulose membrane. Human otoferlin was detected using an anti-OTOF polyclonal antibody (Thermo PA5-52935). Human beta-actin was used as the primary antibody for internal loading control between lanes. Lane 1: CBA.OTOF(AK) with an MOI of 503,000; Lane 2: CBA.OTOF(AK) with an MOI of 1,510,000; Lane 3: CBA.OTOF(AK) with an MOI of 100,000; Lane 4: CBA.OTOF(AK) with an MOI of 303,000; Lane 5: CMV.OTOF(AK) with an MOI of 638,000; Lane 6: CMV.OTOF(AK) with an MOI of 1,910,000; Lane 7: CMV.OTOF(AK) with an MOI of 127,000; Lane 8: CMV.OTOF(AK) with an MOI of 382,000; Lane 9: negative control.

[0115] [Figure 71] A set of immunohistochemistry images of one organ of Corti from a P17 Otof- / - mouse after unilateral intracochlear administration of dual AAV vectors (p105 and 109 vectors) expressing CBA.hOTOF(AK). The ipsilateral cochlea was dissected and analyzed for protein expression using immunohistochemistry in three different frequency regions (basal-apical).

[0116] [Figure 72]Figure 72 is a graph showing the percentage of inner hair cells (IHCs) labeled with otoferlin at the N- and C-terminus in CD1B6F1-Otof- / - mice (aged P18-P30) injected with dual AAV-TS (n=10 mice) and dual AAV-Hyb (n=9 mice). Individual animals are indicated by open symbols. Data are presented as mean ± standard error of the mean (sem). ns P>0.05; *P≦0.05; **P≦0.01; ***P≦0.001, Wilcoxon matched-pairs signed-rank test, and unpaired t-test with Welch's correction.

[0117] [Figure 73] Figure 73 is a graph showing the average N- and C-terminal otoferlin immunofluorescence levels in dual AAV-transduced Otof- / - and wild-type inner hair cells (IHCs) from mice (days P23-30). Otoferlin levels were normalized to the immunofluorescence levels in untransduced B6 wild-type IHCs separately for each antibody. The number of IHCs quantified is indicated inside the bars. Data are presented as mean ± standard error of the mean (sem); ns P>0.05; *P≦0.05; **P≦0.01; ***P≦0.001, Kruskal-Wallis test followed by Dunn's multiple comparison test.

[0118] [Figure 74]Figure 74 is a graph showing the number of synaptic ribbons quantified from inner hair cells (IHCs) in the apical turn of wild-type (B6: n=48 IHCs, CD1B6F1: n=108 IHCs), transduced Otof- / - (dual AAV-TS: n=59 IHCs, dual AAV-Hyb: n=37 IHCs), and untransduced Otof- / - IHCs from injected (-AAV-injected ear, n=65 IHCs) and contralateral uninjected (-AAV-uninjected ear, n=46 IHCs) ears (from mice aged P25-P29). Individual animals are indicated by open symbols. Data are presented as mean ± standard error of the mean (sem); ns P>0.05, **P≦0.001 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

[0119] [Figure 75] Figure 75 is a graph showing synapse number quantified from inner hair cells (IHCs) in the apical turn (c) of B6 wild-type (P6: n=53 IHCs; P14: n=73 IHCs) and B6 Otof- / - (P6: n=62 IHCs; P14: n=65 IHCs) mice at two different developmental stages (P6 and P14). Individual animals are indicated by open symbols. Data are presented as mean ± standard error of the mean (sem); ns P>0.05, **P≦0.001 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

[0120] [Figure 76]Figure 76 is a graph showing the sum of the amplitudes of auditory brainstem response (ABR) waves I-V at different click intensities in Otof- / - mice injected with otoferlin via dual AAV, uninjected Otof- / - mice, and wild-type control mice aged P23-30. Number of mice analyzed: CD1B6F1 wild-type animals (+AAV.eGFP: n=12 mice, dual AAV-TS: n=6 mice) and CD1B6F1 Otof- / - animals (dual AAV-TS: n=16 mice, dual AAV-Hyb: n=8 mice, =AAV: n=38 mice). Data are presented as mean ± standard error of the mean (sem). Individual animals are indicated by open symbols.

[0121] [Figure 77] Figure 77 is a graph showing the sum of the amplitudes of auditory brainstem response (ABR) waves I-V plotted against the inner hair cell (IHC) transduction rate of full-length otoferlin (from Figure 5, C-terminal otoferlin) of individual dual AAV-TS-treated CD1B6F1 Otof- / - animals (n=8 animals; from Figure 76). r: correlation coefficient. = 8 mice, = AAV: n=38 mice. Individual animals are indicated by open symbols. Positive correlation of r≧0.5 (70 decibel sound pressure level (dB SPL) and 90 dB SPL: Pearson's correlation test; 50 dB SPL: Spearman's correlation test).

[0122] [Figure 78] Figure 78 is a schematic diagram of an exemplary dual AAV vector system of the present disclosure, including the "upstream" and "downstream" vectors (AKhOTOF5 and AKhOTOF3, respectively).

[0123] [Figure 79] Figure 79 is a schematic diagram of the "upstream" vector AKhOTOF5.

[0124] [Figure 80] Figure 80 is a schematic diagram of the "downstream" vector AKhOTOF3.

[0125] [Figure 81] FIG. 81 shows a perspective view of a device for delivering fluid to the inner ear according to an embodiment of the present disclosure.

[0126] [Figure 82] FIG. 82 shows a side view of a curved needle subassembly according to an embodiment of the present disclosure.

[0127] [Figure 83] FIG. 83 shows a perspective view of a device for delivering fluid to the inner ear according to an embodiment of the present disclosure.

[0128] [Figure 84] FIG. 84 shows a perspective view of a curved needle subassembly coupled to the distal end of a device, according to an embodiment of the present disclosure. Summary of the Invention [Means for solving the problem]

[0129] overview The present disclosure is based on the discovery that a composition comprising at least two different nucleic acid vectors, each of which comprises a coding sequence encoding a different portion of an otoferlin protein, can be used to produce a sequence encoding an active otoferlin protein (e.g., a full-length otoferlin protein) in mammalian cells, thereby treating non-syndromic sensorineural hearing loss in a subject in need thereof.

[0130] Provided herein is a composition comprising at least two different nucleic acid vectors, each of which contains a coding sequence encoding a different portion of an otoferlin protein, each of which is at least 30 amino acid residues in length, and the amino acid sequence of each of the coded portions optionally overlapping with the amino acid sequence of a different one of the coded portions, wherein no single vector of the at least two different vectors encodes the full-length otoferlin protein, and at least one of the coding sequences contains a nucleotide sequence spanning two adjacent exons of otoferlin genomic DNA and lacks intronic sequence between the two adjacent exons, and wherein, upon introduction into a mammalian cell, the at least two different vectors undergo concatemerization or homologous recombination with each other, thereby forming a recombinant nucleic acid encoding the full-length otoferlin protein. In some embodiments of any of the compositions described herein, each of the at least two different vectors is a plasmid, transposon, cosmid, artificial chromosome, or viral vector. In some embodiments of any of the compositions described herein, each of at least two different vectors is a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC).In some embodiments of any of the compositions described herein, each of at least two different vectors is a viral vector selected from adeno-associated virus (AAV) vector, adenovirus vector, lentivirus vector or retrovirus vector.In some embodiments of any of the compositions described herein, each of at least two different vectors is an AAV vector.

[0131] In some embodiments of any of the compositions described herein, the amino acid sequence of one of the encoded portions overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different encoded portion. In some embodiments of any of the compositions described herein, the overlapping amino acid sequences are from about 30 amino acid residues to about 1000 amino acid residues in length.

[0132] In some embodiments of any of the compositions described herein, the vector comprises two different vectors, each comprising a different segment of an intron, the intron comprising the nucleotide sequence of an intron present in otoferlin genomic DNA, the two different segments overlapping in sequence by at least 100 nucleotides. In some embodiments of any of the compositions described herein, the two different segments overlap in sequence by about 100 nucleotides to about 800 nucleotides. In some embodiments of any of the compositions described herein, the nucleotide sequence of each of the at least two different vectors is about 500 nucleotides to about 10,000 nucleotides in length. In some embodiments of any of the compositions described herein, the nucleotide sequence of each of the at least two different vectors is 500 nucleotides to 5,000 nucleotides in length.

[0133] In some embodiments of any of the compositions described herein, the number of different vectors in the composition is two. In some embodiments of any of the compositions described herein, the first of the two different vectors comprises a coding sequence encoding an N-terminal portion of an otoferlin protein. In some embodiments of any of the compositions described herein, the N-terminal portion of the otoferlin protein is between 30 amino acids and 1600 amino acids in length. In some embodiments of any of the compositions described herein, the N-terminal portion of the otoferlin protein is between 200 amino acids and 1500 amino acids in length. In some embodiments of any of the compositions described herein, the first vector further comprises one or both of a promoter and a Kozak sequence. In some embodiments of any of the compositions described herein, the first vector comprises a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.

[0134] In some embodiments of any of the compositions described herein, one of the two vectors comprises SEQ ID NO:39 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO:39), and the second of the two vectors comprises SEQ ID NO:40 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO:40). In some embodiments of any of the compositions described herein, one of the two vectors comprises SEQ ID NO:41 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO:41), and the second of the two vectors comprises SEQ ID NO:42 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO:42).In some embodiments of any of the compositions described herein, one of the two vectors comprises SEQ ID NO: 84 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 84), and the second of the two vectors comprises SEQ ID NO: 85 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 85).

[0135] In some embodiments of any of the compositions described herein, one of the at least two different vectors comprises a sequence encoding an NTF3 protein.

[0136] In some embodiments of any of the compositions described herein, the sequence encoding the NTF3 protein is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO:78.

[0137] In some embodiments of any of the compositions described herein, the first vector further comprises a coding sequence encoding a destabilizing domain, wherein the destabilizing domain is 3' to the coding sequence encoding the N-terminal portion of the otoferlin protein.

[0138] In some embodiments of any of the compositions described herein, the coding sequence encoding the N-terminal portion of the otoferlin protein comprises exons 1-21 of isoform 5 of the human otoferlin gene.

[0139] In some embodiments of any of the compositions described herein, the second of the two different vectors comprises a coding sequence encoding a C-terminal portion of an otoferlin protein. In some embodiments of any of the compositions described herein, the C-terminal portion of the otoferlin protein is between 30 amino acids and 1600 amino acids in length. In some embodiments of any of the compositions described herein, the C-terminal portion of the otoferlin protein is between 200 amino acids and 1500 amino acids in length. In some embodiments of any of the compositions described herein, the second vector further comprises a poly(dA) signal sequence. In some embodiments of any of the compositions described herein, the coding sequence encoding the C-terminal portion of the otoferlin protein comprises exons 22-48 of isoform 5 of the human otoferlin gene. In some embodiments of any of the compositions described herein, the second vector further comprises a sequence for mRNA stabilization. Some embodiments of any of the compositions described herein further comprise a pharmaceutically acceptable excipient.

[0140] Also provided herein are kits comprising any of the compositions described herein. Some embodiments of any of the kits described herein further comprise a pre-filled syringe containing the composition.

[0141] Also provided herein are methods comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​a mammal. In some embodiments of any of the compositions described herein, the mammal is a human. In some embodiments of any of the compositions described herein, the mammal has previously been identified as having a defective otoferlin gene.

[0142] Also provided herein are methods for increasing expression of active otoferlin protein, e.g., full-length otoferlin protein, in mammalian cells, comprising introducing any of the compositions described herein into the mammalian cells. In some embodiments of any of the methods described herein, the mammalian cells are cochlear inner hair cells. In some embodiments of any of the methods described herein, the mammalian cells are human cells. In some embodiments of any of the methods described herein, the mammalian cells have previously been determined to have a defective otoferlin gene.

[0143] Also provided herein are methods for increasing expression of active otoferlin protein, e.g., full-length otoferlin protein, in inner hair cells of the cochlea of ​​a mammal, comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​the mammal. In some embodiments of any of the methods described herein, the mammal has previously been identified as having a defective otoferlin gene. In some embodiments of any of the methods described herein, the mammal is a human.

[0144] Also provided herein is a method for treating non-syndromic sensorineural hearing loss in a subject identified as having a defective otoferlin gene, the method comprising administering a therapeutically effective amount of any one of the compositions described herein to the cochlea of ​​the subject. In some embodiments of any of the methods described herein, the subject is a human. Some embodiments of any of the methods described herein further comprise determining that the subject has a defective otoferlin gene prior to the administering step.

[0145] A composition comprising two different nucleic acid vectors, wherein a first of the two different nucleic acid vectors comprises a promoter, a first coding sequence encoding an N-terminal portion of an otoferlin protein located 3' of the promoter, and a splicing donor signal sequence located at the 3' end of the first coding sequence, and a second of the two different nucleic acid vectors comprises a splicing acceptor signal sequence, a second coding sequence encoding a C-terminal portion of an otoferlin protein located 3' of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3' end of the second coding sequence. Also provided herein are compositions comprising: each of the encoded portions is at least 30 amino acid residues in length, the amino acid sequences of the encoded portions do not overlap, and no single vector of the two different vectors encodes the full-length otoferlin protein; and when the coding sequences are transcribed in a mammalian cell and RNA transcripts are produced, splicing occurs between a splicing donor signal sequence on one transcript and a splicing acceptor signal sequence on the other transcript, thereby forming a recombinant RNA molecule that encodes the full-length otoferlin protein. In some embodiments of any of the compositions described herein, the coding sequence of at least one of the vectors comprises a nucleotide sequence that spans two adjacent exons of otoferlin genomic DNA and lacks intron sequence between the two adjacent exons.

[0146] The composition includes a first nucleic acid vector including a promoter, a first coding sequence located 3' of the promoter and encoding an N-terminal portion of an otoferlin protein, a splicing donor signal sequence located at the 3' end of the first coding sequence, and a first detectable marker gene located 3' of the splicing donor signal sequence; and a second detectable marker gene, a splicing acceptor signal sequence located 3' of the second detectable marker gene, a second coding sequence located 3' of the splicing acceptor signal sequence and encoding a C-terminal portion of an otoferlin protein, and a polyadenylation sequence located at the 3' end of the second coding sequence. Also provided herein are compositions comprising a first nucleic acid vector and a second nucleic acid vector different from the first nucleic acid vector, each of the encoded portions being at least 30 amino acids in length, the amino acid sequences of the encoded portions not overlapping with each other, and no single vector of the two different vectors encodes the full-length otoferlin protein, but rather, when the coding sequences are transcribed in a mammalian cell to produce RNA transcripts, splicing occurs between a splicing donor signal on one transcript and a splicing acceptor signal on the other transcript, thereby forming a recombinant RNA molecule encoding the full-length otoferlin protein. In some embodiments of any of the compositions described herein, the coding sequence of at least one of the vectors comprises a nucleotide sequence spanning two adjacent exons of otoferlin genomic DNA and lacks intronic sequence between the two adjacent exons. In some embodiments of any of the compositions described herein, the first or second detectable marker gene encodes alkaline phosphatase. In some embodiments of any of the compositions described herein, the first and second detectable marker genes are the same.

[0147] A composition comprising a first nucleic acid vector comprising a promoter, a first coding sequence encoding an N-terminal portion of an otoferlin protein located 3' to the promoter, a splice donor signal sequence located at the 3' end of the first coding sequence, and a highly recombinogenic sequence (e.g., an F1 phage recombinogenic region, e.g., SEQ ID NO: 66) located 3' to the splice donor signal sequence; a second highly recombinogenic sequence (e.g., an F1 phage recombinogenic region, e.g., SEQ ID NO: 67, or an alkaline phosphatase recombinogenic region, e.g., SEQ ID NO: 89), a splice acceptor signal sequence located 3' to the second highly recombinogenic sequence (e.g., the F1 phage recombinogenic region or the alkaline phosphatase recombinogenic region), and a splice acceptor signal sequence located at the 3' end of the splice acceptor signal sequence. Also provided herein is a composition comprising: a second nucleic acid vector different from the first nucleic acid vector, the second coding sequence encoding a C-terminal portion of an otoferlin protein; and a polyadenylation sequence located at the 3' end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acids in length, the amino acid sequences of each of the encoded portions do not overlap with each other; no single vector of the two different vectors encodes the full-length otoferlin protein; and when the coding sequences are transcribed in a mammalian cell and RNA transcripts are produced, splicing occurs between a splicing donor signal on one transcript and a splicing acceptor signal on the other transcript, thereby forming a recombinant RNA molecule encoding the full-length otoferlin protein.

[0148] In some embodiments of any of the compositions described herein, the coding sequence of at least one of the vectors comprises a nucleotide sequence that spans two adjacent exons of otoferlin genomic DNA, and lacks intron sequence between the two adjacent exons.

[0149] Also provided herein are kits comprising any of the compositions described herein. Some embodiments of any of the kits described herein further comprise a pre-filled syringe containing the composition.

[0150] Also provided herein are methods comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​a mammal. In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of the methods described herein, the mammal has previously been identified as having a defective otoferlin gene.

[0151] Also provided herein is a method for increasing expression of full-length otoferlin protein in a mammalian cell, the method comprising introducing any of the compositions described herein into the mammalian cell. In some embodiments of any of the methods described herein, the mammalian cell is a cochlear inner hair cell. In some embodiments of any of the methods described herein, the mammalian cell is a human cell. In some embodiments of any of the methods described herein, the mammalian cell has previously been determined to have a defective otoferlin gene.

[0152] Also provided herein is a method for increasing expression of full-length otoferlin protein in inner hair cells of the cochlea of ​​a mammal, comprising introducing into the cochlea a therapeutically effective amount of any of the compositions described herein. In some embodiments of any of the methods described herein, the mammal has previously been identified as having a defective otoferlin gene. In some embodiments of any of the methods described herein, the mammal is a human.

[0153] Also provided herein is a method of treating asymptomatic sensorineural hearing loss in a subject identified as having a defective otoferlin gene, the method comprising administering a therapeutically effective amount of any of the compositions described herein to the cochlea of ​​the subject. In some embodiments of any of the methods described herein, the subject is human. Some embodiments of any of the methods described herein further comprise determining that the subject has a defective otoferlin gene prior to the administering step.

[0154] Provided herein is a therapeutic composition comprising a plurality of adeno-associated virus (AAV) vectors, wherein the plurality of AAV vectors are capable of constructing auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered.

[0155] In some embodiments of any of the therapeutic compositions described herein, the multiple AAV vectors are capable of constructing full-length auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered.

[0156] Some embodiments of any of the therapeutic compositions described herein further include a first AAV vector and a second AAV vector, wherein the first and second AAV vectors independently contain a packaging capacity of less than about 6 kb.

[0157] In some embodiments of any of the therapeutic compositions described herein, the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group of otoferlin and its orthologs or homologs.

[0158] Some embodiments of any of the therapeutic compositions described herein include Ca vThe auditory polypeptide may further comprise a nucleic acid (e.g., a vector) comprising a nucleic acid sequence encoding an auditory polypeptide messenger RNA encoding an auditory polypeptide selected from the group consisting of a scaffold protein selected from 1.3, bassoon, piccolo, ribeye, and harmonin, Vglut3, synaptotagmin, vesicle binding / docking protein, vesicle priming protein, vesicle fusion protein, GluA2 / 3, and GluA4.

[0159] In some embodiments of any of the therapeutic compositions described herein, the first AAV vector further comprises at least one promoter sequence selected from a CBA, CMV, or CB7 promoter.

[0160] In some embodiments of any of the therapeutic compositions described herein, the first AAV vector further comprises at least one promoter sequence selected from a cochlear-specific promoter.

[0161] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated for intracochlear administration. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include lipid nanoparticles. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include polymer nanoparticles. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include minicircle DNA. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include CELiD DNA. In some embodiments of any of the compositions described herein, the therapeutic composition is formulated to include a synthetic perilymph solution. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include a synthetic perilymph solution comprising 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl2, 1-10 mM glucose, and 2-50 mM HEPES, and having a pH of about 6 to about 9.

[0162] Therapeutic compositions comprising auditory polypeptide messenger RNA are also provided herein.

[0163] Also provided herein is a therapeutic composition comprising one or more adenoviral (AV) vectors, wherein the one or more AV vectors are capable of constructing auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered.

[0164] In some embodiments of any of the therapeutic compositions described herein, the one or more AV vectors are capable of constructing full-length auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered.

[0165] Also provided herein is a therapeutic composition comprising one or more lentiviral vectors, wherein the one or more lentiviral vectors are capable of constructing auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered.

[0166] In some embodiments of any of the therapeutic compositions described herein, the one or more lentiviral vectors are capable of constructing active, e.g., full-length, auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered.

[0167] Also provided herein is a surgical method comprising: i) introducing a first incision into the cochlea of ​​a human subject at a first incision point; and ii) intracochlearly administering an effective dose of a therapeutic composition (e.g., any of the therapeutic compositions described herein).

[0168] In some embodiments of any of the methods described herein, the therapeutic composition is administered to the subject at the first incision point. In some embodiments of any of the methods described herein, the therapeutic composition is administered to the subject in or through the first incision.

[0169] In some embodiments of any of the methods described herein, the therapeutic composition is administered to the subject in or through the oval window membrane of the cochlea. In some embodiments of any of the methods described herein, the therapeutic composition is administered to the subject in or through the round window membrane of the cochlea.

[0170] In some embodiments of any of the methods described herein, the therapeutic composition is administered using a medical device capable of creating multiple incisions in the round window membrane.

[0171] In some embodiments of any of the methods described herein, the medical device includes a plurality of microneedles. In some embodiments of any of the methods described herein, the medical device includes a plurality of microneedles including a substantially circular first aspect, each microneedle having a diameter of at least about 10 microns.

[0172] In some embodiments of any of the methods described herein, the medical device includes a base and / or reservoir capable of holding a therapeutic composition. In some embodiments of any of the methods described herein, the medical device includes a plurality of hollow microneedles, each including a lumen capable of transporting the therapeutic composition.

[0173] In some embodiments of any of the methods described herein, the medical device includes means for generating at least a partial vacuum.

[0174] Also provided herein is a therapeutic delivery system comprising: i) a medical device capable of creating multiple incisions in the round window membrane of the inner ear of a human subject in need thereof; and ii) an effective dose of a therapeutic composition comprising multiple adeno-associated virus (AAV) vectors, wherein the multiple AAV vectors are capable of constructing active, e.g., full-length auditory polypeptide messenger RNA, in target cells of the inner ear.

[0175] Provided herein are means for performing a surgical method comprising the steps of i) intracochlearly administering to a human subject in need thereof an effective dose of a therapeutic composition (e.g., any of the therapeutic compositions described herein), wherein the therapeutic composition can be administered by using a) means for making multiple incisions in the round window membrane, and b) a medical device comprising an effective dose of the therapeutic composition.

[0176] In some embodiments of any of the means for performing the surgical methods described herein, the medical device includes a plurality of microneedles.

[0177] Also provided herein is a therapeutic composition comprising a single adeno-associated virus (AAV) vector, wherein the AAV vector is capable of constructing auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered.

[0178] In some embodiments of any of the therapeutic compositions described herein, a single AAV vector can construct a full-length auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered. In some embodiments of any of the methods described herein, a single vector (e.g., any of the vectors described herein) containing a sequence encoding an active otoferlin protein (e.g., any of the full-length or truncated active otoferlin proteins described herein) can be administered to a subject.

[0179] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition further comprises a second vector other than the AAV vector, and the single AAV vector and the second vector independently contain a packaging capacity of less than about 6 kb.

[0180] In some embodiments of any of the therapeutic compositions described herein, the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group of otoferlin and its orthologs or homologs.

[0181] In some embodiments of any of the therapeutic compositions described herein, the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group of otoferlin and truncation mutants thereof.

[0182] In some embodiments of any of the therapeutic compositions described herein, the otoferlin truncation mutant comprises at least a single C2 domain: [ka]

[0183] In some embodiments of any of the therapeutic compositions described herein, the otoferlin truncation mutant does not include the C-terminal region of the endogenous otoferlin polypeptide.

[0184] In some embodiments of any of the therapeutic compositions described herein, the auditory polypeptide messenger RNA is v 1.3, a scaffold protein selected from bassoon, piccolo, ribey, and harmonin, an auditory polypeptide selected from the group consisting of Vglut3, synaptotagmin, a vesicle binding / docking protein, a vesicle priming protein, a vesicle fusion protein, GluA2 / 3, and GluA4.

[0185] In some embodiments of any of the therapeutic compositions described herein, the single AAV vector further comprises at least one promoter sequence selected from a CBA, CMV, or CB7 promoter.

[0186] In some embodiments of any of the therapeutic compositions described herein, the single AAV vector further comprises at least one promoter sequence selected from a cochlear-specific promoter.

[0187] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated for intracochlear administration.

[0188] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include lipid nanoparticles.

[0189] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include polymeric nanoparticles.

[0190] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include minicircle DNA.

[0191] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include CELiD DNA.

[0192] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include synthetic perilymph fluid.

[0193] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to comprise a synthetic perilymph solution comprising 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl, 1-10 mM glucose, 2-50 mM HEPES, and having a pH of about 6 to about 9.

[0194] Also provided herein are therapeutic compositions comprising auditory polypeptide messenger RNAs encoding otoferlin truncation mutants.

[0195] 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.

[0196] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" includes one element and more than one element.

[0197] The term "about" when referring to a measurable value, e.g., an amount, a time range, etc., is meant to encompass variations of up to ±20%. These variations may be, for example, up to ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0198] The term "signaling domain" refers to a functional portion of a protein that acts to control cellular activity through a given signaling pathway by transmitting information within the cell, either by generating second messengers or by responding to such messengers and functioning as an effector. Otoferlin is thought to be a calcium sensor required for exocytosis in inner hair cells and neurotransmitter release in immature outer hair cells. Five of the six C2 domains of otoferlin protein (C2B-F) bind calcium ions with moderate and low affinity constants in solution (Kd = 25-95 μM and 400-700 μM, respectively) (Padmanarayana et al. 2014 Biochem 53:5023-5033). In the presence of phosphatidylserine (PS), a calcium concentration of 10 μM results in significant C2-liposome interaction with the C2C-C2E domains of otoferlin. Thus, otoferlin possesses domains that appear to operate using an "electrostatic switch" mechanism and calcium-independent binding domains. PI(4,5)P2, a major presynaptic signaling molecule, has been shown to interact with the C2C and C2F domains of otoferlin in a calcium-independent manner (Padmanarayana et al. 2014 Biochem 53:5023-5033).

[0199] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody may also be a tetramer of an immunoglobulin molecule. The term "antibody fragment" refers to at least a portion of an intact antibody, or a recombinant variant thereof, such as an antigen-binding domain of an intact antibody, e.g., an antigenic determining variable region, sufficient to confer recognition and specific binding to the antibody fragment's target, e.g., an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies, e.g., sdAb (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are contiguously linked via a short flexible polypeptide linker and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., with respect to the N- and C-termini of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0200] The term "recombinant polypeptide" refers to a polypeptide produced using recombinant DNA technology, such as, for example, a polypeptide expressed by a viral vector expression system. The term should also be taken to mean a polypeptide produced by synthesis of a DNA molecule encoding the polypeptide, where the DNA molecule expresses a protein or an amino acid sequence specifying the polypeptide, and where the DNA or amino acid sequence was obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0201] The term "mutation in an otoferlin gene" refers to a modification in a wild-type otoferlin gene that results in the production of an otoferlin protein having one or more of the following: one or more amino acid deletions, one or more amino acid substitutions, and one or more amino acid insertions compared to the wild-type otoferlin protein; and / or a reduced expression level of the encoded otoferlin protein in a mammalian cell compared to the expression level of the encoded otoferlin protein in a mammalian cell not having the mutation. In some embodiments, the mutation may 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 may result in a frameshift in the otoferlin gene. The term "frameshift" is known in the art to encompass any mutation in a coding sequence that results in a shift in the reading frame of the coding sequence. In some embodiments, the frameshift may result in a nonfunctional protein. In some embodiments, the point mutation may be a nonsense mutation (i.e., resulting in a premature stop codon in an exon of a gene). The nonsense mutation may 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 may result in the loss of (or a reduction in the level of) expression of otoferlin mRNA or otoferlin protein, or both mRNA and protein. In some embodiments, the mutation may result in the production of an altered otoferlin protein that has a loss or reduction in one or more biological activities (functions) compared to the wild-type otoferlin protein.

[0202] In some embodiments, the mutation is an insertion of one or more nucleotides into the otoferlin gene. In some embodiments, the mutation is in the regulatory sequence of the otoferlin gene, i.e., in a part of the gene that is not the coding sequence. In some embodiments, the mutation in the regulatory sequence may be in the promoter or enhancer region, which may prevent or reduce proper transcription of the otoferlin gene.

[0203] Modifications can be introduced into a nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0204] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an 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 those 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 have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar 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).

[0205] The term "encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, e.g., a gene, cDNA, or mRNA, to serve as a template for the synthesis of a defined amino acid sequence according to the genetic code. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced by transcription and translation of the mRNA corresponding to that gene, cDNA, or RNA. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription can be said to encode a protein product.

[0206] The term "homologous" or "identity" refers to the sequence identity of subunits between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both molecules is occupied by the same monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half the positions in two sequences (e.g., 5 positions in a polymer 10 subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0207] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and therefore encode the same amino acid sequence. A nucleotide sequence that encodes a protein may also contain introns.

[0208] The term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system.

[0209] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0210] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide is "isolated" if it is partially or completely separated from the coexisting materials of its natural state.

[0211] An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0212] The term "substantially purified cells" refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which the cells are normally associated in their naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous cell population. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells have been cultured in vitro. In other aspects, the cells have not been cultured in vitro.

[0213] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0214] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0215] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days, or weeks, where the period of expression is shorter than the period of expression of the gene when integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0216] The term "subject" is intended to include organisms (e.g., mammals, humans) in which an immune response can be elicited. In some embodiments, the subject is a rodent (e.g., a rat or mouse), rabbit, sheep, dog, cat, horse, non-human primate, or human. In some embodiments, the subject has or is at risk of developing non-syndromic 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.

[0217] In some embodiments, the subject has been identified as having asymptomatic sensorineural hearing loss.

[0218] The term "therapeutic" as used herein means treatment. A therapeutic effect is achieved by the reduction, suppression, amelioration, or eradication of a disease state.

[0219] The term "prophylactic," as used herein, refers to the prevention or protective treatment of a disease or disease state. "Prevention" in this context includes reducing the likelihood that a subject will experience the disease.

[0220] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an 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., a wild-type full-length otoferlin protein, or an otoferlin protein variant having a desired activity) (e.g., compared to the expression level before 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., a wild-type full-length otoferlin protein or an active variant) in target cells (e.g., cochlear inner hair cells). In some embodiments, a therapeutically effective amount of a composition can result in a different cellular localization of an active otoferlin protein (e.g., a wild-type full-length otoferlin protein or an active variant) in target cells (e.g., cochlear inner hair cells). In some embodiments, a therapeutically effective amount of the composition may result in an increase in the expression level of 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 otoferlin protein in the target cell (e.g., compared to a reference level, e.g., a level in a subject before treatment, a level in a subject with a mutation in the otoferlin gene, or a level in a subject or population of subjects with asymptomatic sensorineural hearing loss).

[0221] The term "parenteral" administration of a composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0222] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof, in single- or double-stranded form. Unless specifically limited, this term encompasses nucleic acids containing known analogs of natural nucleotides that 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 also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. 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-base 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)).

[0223] In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.

[0224] In the context of the present disclosure, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0225] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, synthesized in vitro. Generally, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector contains a template used to generate the in vitro transcribed RNA.

[0226] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, but there is no limit regarding the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, as well as longer chains, of which there are numerous varieties and commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes a naturally occurring peptide, a recombinant peptide, or a combination thereof.

[0227] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across a cell membrane.

[0228] The term "active otoferlin protein" refers to 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 an otherwise wild-type mammal, and expressed in the auditory hair cells of that mammal, results in that mammal having a hearing level that approximates the normal hearing level of a similar mammal that is completely wild-type. A non-limiting example of an active otoferlin protein is a full-length otoferlin protein (e.g., any of the full-length otoferlin proteins described herein).

[0229] For example, an active otoferlin protein may have from 1 to about 240 amino acid substitutions, from 1 to about 235 amino acid substitutions, from 1 to about 230 amino acid substitutions, from 1 to about 225 amino acid substitutions, from 1 to about 220 amino acid substitutions, from 1 to about 215 amino acid substitutions, from 1 to about 210 amino acid substitutions, from 1 to about 205 amino acid substitutions, from 1 to about 200 amino acid substitutions, from 1 to about 195 amino acid substitutions, from 1 to about 190 amino acid substitutions, from 1 to about 185 ...215 amino acid substitutions, from 1 to about 210 amino acid substitutions, from 1 to about 205 amino acid substitutions, from 1 to about 200 amino acid substitutions, from 1 to about 215 amino acid substitutions, from 1 to about 210 Amino acid substitutions up to about 180 amino acid substitutions, 1 amino acid substitution to about 175 amino acid substitutions, 1 amino acid substitution to about 170 amino acid substitutions, 1 amino acid substitution to about 165 amino acid substitutions, 1 amino acid substitution to about 160 amino acid substitutions, 1 amino acid substitution to about 155 amino acid substitutions, 1 amino acid substitution to about 150 amino acid substitutions, 1 amino acid substitution to about 145 amino acid substitutions, 1 amino acid substitution to about 140 amino acid substitutions, 1 amino acid substitution to about 135 amino acid substitutions, 1 amino acid substitution to about 130 amino acid substitutions, 1 amino acid substitution to about 125 amino acid substitutions, 1 amino acid substitution to about 12 ... 1 to about 115 amino acid substitutions, 1 to about 110 amino acid substitutions, 1 to about 105 amino acid substitutions, 1 to about 100 amino acid substitutions, 1 to about 95 amino acid substitutions, 1 to about 90 amino acid substitutions, 1 to about 85 amino acid substitutions, 1 to about 80 amino acid substitutions, 1 to about 75 amino acid substitutions, 1 to about 70 amino acid substitutions, 1 to about 65 amino acid substitutions, 1 to about 60 amino acid substitutions, 1 to about 55 amino acid substitutions, 1 to about 50 amino acid substitutions amino acid substitution, 1 to about 45 amino acid substitutions, 1 to about 40 amino acid substitutions, 1 to about 35 amino acid substitutions, 1 to about 30 amino acid substitutions, 1 to about 25 amino acid substitutions, 1 to about 20 amino acid substitutions, 1 to about 15 amino acid substitutions, 1 to about 10 amino acid substitutions, 1 to about 9 amino acid substitutions, 1 to about 8 amino acid substitutions, 1 to about 7 amino acid substitutions, 1 to about 6 amino acid substitutions, 1 to about 5 amino acid substitutions, 1 to about 4 amino acid substitutions,1 amino acid substitution to about 3 amino acid substitutions, about 2 amino acid substitutions to about 240 amino acid substitutions, about 2 amino acid substitutions to about 235 amino acid substitutions, about 2 amino acid substitutions to about 230 amino acid substitutions, about 2 amino acid substitutions to about 225 amino acid substitutions, about 2 amino acid substitutions to about 220 amino acid substitutions, about 2 amino acid substitutions to about 215 amino acid substitutions, about 2 amino acid substitutions to about 210 amino acid substitutions, about 2 amino acid substitutions to about 205 amino acid substitutions, about 2 amino acid substitutions to about 200 amino acid substitutions, about 2 amino acid substitutions to about 195 amino acid substitutions, about 2 amino acid substitutions to about 190 amino acid substitutions, about 2 amino acid substitutions Approximately 185 amino acid substitutions, approximately 2 to approximately 180 amino acid substitutions, approximately 2 to approximately 175 amino acid substitutions, approximately 2 to approximately 170 amino acid substitutions, approximately 2 to approximately 165 amino acid substitutions, approximately 2 to approximately 160 amino acid substitutions, approximately 2 to approximately 155 amino acid substitutions, approximately 2 to approximately 150 amino acid substitutions, approximately 2 to approximately 145 amino acid substitutions, approximately 2 to approximately 140 amino acid substitutions, approximately 2 to approximately 135 amino acid substitutions, approximately 2 to approximately 130 amino acid substitutions, approximately 2 to approximately 125 amino acid substitutions substitutions, about 2 amino acid substitutions to about 120 amino acid substitutions, about 2 amino acid substitutions to about 115 amino acid substitutions, about 2 amino acid substitutions to about 110 amino acid substitutions, about 2 amino acid substitutions to about 105 amino acid substitutions, about 2 amino acid substitutions to about 100 amino acid substitutions, about 2 amino acid substitutions to about 95 amino acid substitutions, about 2 amino acid substitutions to about 90 amino acid substitutions, about 2 amino acid substitutions to about 85 amino acid substitutions, about 2 amino acid substitutions to about 80 amino acid substitutions, about 2 amino acid substitutions to about 75 amino acid substitutions, about 2 amino acid substitutions to about 70 amino acid substitutions, about 2 amino acid substitutions to about 65 amino acid substitutions, about 2 amino acid substitutions to about 60 amino acid substitutions amino acid substitutions, about 2 to about 55 amino acid substitutions, about 2 to about 50 amino acid substitutions, about 2 to about 45 amino acid substitutions, about 2 to about 40 amino acid substitutions, about 2 to about 35 amino acid substitutions, about 2 to about 30 amino acid substitutions, about 2 to about 25 amino acid substitutions, about 2 to about 20 amino acid substitutions, about 2 to about 15 amino acid substitutions, about 2 to about 10 amino acid substitutions, about 2 to about 9 amino acid substitutions, about 2 to about 8 amino acid substitutions, about 2 to about 7 amino acid substitutions,About 2 to about 6 amino acid substitutions, about 2 to about 5 amino acid substitutions, about 2 to about 4 amino acid substitutions, about 3 to about 240 amino acid substitutions, about 3 to about 235 amino acid substitutions, about 3 to about 230 amino acid substitutions, about 3 to about 225 amino acid substitutions, about 3 to about 220 amino acid substitutions, about 3 to about 215 amino acid substitutions, about 3 to about 210 amino acid substitutions, about 3 to about 205 amino acid substitutions, about 3 to about 200 amino acid substitutions, about 3 to about 195 Amino acid substitutions, about 3 amino acid substitutions to about 190 amino acid substitutions, about 3 amino acid substitutions to about 185 amino acid substitutions, about 3 amino acid substitutions to about 180 amino acid substitutions, about 3 amino acid substitutions to about 175 amino acid substitutions, about 3 amino acid substitutions to about 170 amino acid substitutions, about 3 amino acid substitutions to about 165 amino acid substitutions, about 3 amino acid substitutions to about 160 amino acid substitutions, about 3 amino acid substitutions to about 155 amino acid substitutions, about 3 amino acid substitutions to about 150 amino acid substitutions, about 3 amino acid substitutions to about 145 amino acid substitutions, about 3 amino acid substitutions to about 140 amino acid substitutions, about 3 amino acid substitutions to about 135 amino acid substitutions, about 3 Amino acid substitutions up to about 130 amino acid substitutions, about 3 amino acid substitutions to about 125 amino acid substitutions, about 3 amino acid substitutions to about 120 amino acid substitutions, about 3 amino acid substitutions to about 115 amino acid substitutions, about 3 amino acid substitutions to about 110 amino acid substitutions, about 3 amino acid substitutions to about 105 amino acid substitutions, about 3 amino acid substitutions to about 100 amino acid substitutions, about 3 amino acid substitutions to about 95 amino acid substitutions, about 3 amino acid substitutions to about 90 amino acid substitutions, about 3 amino acid substitutions to about 85 amino acid substitutions, about 3 amino acid substitutions to about 80 amino acid substitutions, about 3 amino acid substitutions to about 75 amino acid substitutions, about 3 amino acid substitutions to about 70 amino acid substitutions substitutions, about 3 amino acid substitutions to about 65 amino acid substitutions, about 3 amino acid substitutions to about 60 amino acid substitutions, about 3 amino acid substitutions to about 55 amino acid substitutions, about 3 amino acid substitutions to about 50 amino acid substitutions, about 3 amino acid substitutions to about 45 amino acid substitutions, about 3 amino acid substitutions to about 40 amino acid substitutions, about 3 amino acid substitutions to about 35 amino acid substitutions, about 3 amino acid substitutions to about 30 amino acid substitutions, about 3 amino acid substitutions to about 25 amino acid substitutions, about 3 amino acid substitutions to about 20 amino acid substitutions, about 3 amino acid substitutions to about 15 amino acid substitutions, about 3 amino acid substitutions to about 10 amino acid substitutions, about 3 amino acid substitutions to about 9 amino acid substitutions,About 3 to about 8 amino acid substitutions, about 3 to about 7 amino acid substitutions, about 3 to about 6 amino acid substitutions, about 3 to about 5 amino acid substitutions, about 4 to about 240 amino acid substitutions, about 4 to about 235 amino acid substitutions, about 4 to about 230 amino acid substitutions, about 4 to about 225 amino acid substitutions, about 4 to about 220 amino acid substitutions, about 4 to about 215 amino acid substitutions, about 4 to about 210 amino acid substitutions, about 4 to about 205 amino acid substitutions, about 4 to about 200 amino acid substitutions amino acid substitutions, about 4 amino acid substitutions to about 195 amino acid substitutions, about 4 amino acid substitutions to about 190 amino acid substitutions, about 4 amino acid substitutions to about 185 amino acid substitutions, about 4 amino acid substitutions to about 180 amino acid substitutions, about 4 amino acid substitutions to about 175 amino acid substitutions, about 4 amino acid substitutions to about 170 amino acid substitutions, about 4 amino acid substitutions to about 165 amino acid substitutions, about 4 amino acid substitutions to about 160 amino acid substitutions, about 4 amino acid substitutions to about 155 amino acid substitutions, about 4 amino acid substitutions to about 150 amino acid substitutions, about 4 amino acid substitutions to about 145 amino acid substitutions, about 4 amino acid substitutions to about 14 ...0 amino acid substitutions, about 4 amino acid substitutions to about 145 amino acid substitutions, about 4 amino acid substitutions to about 140 amino acid substitutions, about 4 amino acid substitutions to about 165 amino acid substitutions, about 4 amino acid substitutions to about 160 amino acid substitutions, about 4 amino acid substitutions to about 150 amino acid substitutions, about 4 amino acid substitutions to about 140 amino acid substitutions, about 4 amino acid substitutions to about 165 amino acid substitutions, about about 4 amino acid substitutions to about 135 amino acid substitutions, about 4 amino acid substitutions to about 130 amino acid substitutions, about 4 amino acid substitutions to about 125 amino acid substitutions, about 4 amino acid substitutions to about 120 amino acid substitutions, about 4 amino acid substitutions to about 115 amino acid substitutions, about 4 amino acid substitutions to about 110 amino acid substitutions, about 4 amino acid substitutions to about 105 amino acid substitutions, about 4 amino acid substitutions to about 100 amino acid substitutions, about 4 amino acid substitutions to about 95 amino acid substitutions, about 4 amino acid substitutions to about 90 amino acid substitutions, about 4 amino acid substitutions to about 85 amino acid substitutions, about 4 amino acid substitutions to about 80 amino acid substitutions, about 4 amino acid substitutions to about 75 amino acid substitutions substitutions, about 4 amino acid substitutions to about 70 amino acid substitutions, about 4 amino acid substitutions to about 65 amino acid substitutions, about 4 amino acid substitutions to about 60 amino acid substitutions, about 4 amino acid substitutions to about 55 amino acid substitutions, about 4 amino acid substitutions to about 50 amino acid substitutions, about 4 amino acid substitutions to about 45 amino acid substitutions, about 4 amino acid substitutions to about 40 amino acid substitutions, about 4 amino acid substitutions to about 35 amino acid substitutions, about 4 amino acid substitutions to about 30 amino acid substitutions, about 4 amino acid substitutions to about 25 amino acid substitutions, about 4 amino acid substitutions to about 20 amino acid substitutions, about 4 amino acid substitutions to about 15 amino acid substitutions, about 4 amino acid substitutions to about 10 amino acid substitutions,About 4 to about 9 amino acid substitutions, about 4 to about 8 amino acid substitutions, about 4 to about 7 amino acid substitutions, about 4 to about 6 amino acid substitutions, about 5 to about 240 amino acid substitutions, about 5 to about 235 amino acid substitutions, about 5 to about 230 amino acid substitutions, about 5 to about 225 amino acid substitutions, about 5 to about 220 amino acid substitutions, about 5 to about 215 amino acid substitutions, about 5 to about 210 amino acid substitutions, about 5 to about 205 amino acid substitutions, about 5 to about 200 amino acid substitutions amino acid substitutions, about 5 to about 195 amino acid substitutions, about 5 to about 190 amino acid substitutions, about 5 to about 185 amino acid substitutions, about 5 to about 180 amino acid substitutions, about 5 to about 175 amino acid substitutions, about 5 to about 170 amino acid substitutions, about 5 to about 165 amino acid substitutions, about 5 to about 160 amino acid substitutions, about 5 to about 155 amino acid substitutions, about 5 to about 150 amino acid substitutions, about 5 to about 145 amino acid substitutions, about 5 to about 140 amino acid substitutions, about 5 about 5 amino acid substitutions to about 135 amino acid substitutions, about 5 amino acid substitutions to about 130 amino acid substitutions, about 5 amino acid substitutions to about 125 amino acid substitutions, about 5 amino acid substitutions to about 120 amino acid substitutions, about 5 amino acid substitutions to about 115 amino acid substitutions, about 5 amino acid substitutions to about 110 amino acid substitutions, about 5 amino acid substitutions to about 105 amino acid substitutions, about 5 amino acid substitutions to about 100 amino acid substitutions, about 5 amino acid substitutions to about 95 amino acid substitutions, about 5 amino acid substitutions to about 90 amino acid substitutions, about 5 amino acid substitutions to about 85 amino acid substitutions, about 5 amino acid substitutions to about 80 amino acid substitutions, about 5 amino acid substitutions to about 75 amino acid substitutions substitutions, about 5 to about 70 amino acid substitutions, about 5 to about 65 amino acid substitutions, about 5 to about 60 amino acid substitutions, about 5 to about 55 amino acid substitutions, about 5 to about 50 amino acid substitutions, about 5 to about 45 amino acid substitutions, about 5 to about 40 amino acid substitutions, about 5 to about 35 amino acid substitutions, about 5 to about 30 amino acid substitutions, about 5 to about 25 amino acid substitutions, about 5 to about 20 amino acid substitutions, about 5 to about 15 amino acid substitutions, about 5 to about 10 amino acid substitutions,About 5 to about 9 amino acid substitutions, about 5 to about 8 amino acid substitutions, about 5 to about 7 amino acid substitutions, about 6 to about 240 amino acid substitutions, about 6 to about 235 amino acid substitutions, about 6 to about 230 amino acid substitutions, about 6 to about 225 amino acid substitutions, about 6 amino acid substitutions, Substitutions up to about 220 amino acid substitutions, about 6 amino acid substitutions to about 215 amino acid substitutions, about 6 amino acid substitutions to about 210 amino acid substitutions, about 6 amino acid substitutions to about 205 amino acid substitutions, about 6 amino acid substitutions to about 200 amino acid substitutions, about 6 amino acid substitutions to about 195 amino acid substitutions, about 6 amino acid substitutions to about 190 amino acid substitutions, about 6 amino acid substitutions to about 185 amino acid substitutions, about 6 amino acid substitutions to about 180 amino acid substitutions, about 6 amino acid substitutions to about 175 amino acid substitutions, about 6 amino acid substitutions to about 170 amino acid substitutions, about 6 amino acid substitutions to about 165 amino acid substitutions, about 6 amino acid substitutions to about 160 Amino acid substitutions, about 6 amino acid substitutions to about 155 amino acid substitutions, about 6 amino acid substitutions to about 150 amino acid substitutions, about 6 amino acid substitutions to about 145 amino acid substitutions, about 6 amino acid substitutions to about 140 amino acid substitutions, about 6 amino acid substitutions to about 135 amino acid substitutions, about 6 amino acid substitutions to about 130 amino acid substitutions, about 6 amino acid substitutions to about 125 amino acid substitutions, about 6 amino acid substitutions to about 120 amino acid substitutions, about 6 amino acid substitutions to about 115 amino acid substitutions, about 6 amino acid substitutions to about 110 amino acid substitutions, about 6 amino acid substitutions to about 105 amino acid substitutions, about 6 amino acid substitutions to about 100 amino acid substitutions, about 6 Amino acid substitutions up to about 95 amino acid substitutions, about 6 amino acid substitutions to about 90 amino acid substitutions, about 6 amino acid substitutions to about 85 amino acid substitutions, about 6 amino acid substitutions to about 80 amino acid substitutions, about 6 amino acid substitutions to about 75 amino acid substitutions, about 6 amino acid substitutions to about 70 amino acid substitutions, about 6 amino acid substitutions to about 65 amino acid substitutions, about 6 amino acid substitutions to about 60 amino acid substitutions, about 6 amino acid substitutions to about 55 amino acid substitutions, about 6 amino acid substitutions to about 50 amino acid substitutions, about 6 amino acid substitutions to about 45 amino acid substitutions, about 6 amino acid substitutions to about 40 amino acid substitutions, about 6 amino acid substitutions to about 35 amino acid substitutions, about 6 amino acid substitutions amino acid substitutions to about 30 amino acid substitutions, about 6 amino acid substitutions to about 25 amino acid substitutions, about 6 amino acid substitutions to about 20 amino acid substitutions, about 6 amino acid substitutions to about 15 amino acid substitutions, about 6 amino acid substitutions to about 10 amino acid substitutions, about 6 amino acid substitutions to about 9 amino acid substitutions, about 6 amino acid substitutions to about 8 amino acid substitutions, about 7 amino acid substitutions to about 240 amino acid substitutions, about 7 amino acid substitutions to about 235 amino acid substitutions, about 7 amino acid substitutions to about 230 amino acid substitutions, about 7 amino acid substitutions to about 225 amino acid substitutions, about 7 amino acid substitutions to about 220 amino acid substitutions, about 7 amino acid substitutions to about 215 amino acid substitutions,about 7 amino acid substitutions to about 210 amino acid substitutions, about 7 amino acid substitutions to about 205 amino acid substitutions, about 7 amino acid substitutions to about 200 amino acid substitutions, about 7 amino acid substitutions to about 195 amino acid substitutions, about 7 amino acid substitutions to about 190 amino acid substitutions, about 7 amino acid substitutions to about 185 amino acid substitutions, about 7 amino acid substitutions to about 180 amino acid substitutions, about 7 amino acid substitutions to about 175 amino acid substitutions, about 7 amino acid substitutions to about 170 amino acid substitutions, about 7 amino acid substitutions to about 165 amino acid substitutions, about 7 amino acid substitutions to about 160 amino acid substitutions, about 7 amino acid substitutions to about 15 ...65 amino acid substitutions, about 7 amino acid substitutions to about 160 amino acid substitutions, about 7 amino acid substitutions to about 155 amino acid substitutions, about 7 amino acid substitutions to about 195 amino acid substitutions, about 7 amino acid substitutions to about 190 amino acid substitutions, about 7 amino acid substitutions about 7 amino acid substitutions to about 150 amino acid substitutions, about 7 amino acid substitutions to about 145 amino acid substitutions, about 7 amino acid substitutions to about 140 amino acid substitutions, about 7 amino acid substitutions to about 135 amino acid substitutions, about 7 amino acid substitutions to about 130 amino acid substitutions, about 7 amino acid substitutions to about 125 amino acid substitutions, about 7 amino acid substitutions to about 120 amino acid substitutions, about 7 amino acid substitutions to about 115 amino acid substitutions, about 7 amino acid substitutions to about 110 amino acid substitutions, about 7 amino acid substitutions to about 105 amino acid substitutions, about 7 amino acid substitutions to about 100 amino acid substitutions, about 7 amino acid substitutions to about 95 amino acid substitutions, about 7 amino acid substitutions to about 90 Amino acid substitutions, about 7 to about 85 amino acid substitutions, about 7 to about 80 amino acid substitutions, about 7 to about 75 amino acid substitutions, about 7 to about 70 amino acid substitutions, about 7 to about 65 amino acid substitutions, about 7 to about 60 amino acid substitutions, about 7 to about 55 amino acid substitutions, about 7 to about 50 amino acid substitutions, about 7 to about 45 amino acid substitutions, about 7 to about 40 amino acid substitutions, about 7 to about 35 amino acid substitutions, about 7 to about 30 amino acid substitutions, about 7 to about 25 amino acid substitutions, about 7 to about 20 amino acid substitutions, about 7 to about 15 amino acid substitutions, about 7 to about 10 amino acid substitutions, about 7 to about 9 amino acid substitutions, about 8 to about 240 amino acid substitutions, about 8 to about 235 amino acid substitutions, about 8 to about 230 amino acid substitutions, about 8 to about 225 amino acid substitutions, about 8 to about 220 amino acid substitutions, about 8 to about 215 amino acid substitutions, about 8 to about 210 amino acid substitutions, about 8 to about 205 amino acid substitutions,About 8 amino acid substitutions to about 200 amino acid substitutions, about 8 amino acid substitutions to about 195 amino acid substitutions, about 8 amino acid substitutions to about 190 amino acid substitutions, about 8 amino acid substitutions to about 185 amino acid substitutions, about 8 amino acid substitutions to about 180 amino acid substitutions, about 8 amino acid substitutions to about 175 amino acid substitutions, about 8 amino acid substitutions to about 170 amino acid substitutions, about 8 amino acid substitutions to about 165 amino acid substitutions, about 8 amino acid substitutions to about 160 amino acid substitutions, about 8 amino acid substitutions to about 155 amino acid substitutions, about 8 amino acid substitutions to about 150 amino acid substitutions, about 8 amino acid substitutions to about 145 amino acid substitutions, about 8 amino acid substitutions to about 140 amino acid substitutions, about 8 amino acid substitutions to about 135 amino acid substitutions, about 8 amino acid substitutions to about 130 amino acid substitutions, about 8 amino acid substitutions to about 125 amino acid substitutions, about 8 amino acid substitutions to about 120 amino acid substitutions, about 8 amino acid substitutions to about 115 amino acid substitutions, about 8 amino acid substitutions to about 110 amino acid substitutions, about 8 amino acid substitutions to about 105 amino acid substitutions, about 8 amino acid substitutions to about 100 amino acid substitutions, about 8 amino acid substitutions to about 95 amino acid substitutions, about 8 amino acid substitutions to about 90 amino acid substitutions, about 8 amino acid substitutions to about 85 amino acid substitutions, about 8 amino acid substitutions to about 80 amino acid substitutions, 8 to about 75 amino acid substitutions, about 8 to about 70 amino acid substitutions, about 8 to about 65 amino acid substitutions, about 8 to about 60 amino acid substitutions, about 8 to about 55 amino acid substitutions, about 8 to about 50 amino acid substitutions, about 8 to about 45 amino acid substitutions, about 8 to about 40 amino acid substitutions, about 8 to about 35 amino acid substitutions, about 8 to about 30 amino acid substitutions, about 8 to about 25 amino acid substitutions, about 8 to about 20 amino acid substitutions, about 8 to about 15 amino acid substitutions, about 8 amino acid substitutions to about 10 amino acid substitutions, about 10 amino acid substitutions to about 240 amino acid substitutions, about 10 amino acid substitutions to about 235 amino acid substitutions, about 10 amino acid substitutions to about 230 amino acid substitutions, about 10 amino acid substitutions to about 225 amino acid substitutions, about 10 amino acid substitutions to about 220 amino acid substitutions, about 10 amino acid substitutions to about 215 amino acid substitutions, about 10 amino acid substitutions to about 210 amino acid substitutions, about 10 amino acid substitutions to about 205 amino acid substitutions, about 10 amino acid substitutions to about 200 amino acid substitutions, about 10 amino acid substitutions to about 195 amino acid substitutions, about 10 amino acid substitutions to about 190 amino acid substitutions,About 10 to about 185 amino acid substitutions, about 10 to about 180 amino acid substitutions, about 10 to about 175 amino acid substitutions, about 10 to about 170 amino acid substitutions, about 10 to about 165 amino acid substitutions, about 10 to about 160 amino acid substitutions, about 10 to about 155 amino acid substitutions, about 10 to about 150 amino acid substitutions, about 10 to about 145 amino acid substitutions, about 10 to about 140 amino acid substitutions, about 10 to about 135 amino acid substitutions, about 10 to about 130 amino acid substitutions amino acid substitutions, about 10 to about 125 amino acid substitutions, about 10 to about 120 amino acid substitutions, about 10 to about 115 amino acid substitutions, about 10 to about 110 amino acid substitutions, about 10 to about 105 amino acid substitutions, about 10 to about 100 amino acid substitutions, about 10 to about 95 amino acid substitutions, about 10 to about 90 amino acid substitutions, about 10 to about 85 amino acid substitutions, about 10 to about 80 amino acid substitutions, about 10 to about 75 amino acid substitutions, about 10 to about 70 amino acid substitutions substitutions, about 10 to about 65 amino acid substitutions, about 10 to about 60 amino acid substitutions, about 10 to about 55 amino acid substitutions, about 10 to about 50 amino acid substitutions, about 10 to about 45 amino acid substitutions, about 10 to about 40 amino acid substitutions, about 10 to about 35 amino acid substitutions, about 10 to about 30 amino acid substitutions, about 10 to about 25 amino acid substitutions, about 10 to about 20 amino acid substitutions, about 10 to about 15 amino acid substitutions, about 15 to about 240 amino acid substitutions, about 15 ... amino acid substitutions to about 235 amino acid substitutions, about 15 amino acid substitutions to about 230 amino acid substitutions, about 15 amino acid substitutions to about 225 amino acid substitutions, about 15 amino acid substitutions to about 220 amino acid substitutions, about 15 amino acid substitutions to about 215 amino acid substitutions, about 15 amino acid substitutions to about 210 amino acid substitutions, about 15 amino acid substitutions to about 205 amino acid substitutions, about 15 amino acid substitutions to about 200 amino acid substitutions, about 15 amino acid substitutions to about 195 amino acid substitutions, about 15 amino acid substitutions to about 190 amino acid substitutions, about 15 amino acid substitutions to about 185 amino acid substitutions, about 15 amino acid substitutions to about 180 amino acid substitutions,About 15 to about 175 amino acid substitutions, about 15 to about 170 amino acid substitutions, about 15 to about 165 amino acid substitutions, about 15 to about 160 amino acid substitutions, about 15 to about 155 amino acid substitutions, about 15 to about 150 amino acid substitutions, about 15 to about 145 amino acid substitutions, about 15 to about 140 amino acid substitutions, about 15 to about 135 amino acid substitutions, about 15 to about 130 amino acid substitutions, about 15 to about 125 amino acid substitutions, about 15 to about 120 amino acid substitutions amino acid substitutions, about 15 to about 115 amino acid substitutions, about 15 to about 110 amino acid substitutions, about 15 to about 105 amino acid substitutions, about 15 to about 100 amino acid substitutions, about 15 to about 95 amino acid substitutions, about 15 to about 90 amino acid substitutions, about 15 to about 85 amino acid substitutions, about 15 to about 80 amino acid substitutions, about 15 to about 75 amino acid substitutions, about 15 to about 70 amino acid substitutions, about 15 to about 65 amino acid substitutions, about 15 to about 60 amino acid substitutions, 15 to about 55 amino acid substitutions, about 15 to about 50 amino acid substitutions, about 15 to about 45 amino acid substitutions, about 15 to about 40 amino acid substitutions, about 15 to about 35 amino acid substitutions, about 15 to about 30 amino acid substitutions, about 15 to about 25 amino acid substitutions, about 15 to about 20 amino acid substitutions, about 20 to about 240 amino acid substitutions, about 20 to about 235 amino acid substitutions, about 20 to about 230 amino acid substitutions, about 20 to about 225 amino acid substitutions, about 20 amino acid substitutions amino acid substitutions to about 220 amino acid substitutions, about 20 amino acid substitutions to about 215 amino acid substitutions, about 20 amino acid substitutions to about 210 amino acid substitutions, about 20 amino acid substitutions to about 205 amino acid substitutions, about 20 amino acid substitutions to about 200 amino acid substitutions, about 20 amino acid substitutions to about 195 amino acid substitutions, about 20 amino acid substitutions to about 190 amino acid substitutions, about 20 amino acid substitutions to about 185 amino acid substitutions, about 20 amino acid substitutions to about 180 amino acid substitutions, about 20 amino acid substitutions to about 175 amino acid substitutions, about 20 amino acid substitutions to about 170 amino acid substitutions, about 20 amino acid substitutions to about 165 amino acid substitutions,about 20 amino acid substitutions to about 160 amino acid substitutions, about 20 amino acid substitutions to about 155 amino acid substitutions, about 20 amino acid substitutions to about 150 amino acid substitutions, about 20 amino acid substitutions to about 145 amino acid substitutions, about 20 amino acid substitutions to about 140 amino acid substitutions, about 20 amino acid substitutions to about 135 amino acid substitutions, about 20 amino acid substitutions to about 130 amino acid substitutions, about 20 amino acid substitutions to about 125, Amino acid substitutions, about 20 to about 120 amino acid substitutions, about 20 to about 115 amino acid substitutions, about 20 to about 110 amino acid substitutions, about 20 to about 105 amino acid substitutions, about 20 to about 100 amino acid substitutions, about 20 to about 95 amino acid substitutions, about 20 to about 90 amino acid substitutions, about 20 to about 85 amino acid substitutions, about 20 to about 80 amino acid substitutions, about 20 to about 75 amino acid substitutions, about 20 to about 70 amino acid substitutions, about 20 to about 65 Amino acid substitution, about 20 to about 60 amino acid substitutions, about 20 to about 55 amino acid substitutions, about 20 to about 50 amino acid substitutions, about 20 to about 45 amino acid substitutions, about 20 to about 40 amino acid substitutions, about 20 to about 35 amino acid substitutions, about 20 to about 30 amino acid substitutions, about 20 to about 25 amino acid substitutions, about 25 to about 240 amino acid substitutions, about 25 to about 235 amino acid substitutions, about 25 to about 230 amino acid substitutions, about 25 to about 225 amino acid substitutions amino acid substitutions, about 25 to about 220 amino acid substitutions, about 25 to about 215 amino acid substitutions, about 25 to about 210 amino acid substitutions, about 25 to about 205 amino acid substitutions, about 25 to about 200 amino acid substitutions, about 10 to about 195 amino acid substitutions, about 25 to about 190 amino acid substitutions, about 25 to about 185 amino acid substitutions, about 25 to about 180 amino acid substitutions, about 25 to about 175 amino acid substitutions, about 25 to about 170 amino acid substitutions, about 25 amino acid substitutions amino acid substitutions to about 165 amino acid substitutions, about 25 amino acid substitutions to about 160 amino acid substitutions, about 25 amino acid substitutions to about 155 amino acid substitutions, about 25 amino acid substitutions to about 150 amino acid substitutions, about 25 amino acid substitutions to about 145 amino acid substitutions, about 25 amino acid substitutions to about 140 amino acid substitutions, about 25 amino acid substitutions to about 135 amino acid substitutions, about 25 amino acid substitutions to about 130 amino acid substitutions, about 25 amino acid substitutions to about 125 amino acid substitutions, about 25 amino acid substitutions to about 120 amino acid substitutions, about 25 amino acid substitutions to about 115 amino acid substitutions, about 25 amino acid substitutions to about 110 amino acid substitutions,about 25 to about 105 amino acid substitutions, about 25 to about 100 amino acid substitutions, about 25 to about 95 amino acid substitutions, about 25 to about 90 amino acid substitutions, about 25 to about 85 amino acid substitutions, about 25 to about 80 amino acid substitutions, about 25 to about 75 amino acid substitutions, about 25 to about 70 amino acid substitutions, about 25 to about 65 amino acid substitutions, about 25 to about 60 amino acid substitutions, about 25 to about 55 amino acid substitutions, about 25 to about 50 amino acid substitutions, about 25 amino acid substitutions amino acid substitutions to about 45 amino acid substitutions, about 25 amino acid substitutions to about 40 amino acid substitutions, about 25 amino acid substitutions to about 35 amino acid substitutions, about 25 amino acid substitutions to about 30 amino acid substitutions, about 30 amino acid substitutions to about 240 amino acid substitutions, about 30 amino acid substitutions to about 235 amino acid substitutions, about 30 amino acid substitutions to about 230 amino acid substitutions, about 30 amino acid substitutions to about 225 amino acid substitutions, about 30 amino acid substitutions to about 220 amino acid substitutions, about 30 amino acid substitutions to about 215 amino acid substitutions, about 30 amino acid substitutions to about 210 amino acid substitutions, about 30 amino acid substitutions to about 205 ... amino acid substitutions to about 200 amino acid substitutions, about 30 amino acid substitutions to about 195 amino acid substitutions, about 30 amino acid substitutions to about 190 amino acid substitutions, about 30 amino acid substitutions to about 185 amino acid substitutions, about 30 amino acid substitutions to about 180 amino acid substitutions, about 30 amino acid substitutions to about 175 amino acid substitutions, about 30 amino acid substitutions to about 170 amino acid substitutions, about 30 amino acid substitutions to about 165 amino acid substitutions, about 30 amino acid substitutions to about 160 amino acid substitutions, about 30 amino acid substitutions to about 155 amino acid substitutions, about 30 amino acid substitutions to about 150 amino acid substitutions, about 30 amino acid substitutions to about 145 amino acid substitutions, about 30 amino acid substitutions to about 140 amino acid substitutions, about 30 amino acid substitutions to about 135 amino acid substitutions, about 30 amino acid substitutions to about 130 amino acid substitutions, about 30 amino acid substitutions to about 125 amino acid substitutions, about 30 amino acid substitutions to about 120 amino acid substitutions, about 30 amino acid substitutions to about 115 amino acid substitutions, about 30 amino acid substitutions to about 110 amino acid substitutions, about 30 amino acid substitutions to about 105 amino acid substitutions, about 30 amino acid substitutions to about 100 amino acid substitutions, about 30 amino acid substitutions to about 95 amino acid substitutions, about 30 amino acid substitutions to about 90 amino acid substitutions, about 30 amino acid substitutions to about 85 amino acid substitutions,About 30 to about 80 amino acid substitutions, about 30 to about 75 amino acid substitutions, about 30 to about 70 amino acid substitutions, about 30 to about 65 amino acid substitutions, about 30 to about 60 amino acid substitutions, about 30 to about 55 amino acid substitutions, about 30 to about 50 amino acid substitutions, about 30 to about 45 amino acid substitutions, about 30 to about 40 amino acid substitutions, about 30 to about 35 amino acid substitutions, about 35 to about 240 amino acid substitutions, about 35 to about 235 amino acid substitutions, about 35 amino acid substitutions amino acid substitutions to about 230 amino acid substitutions, about 35 amino acid substitutions to about 225 amino acid substitutions, about 35 amino acid substitutions to about 220 amino acid substitutions, about 35 amino acid substitutions to about 215 amino acid substitutions, about 35 amino acid substitutions to about 210 amino acid substitutions, about 35 amino acid substitutions to about 205 amino acid substitutions, about 35 amino acid substitutions to about 200 amino acid substitutions, about 35 amino acid substitutions to about 195 amino acid substitutions, about 35 amino acid substitutions to about 190 amino acid substitutions, about 35 amino acid substitutions to about 185 amino acid substitutions, about 35 amino acid substitutions to about 180 amino acid substitutions, about 35 amino acid substitutions to about 175 amino acid substitutions , about 35 amino acid substitutions to about 170 amino acid substitutions, about 35 amino acid substitutions to about 165 amino acid substitutions, about 35 amino acid substitutions to about 160 amino acid substitutions, about 35 amino acid substitutions to about 155 amino acid substitutions, about 35 amino acid substitutions to about 150 amino acid substitutions, about 35 amino acid substitutions to about 145 amino acid substitutions, about 35 amino acid substitutions to about 140 amino acid substitutions, about 35 amino acid substitutions to about 135 amino acid substitutions, about 35 amino acid substitutions to about 130 amino acid substitutions, about 35 amino acid substitutions to about 125 amino acid substitutions, about 35 amino acid substitutions to about 120 amino acid substitutions, about 35 amino acid substitutions to about 115 amino acid substitutions amino acid substitutions, about 35 amino acid substitutions to about 110 amino acid substitutions, about 35 amino acid substitutions to about 105 amino acid substitutions, about 35 amino acid substitutions to about 100 amino acid substitutions, about 35 amino acid substitutions to about 95 amino acid substitutions, about 35 amino acid substitutions to about 90 amino acid substitutions, about 35 amino acid substitutions to about 85 amino acid substitutions, about 35 amino acid substitutions to about 80 amino acid substitutions, about 35 amino acid substitutions to about 75 amino acid substitutions, about 35 amino acid substitutions to about 70 amino acid substitutions, about 35 amino acid substitutions to about 65 amino acid substitutions, about 35 amino acid substitutions to about 60 amino acid substitutions, about 35 amino acid substitutions to about 55 amino acid substitutions,About 35 to about 50 amino acid substitutions, about 35 to about 45 amino acid substitutions, about 35 to about 40 amino acid substitutions, about 40 to about 240 amino acid substitutions, about 40 to about 235 amino acid substitutions, about 40 to about 230 amino acid substitutions, about 40 to about 225 amino acid substitutions, about 40 to about 220 amino acid substitutions, about 40 to about 215 amino acid substitutions, about 40 to about 210 amino acid substitutions, about 40 to about 205 amino acid substitutions, about 40 to about 200 amino acid substitutions substitutions, about 40 amino acid substitutions to about 195 amino acid substitutions, about 40 amino acid substitutions to about 190 amino acid substitutions, about 40 amino acid substitutions to about 185 amino acid substitutions, about 40 amino acid substitutions to about 180 amino acid substitutions, about 40 amino acid substitutions to about 175 amino acid substitutions, about 40 amino acid substitutions to about 170 amino acid substitutions, about 40 amino acid substitutions to about 165 amino acid substitutions, about 40 amino acid substitutions to about 160 amino acid substitutions, about 40 amino acid substitutions to about 155 amino acid substitutions, about 40 amino acid substitutions to about 150 amino acid substitutions, about 40 amino acid substitutions to about 145 amino acid substitutions, about 40 amino acid substitutions to about 140 amino acid substitutions amino acid substitutions, about 40 to about 135 amino acid substitutions, about 40 to about 130 amino acid substitutions, about 40 to about 125 amino acid substitutions, about 40 to about 120 amino acid substitutions, about 40 to about 115 amino acid substitutions, about 40 to about 110 amino acid substitutions, about 40 to about 105 amino acid substitutions, about 40 to about 100 amino acid substitutions, about 40 to about 95 amino acid substitutions, about 40 to about 90 amino acid substitutions, about 40 to about 85 amino acid substitutions, about 40 to about 80 amino acid substitutions substitutions, about 40 amino acid substitutions to about 75 amino acid substitutions, about 40 amino acid substitutions to about 70 amino acid substitutions, about 40 amino acid substitutions to about 65 amino acid substitutions, about 40 amino acid substitutions to about 60 amino acid substitutions, about 40 amino acid substitutions to about 55 amino acid substitutions, about 40 amino acid substitutions to about 50 amino acid substitutions, about 40 amino acid substitutions to about 45 amino acid substitutions, about 45 amino acid substitutions to about 240 amino acid substitutions, about 45 amino acid substitutions to about 235 amino acid substitutions, about 45 amino acid substitutions to about 230 amino acid substitutions, about 45 amino acid substitutions to about 225 amino acid substitutions, about 45 amino acid substitutions to about 220 amino acid substitutions,Approximately 45 to 215 amino acid substitutions, approximately 45 to 210 amino acid substitutions, approximately 45 to 205 amino acid substitutions, approximately 45 to 200 amino acid substitutions, approximately 45 to 195 amino acid substitutions, approximately 45 to 190 amino acid substitutions, approximately 45 to 185 amino acid substitutions, approximately 45 to 180 amino acid substitutions, approximately 45 to 175 amino acid substitutions, approximately 45 to 170 amino acid substitutions, approximately 45 to 165 amino acid substitutions, approximately 45 to 160 amino acid substitutions substitutions, about 45 amino acid substitutions to about 155 amino acid substitutions, about 45 amino acid substitutions to about 150 amino acid substitutions, about 45 amino acid substitutions to about 145 amino acid substitutions, about 45 amino acid substitutions to about 140 amino acid substitutions, about 45 amino acid substitutions to about 135 amino acid substitutions, about 45 amino acid substitutions to about 130 amino acid substitutions, about 45 amino acid substitutions to about 125 amino acid substitutions, about 45 amino acid substitutions to about 120 amino acid substitutions, about 45 amino acid substitutions to about 115 amino acid substitutions, about 45 amino acid substitutions to about 110 amino acid substitutions, about 45 amino acid substitutions to about 105 amino acid substitutions, about 45 amino acid substitutions to about 100 amino acid substitutions amino acid substitutions, about 45 to about 95 amino acid substitutions, about 45 to about 90 amino acid substitutions, about 45 to about 85 amino acid substitutions, about 45 to about 80 amino acid substitutions, about 45 to about 75 amino acid substitutions, about 45 to about 70 amino acid substitutions, about 45 to about 65 amino acid substitutions, about 45 to about 60 amino acid substitutions, about 45 to about 55 amino acid substitutions, about 45 to about 50 amino acid substitutions, about 50 to about 240 amino acid substitutions, about 50 to about 235 amino acid substitutions, about 50 amino acid substitutions to about 230 amino acid substitutions, about 50 amino acid substitutions to about 225 amino acid substitutions, about 50 amino acid substitutions to about 220 amino acid substitutions, about 50 amino acid substitutions to about 215 amino acid substitutions, about 50 amino acid substitutions to about 210 amino acid substitutions, about 50 amino acid substitutions to about 205 amino acid substitutions, about 50 amino acid substitutions to about 200 amino acid substitutions, about 50 amino acid substitutions to about 195 amino acid substitutions, about 50 amino acid substitutions to about 190 amino acid substitutions, about 50 amino acid substitutions to about 185 amino acid substitutions, about 50 amino acid substitutions to about 180 amino acid substitutions, about 50 amino acid substitutions to about 175 amino acid substitutions,About 50 amino acid substitutions to about 170 amino acid substitutions, about 50 amino acid substitutions to about 165 amino acid substitutions, about 50 amino acid substitutions to about 160 amino acid substitutions, about 50 amino acid substitutions to about 155 amino acid substitutions, about 50 amino acid substitutions to about 150 amino acid substitutions, about 50 amino acid substitutions to about 145 amino acid substitutions, about 50 amino acid substitutions to about 140 amino acid substitutions, about 50 amino acid substitutions to about, 135 amino acid substitutions, about 50 amino acid substitutions to about 130 amino acid substitutions, about 50 amino acid substitutions to about 125 amino acid substitutions, about 50 amino acid substitutions to about 120 amino acid substitutions, about 50 amino acid substitutions to about 115 amino acid substitutions, about 50 amino acid substitutions to about 110 amino acid substitutions, about 50 amino acid substitutions to about 105 amino acid substitutions, about 50 amino acid substitutions to about 100 amino acid substitutions, about 50 amino acid substitutions to about 95 amino acid substitutions, about 50 amino acid substitutions to about 90 amino acid substitutions, about 50 amino acid substitutions to about 85 amino acid substitutions, about 50 amino acid substitutions to about 80 amino acid substitutions, about 50 amino acid substitutions Approximately 75 amino acid substitutions, approximately 50 to approximately 70 amino acid substitutions, approximately 50 to approximately 65 amino acid substitutions, approximately 50 to approximately 60 amino acid substitutions, approximately 50 to approximately 55 amino acid substitutions, approximately 60 to approximately 240 amino acid substitutions, approximately 60 to approximately 235 amino acid substitutions, approximately 60 to approximately 230 amino acid substitutions, approximately 60 to approximately 225 amino acid substitutions, approximately 60 to approximately 220 amino acid substitutions, approximately 60 to approximately 215 amino acid substitutions, approximately 60 to approximately 210 amino acid substitutions, approximately 60 amino acid substitutions about 60 to about 205 amino acid substitutions, about 60 to about 200 amino acid substitutions, about 60 to about 195 amino acid substitutions, about 60 to about 190 amino acid substitutions, about 60 to about 185 amino acid substitutions, about 60 to about 180 amino acid substitutions, about 60 to about 175 amino acid substitutions, about 60 to about 170 amino acid substitutions, about 60 to about 165 amino acid substitutions, about 60 to about 160 amino acid substitutions, about 60 to about 155 amino acid substitutions, about 60 to about 150 amino acid substitutions, about 60 Amino acid substitutions to about 145 amino acid substitutions, about 60 amino acid substitutions to about 140 amino acid substitutions, about 60 amino acid substitutions to about 135 amino acid substitutions, about 60 amino acid substitutions to about 130 amino acid substitutions, about 60 amino acid substitutions to about 125 amino acid substitutions, about 60 amino acid substitutions to about 120 amino acid substitutions, about 60 amino acid substitutions to about 115 amino acid substitutions, about 60 amino acid substitutions to about 110 amino acid substitutions, about 60 amino acid substitutions to about 105 amino acid substitutions, about 60 amino acid substitutions to about 100 amino acid substitutions, about 60 amino acid substitutions to about 95 amino acid substitutions, about 60 amino acid substitutions to about 90 amino acid substitutions,About 60 to about 85 amino acid substitutions, about 60 to about 80 amino acid substitutions, about 60 to about 75 amino acid substitutions, about 60 to about 70 amino acid substitutions, about 60 to about 65 amino acid substitutions, about 70 to about 240 amino acid substitutions, about 70 to about 235 amino acid substitutions, about 70 to about 230 amino acid substitutions, about 70 to about 225 amino acid substitutions, about 70 to about 220 amino acid substitutions, about 70 to about 215 amino acid substitutions, about 70 to about 210 amino acid substitutions amino acid substitutions, about 10 to about 205 amino acid substitutions, about 70 to about 200 amino acid substitutions, about 70 to about 195 amino acid substitutions, about 70 to about 190 amino acid substitutions, about 70 to about 185 amino acid substitutions, about 70 to about 180 amino acid substitutions, about 70 to about 175 amino acid substitutions, about 70 to about 170 amino acid substitutions, about 70 to about 165 amino acid substitutions, about 70 to about 160 amino acid substitutions, about 70 to about 155 amino acid substitutions, about 70 to about Approximately 150 amino acid substitutions, approximately 70 to approximately 145 amino acid substitutions, approximately 70 to approximately 140 amino acid substitutions, approximately 70 to approximately 135 amino acid substitutions, approximately 70 to approximately 130 amino acid substitutions, approximately 70 to approximately 125 amino acid substitutions, approximately 70 to approximately 120 amino acid substitutions, approximately 70 to approximately 115 amino acid substitutions, approximately 70 to approximately 110 amino acid substitutions, approximately 70 to approximately 105 amino acid substitutions, approximately 70 to approximately 100 amino acid substitutions, approximately 70 to approximately 95 ... amino acid substitutions to about 90 amino acid substitutions, about 70 amino acid substitutions to about 85 amino acid substitutions, about 70 amino acid substitutions to about 80 amino acid substitutions, about 70 amino acid substitutions to about 75 amino acid substitutions, about 80 amino acid substitutions to about 240 amino acid substitutions, about 80 amino acid substitutions to about 235 amino acid substitutions, about 80 amino acid substitutions to about 230 amino acid substitutions, about 80 amino acid substitutions to about 225 amino acid substitutions, about 80 amino acid substitutions to about 220 amino acid substitutions, about 80 amino acid substitutions to about 215 amino acid substitutions, about 80 amino acid substitutions to about 210 amino acid substitutions, about 80 amino acid substitutions to about 205 amino acid substitutions,Approximately 80 to 200 amino acid substitutions, approximately 80 to 195 amino acid substitutions, approximately 80 to 190 amino acid substitutions, approximately 80 to 185 amino acid substitutions, approximately 80 to 180 amino acid substitutions, approximately 80 to 175 amino acid substitutions, approximately 80 to 170 amino acid substitutions, approximately 80 to 165 amino acid substitutions, approximately 80 to 160 amino acid substitutions, approximately 80 to 155 amino acid substitutions, approximately 80 to 150 amino acid substitutions, approximately 80 to 145 amino acid substitutions amino acid substitutions, about 80 amino acid substitutions to about 140 amino acid substitutions, about 80 amino acid substitutions to about 135 amino acid substitutions, about 80 amino acid substitutions to about 130 amino acid substitutions, about 80 amino acid substitutions to about 125 amino acid substitutions, about 80 amino acid substitutions to about 120 amino acid substitutions, about 80 amino acid substitutions to about 115 amino acid substitutions, about 80 amino acid substitutions to about 110 amino acid substitutions, about 80 amino acid substitutions to about 105 amino acid substitutions, about 80 amino acid substitutions to about 100 amino acid substitutions, about 80 amino acid substitutions to about 95 amino acid substitutions, about 80 amino acid substitutions to about 90 amino acid substitutions, about 80 amino acid substitutions to about 8 5 amino acid substitutions, about 90 to about 240 amino acid substitutions, about 90 to about 235 amino acid substitutions, about 90 to about 230 amino acid substitutions, about 90 to about 225 amino acid substitutions, about 90 to about 220 amino acid substitutions, about 90 to about 215 amino acid substitutions, about 90 to about 210 amino acid substitutions, about 90 to about 205 amino acid substitutions, about 90 to about 200 amino acid substitutions, about 90 to about 195 amino acid substitutions, about 90 to about 190 amino acid substitutions, about 90 amino acid substitutions amino acid substitutions to about 185 amino acid substitutions, about 90 amino acid substitutions to about 180 amino acid substitutions, about 90 amino acid substitutions to about 175 amino acid substitutions, about 90 amino acid substitutions to about 170 amino acid substitutions, about 90 amino acid substitutions to about 165 amino acid substitutions, about 90 amino acid substitutions to about 160 amino acid substitutions, about 90 amino acid substitutions to about 155 amino acid substitutions, about 90 amino acid substitutions to about 150 amino acid substitutions, about 90 amino acid substitutions to about 145 amino acid substitutions, about 90 amino acid substitutions to about 140 amino acid substitutions, about 90 amino acid substitutions to about 135 amino acid substitutions, about 90 amino acid substitutions to about 130 amino acid substitutions,About 90 to about 125 amino acid substitutions, about 90 to about 120 amino acid substitutions, about 90 to about 115 amino acid substitutions, about 90 to about 110 amino acid substitutions, about 90 to about 105 amino acid substitutions, about 90 to about 100 amino acid substitutions, about 90 to about 95 amino acid substitutions, about 100 to about 240 amino acid substitutions, about 100 to about 235 amino acid substitutions, about 100 to about 230 amino acid substitutions, about 100 to about 225 amino acid substitutions, about 100 amino acid substitutions to about 220 amino acid substitutions, about 100 amino acid substitutions to about 215 amino acid substitutions, about 100 amino acid substitutions to about 210 amino acid substitutions, about 100 amino acid substitutions to about 205 amino acid substitutions, about 100 amino acid substitutions to about 200 amino acid substitutions, about 100 amino acid substitutions to about 195 amino acid substitutions, about 100 amino acid substitutions to about 190 amino acid substitutions, about 100 amino acid substitutions to about 185 amino acid substitutions, about 100 amino acid substitutions to about 180 amino acid substitutions, about 100 amino acid substitutions to about 175 amino acid substitutions, about 100 amino acid substitutions to about 170 amino acid substitutions, about 100 amino acid substitutions to about 165 Amino acid substitution, about 100 to about 160 amino acid substitutions, about 100 to about 155 amino acid substitutions, about 100 to about 150 amino acid substitutions, about 100 to about 145 amino acid substitutions, about 100 to about 140 amino acid substitutions, about 100 to about 135 amino acid substitutions, about 100 to about 130 amino acid substitutions, about 100 to about 125 amino acid substitutions, about 100 to about 120 amino acid substitutions, about 100 to about 115 amino acid substitutions, about 100 to about 110 amino acid substitutions substitutions, about 100 amino acid substitutions to about 105 amino acid substitutions, about 110 amino acid substitutions to about 240 amino acid substitutions, about 110 amino acid substitutions to about 235 amino acid substitutions, about 110 amino acid substitutions to about 230 amino acid substitutions, about 110 amino acid substitutions to about 225 amino acid substitutions, about 110 amino acid substitutions to about 220 amino acid substitutions, about 110 amino acid substitutions to about 215 amino acid substitutions, about 110 amino acid substitutions to about 210 amino acid substitutions, about 110 amino acid substitutions to about 205 amino acid substitutions, about 110 amino acid substitutions to about 200 amino acid substitutions, about 110 amino acid substitutions to about 195 amino acid substitutions,About 110 amino acid substitutions to about 190 amino acid substitutions, about 110 amino acid substitutions to about 185 amino acid substitutions, about 110 amino acid substitutions to about 180 amino acid substitutions, about 110 amino acid substitutions to about 175 amino acid substitutions, about 110 amino acid substitutions to about 170 amino acid substitutions, about 110 amino acid substitutions to about 165 amino acid substitutions, about 110 amino acid substitutions to about 160 amino acid substitutions, about 110 amino acid substitutions to about 155 amino acid substitutions, about 110 amino acid substitutions to about 150 amino acid substitutions, about 110 amino acid substitutions to about 145 amino acid substitutions, about 110 amino acid substitutions to about 14 ...50 amino acid substitutions, about 110 amino acid substitutions to about 145 amino acid substitutions, about 110 amino acid substitutions to about amino acid substitutions to about 135 amino acid substitutions, about 110 amino acid substitutions to about 130 amino acid substitutions, about 110 amino acid substitutions to about 125 amino acid substitutions, about 110 amino acid substitutions to about 120 amino acid substitutions, about 110 amino acid substitutions to about 115 amino acid substitutions, about 120 amino acid substitutions to about 240 amino acid substitutions, about 120 amino acid substitutions to about 235 amino acid substitutions, about 120 amino acid substitutions to about 230 amino acid substitutions, about 120 amino acid substitutions to about 225 amino acid substitutions, about 120 amino acid substitutions to about 220 amino acid substitutions, about 120 amino acid substitutions to about 215 amino acid substitutions, about 120 amino acid substitutions Approximately 210 amino acid substitutions, approximately 120 to approximately 205 amino acid substitutions, approximately 120 to approximately 200 amino acid substitutions, approximately 120 to approximately 195 amino acid substitutions, approximately 120 to approximately 190 amino acid substitutions, approximately 120 to approximately 185 amino acid substitutions, approximately 120 to approximately 180 amino acid substitutions, approximately 120 to approximately 175 amino acid substitutions, approximately 120 to approximately 170 amino acid substitutions, approximately 100 to approximately 165 amino acid substitutions, approximately 120 to approximately 160 amino acid substitutions, approximately 120 to approximately 155 amino acid substitutions amino acid substitutions, about 120 amino acid substitutions to about 150 amino acid substitutions, about 120 amino acid substitutions to about 145 amino acid substitutions, about 120 amino acid substitutions to about 140 amino acid substitutions, about 120 amino acid substitutions to about 135 amino acid substitutions, about 120 amino acid substitutions to about 130 amino acid substitutions, about 120 amino acid substitutions to about 125 amino acid substitutions, about 130 amino acid substitutions to about 240 amino acid substitutions, about 130 amino acid substitutions to about 235 amino acid substitutions, about 130 amino acid substitutions to about 230 amino acid substitutions, about 130 amino acid substitutions to about 225 amino acid substitutions, about 130 amino acid substitutions to about 220 amino acid substitutions,about 130 amino acid substitutions to about 215 amino acid substitutions, about 130 amino acid substitutions to about 210 amino acid substitutions, about 130 amino acid substitutions to about 205 amino acid substitutions, about 130 amino acid substitutions to about 200 amino acid substitutions, about 130 amino acid substitutions to about 195 amino acid substitutions, about 130 amino acid substitutions to about 190 amino acid substitutions, about 130 amino acid substitutions to about 185 amino acid substitutions, about 130 amino acid substitutions to about 180 amino acid substitutions, about 130 amino acid substitutions, about 175 amino acid substitutions, about 130 amino acid substitutions to about 170 amino acid substitutions, about 130 amino acid substitutions to about 165 amino acid substitutions, about 130 amino acid substitutions to about 160 amino acid substitutions, about 130 amino acid substitutions to about 155 amino acid substitutions, about 130 amino acid substitutions to about 150 amino acid substitutions, about 130 amino acid substitutions to about 145 amino acid substitutions, about 130 amino acid substitutions to about 140 amino acid substitutions, about 130 amino acid substitutions to about 135 amino acid substitutions, about 140 amino acid substitutions to about 240 amino acid substitutions, about 140 amino acid substitutions to about 235 amino acid substitutions, about 140 amino acid substitutions to about 23 0 amino acid substitutions, about 140 amino acid substitutions to about 225 amino acid substitutions, about 140 amino acid substitutions to about 220 amino acid substitutions, about 140 amino acid substitutions to about 215 amino acid substitutions, about 140 amino acid substitutions to about 210 amino acid substitutions, about 140 amino acid substitutions to about 205 amino acid substitutions, about 140 amino acid substitutions to about 200 amino acid substitutions, about 140 amino acid substitutions to about 195 amino acid substitutions, about 140 amino acid substitutions to about 190 amino acid substitutions, about 140 amino acid substitutions to about 185 amino acid substitutions, about 140 amino acid substitutions to about 180 amino acid substitutions, about 140 amino acid substitutions to about 175 amino acid substitutions amino acid substitutions, about 140 amino acid substitutions to about 170 amino acid substitutions, about 140 amino acid substitutions to about 165 amino acid substitutions, about 140 amino acid substitutions to about 160 amino acid substitutions, about 140 amino acid substitutions to about 155 amino acid substitutions, about 140 amino acid substitutions to about 150 amino acid substitutions, about 140 amino acid substitutions to about 145 amino acid substitutions, about 150 amino acid substitutions to about 240 amino acid substitutions, about 150 amino acid substitutions to about 235 amino acid substitutions, about 150 amino acid substitutions to about 230 amino acid substitutions, about 150 amino acid substitutions to about 225 amino acid substitutions, about 150 amino acid substitutions to about 220 amino acid substitutions amino acid substitutions, about 150 to about 215 amino acid substitutions, about 150 to about 210 amino acid substitutions, about 150 to about 205 amino acid substitutions, about 150 to about 200 amino acid substitutions, about 150 to about 195 amino acid substitutions, about 150 to about 190 amino acid substitutions, about 150 to about 185 amino acid substitutions, about 150 to about 180 amino acid substitutions, about 150 to about 175 amino acid substitutions, about 150 to about 170 amino acid substitutions, about 150 to about 165 amino acid substitutions,about 150 amino acid substitutions to about 160 amino acid substitutions, or about 150 amino acid substitutions to about 155 amino acid substitutions, about 160 amino acid substitutions to about 240 amino acid substitutions, about 160 amino acid substitutions to about 235 amino acid substitutions, about 160 amino acid substitutions to about 230 amino acid substitutions, about 160 amino acid substitutions to about 225 amino acid substitutions, about 160 amino acid substitutions to about 220 amino acid substitutions, about 160 amino acid substitutions to about 215 amino acid substitutions, about 160 amino acid substitutions to about 210 amino acid substitutions, about 160 amino acid substitutions to about 205 amino acid substitutions, about 160 amino acid substitutions to about 200 amino acid substitutions, about 1 60 to about 195 amino acid substitutions, about 160 to about 190 amino acid substitutions, about 160 to about 185 amino acid substitutions, about 160 to about 180 amino acid substitutions, about 160 to about 175 amino acid substitutions, about 160 to about 170 amino acid substitutions, about 160 to about 165 amino acid substitutions, about 170 to about 240 amino acid substitutions, about 170 to about 235 amino acid substitutions, about 170 to about 230 amino acid substitutions, about 170 to about 225 amino acid substitutions, about 170 amino acid substitutions substitutions to about 220 amino acid substitutions, about 170 amino acid substitutions to about 215 amino acid substitutions, about 170 amino acid substitutions to about 210 amino acid substitutions, about 170 amino acid substitutions to about 205 amino acid substitutions, about 170 amino acid substitutions to about 200 amino acid substitutions, about 170 amino acid substitutions to about 195 amino acid substitutions, about 170 amino acid substitutions to about 190 amino acid substitutions, about 170 amino acid substitutions to about 185 amino acid substitutions, about 170 amino acid substitutions to about 180 amino acid substitutions, about 170 amino acid substitutions to about 175 amino acid substitutions, about 180 amino acid substitutions to about 240 amino acid substitutions, about 180 amino acid substitutions to about 235 amino acid substitutions amino acid substitutions, about 180 amino acid substitutions to about 230 amino acid substitutions, about 180 amino acid substitutions to about 225 amino acid substitutions, about 180 amino acid substitutions to about 220 amino acid substitutions, about 180 amino acid substitutions to about 215 amino acid substitutions, about 180 amino acid substitutions to about 210 amino acid substitutions, about 180 amino acid substitutions to about 205 amino acid substitutions, about 180 amino acid substitutions to about 200 amino acid substitutions, about 180 amino acid substitutions to about 195 amino acid substitutions, about 180 amino acid substitutions to about 190 amino acid substitutions, about 180 amino acid substitutions to about 185 amino acid substitutions, about 190 amino acid substitutions to about 240 amino acid substitutions,about 190 amino acid substitutions to about 235 amino acid substitutions, about 190 amino acid substitutions to about 230 amino acid substitutions, about 190 amino acid substitutions to about 225 amino acid substitutions, about 190 amino acid substitutions to about 220 amino acid substitutions, about 190 amino acid substitutions to about 215 amino acid substitutions, about 190 amino acid substitutions to about 210 amino acid substitutions, about 190 amino acid substitutions to about 205 amino acid substitutions, about 190 amino acid substitutions to about 200 amino acid substitutions, about 190 amino acid substitutions to about 195 amino acid substitutions, about 200 amino acid substitutions to about 240 amino acid substitutions, about 200 amino acid substitutions to about 235 amino acid substitutions, about 2 00 amino acid substitutions to about 230 amino acid substitutions, about 200 amino acid substitutions to about 225 amino acid substitutions, about 200 amino acid substitutions to about 220 amino acid substitutions, about 200 amino acid substitutions to about 215 amino acid substitutions, about 200 amino acid substitutions to about 210 amino acid substitutions, about 200 amino acid substitutions to about 205 amino acid substitutions, about 205 amino acid substitutions to about 240 amino acid substitutions, about 205 amino acid substitutions to about 235 amino acid substitutions, about 205 amino acid substitutions to about 230 amino acid substitutions, about 205 amino acid substitutions to about 225 amino acid substitutions, about 205 amino acid substitutions to about 220 amino acid substitutions, 05 amino acid substitutions to about 215 amino acid substitutions, about 205 amino acid substitutions to about 210 amino acid substitutions, about 210 amino acid substitutions to about 240 amino acid substitutions, about 210 amino acid substitutions to about 235 amino acid substitutions, about 210 amino acid substitutions to about 230 amino acid substitutions, about 210 amino acid substitutions to about 225 amino acid substitutions, about 210 amino acid substitutions to about 220 amino acid substitutions, about 210 amino acid substitutions to about 215 amino acid substitutions, about 215 amino acid substitutions to about 240 amino acid substitutions, about 215 amino acid substitutions to about 235 amino acid substitutions, about 215 amino acid substitutions to about 230 amino acid substitutions, about 215 a wild-type containing up to about 225 amino acid substitutions, up to about 215 amino acid substitutions, up to about 220 amino acid substitutions, up to about 220 amino acid substitutions, up to about 240 amino acid substitutions, up to about 220 amino acid substitutions, up to about 235 amino acid substitutions, up to about 220 amino acid substitutions, up to about 230 amino acid substitutions, up to about 220 amino acid substitutions, up to about 225 amino acid substitutions, up to about 240 amino acid substitutions, up to about 225 amino acid substitutions, up to about 235 amino acid substitutions, up to about 225 amino acid substitutions, up to about 230 amino acid substitutions, up to about 240 amino acid substitutions, up to about 230 amino acid substitutions,The sequence may include the sequence of a full-length otoferlin protein (e.g., wild-type, human, full-length otoferlin protein). One skilled in the art will understand that amino acids that are conserved between wild-type otoferlin proteins from different species are more likely to be involved in activity (than amino acids that are not conserved between different species) and should not be mutated, whereas amino acids that are not conserved between wild-type otoferlin proteins from different species can be mutated without losing activity.

[0230] Active feline proteins include, for example, 1 to about 200 amino acids, 1 to about 195 amino acids, 1 to about 190 amino acids, 1 to about 185 amino acids, 1 to about 180 amino acids, 1 to about 175 amino acids, 1 to about 170 amino acids, 1 to about 165 amino acids, 1 to about 160 amino acids, 1 to about 155 amino acids, 1 to about 150 amino acids, 1 to about 145 amino acids, 1 to about 140 amino acids, 1 to about 135 amino acids, 1 to about 130 amino acids, 1 to about 15 ...0 amino acids, 1 to about 165 amino acids, 1 to about 160 amino acids, 1 to about 150 amino acids, 1 to about 165 amino acids, 1 to about 160 amino acids, 1 to about 160 amino acids, 1 to about 16 About 125 amino acids, 1 to about 120 amino acids, 1 to about 115 amino acids, 1 to about 110 amino acids, 1 to about 105 amino acids, 1 to about 100 amino acids, 1 to about 95 amino acids, 1 to about 90 amino acids, 1 to about 85 amino acids, 1 to about 80 amino acids, 1 to about 75 amino acids, 1 to about 70 amino acids, 1 to about 65 amino acids, 1 to about 60 amino acids, 1 to about 55 amino acids, 1 to about 50 amino acids, 1 to about 45 amino acids, 1 to about 40 amino acids amino acids, 1 to about 35 amino acids, 1 to about 30 amino acids, 1 to about 25 amino acids, 1 to about 20 amino acids, 1 to about 15 amino acids, 1 to about 10 amino acids, 1 to about 9 amino acids, 1 to about 8 amino acids, 1 to about 7 amino acids, 1 to about 6 amino acids, 1 to about 5 amino acids, 1 to about 4 amino acids, 1 to about 3 amino acids, about 2 to about 200 amino acids, about 2 to about 195 amino acids, about 2 to about 190 amino acids, about 2 to about 185 amino acids, about 2 to about 20 amino acids, about 180 amino acids, about 2 amino acids to about 175 amino acids, about 2 amino acids to about 170 amino acids, about 2 amino acids to about 165 amino acids, about 2 amino acids to about 160 amino acids, about 2 amino acids to about 155 amino acids, about 2 amino acids to about 150 amino acids, about 2 amino acids to about 145 amino acids, about 2 amino acids to about 140 amino acids, about 2 amino acids to about 135 amino acids, about 2 amino acids to about 130 amino acids, about 2 amino acids to about 125 amino acids, about 2 amino acids to about 120 amino acids, about 2 amino acids to about 115 amino acids, about 2 amino acids to about 110 amino acids, about 2 amino acids to about 105 amino acids,About 2 to about 100 amino acids, about 2 to about 95 amino acids, about 2 to about 90 amino acids, about 2 to about 85 amino acids, about 2 to about 80 amino acids, about 2 to about 75 amino acids, about 2 to about 70 amino acids, about 2 to about 65 amino acids, about 2 to about 60 amino acids, about 2 to about 55 amino acids, about 2 to about 50 amino acids, about 2 to about 45 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 to about 25 amino acids, about 2 amino acids about 20 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 9 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 7 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 3 amino acids to about 200 amino acids, about 3 amino acids to about 195 amino acids, about 3 amino acids to about 190 amino acids, about 3 amino acids to about 185 amino acids, about 3 amino acids to about 180 amino acids, about 3 amino acids to about 175 amino acids, about 3 amino acids to about 170 amino acids, about 3 amino acids to about 165 amino acids amino acids, about 3 amino acids to about 160 amino acids, about 3 amino acids to about 155 amino acids, about 3 amino acids to about 150 amino acids, about 3 amino acids to about 145 amino acids, about 3 amino acids to about 140 amino acids, about 3 amino acids to about 135 amino acids, about 3 amino acids to about 130 amino acids, about 3 amino acids to about 125 amino acids, about 3 amino acids to about 120 amino acids, about 3 amino acids to about 115 amino acids, about 3 amino acids to about 110 amino acids, about 3 amino acids to about 105 amino acids, about 3 amino acids to about 100 amino acids, about 3 amino acids to about 95 amino acids, about 3 amino acids to about 90 amino acids, about 3 amino acids to about 85 amino acids, about 3 amino acids to about 80 amino acids, about 3 amino acids to about 75 amino acids, about 3 amino acids to about 70 amino acids, about 3 amino acids to about 65 amino acids, about 3 amino acids to about 60 amino acids, about 3 amino acids to about 55 amino acids, about 3 amino acids to about 50 amino acids, about 3 amino acids to about 45 amino acids, about 3 amino acids to about 40 amino acids, about 3 amino acids to about 35 amino acids, about 3 amino acids to about 30 amino acids, about 3 amino acids to about 25 amino acids, about 3 amino acids to about 20 amino acids, about 3 amino acids to about 15 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 9 amino acids,About 3 to about 8 amino acids, about 3 to about 7 amino acids, about 3 to about 6 amino acids, about 3 to about 5 amino acids, about 4 to about 200 amino acids, about 4 to about 195 amino acids, about 4 to about 190 amino acids, about 4 to about 185 amino acids, about 4 to about 180 amino acids, about 4 to about 175 amino acids, about 4 to about 170 amino acids, about 4 to about 165 amino acids, about 4 to about 160 amino acids, about 4 to about 155 amino acids, about 4 to about 150 amino acids, about 4 to about 145 amino acids , about 4 amino acids to about 140 amino acids, about 4 amino acids to about 135 amino acids, about 4 amino acids to about 130 amino acids, about 4 amino acids to about 125 amino acids, about 4 amino acids to about 120 amino acids, about 4 amino acids to about 115 amino acids, about 4 amino acids to about 110 amino acids, about 4 amino acids to about 105 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about 95 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 85 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 75 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 65 amino acids acid, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 55 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 9 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 7 amino acids, about 4 amino acids to about 6 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 195 amino acids, about 5 amino acids to about 190 amino acids, about 5 amino acids to about 185 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 175 amino acids, about 5 amino acids to about 170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids,About 5 to about 115 amino acids, about 5 to about 110 amino acids, about 5 to about 105 amino acids, about 5 to about 100 amino acids, about 5 to about 95 amino acids, about 5 to about 90 amino acids, about 5 to about 85 amino acids, about 5 to about 80 amino acids, about 5 to about 75 amino acids, about 5 to about 70 amino acids, about 5 to about 65 amino acids, about 5 to about 60 amino acids, about 5 to about 55 amino acids, about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 amino acids about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 5 to about 9 amino acids, about 5 to about 8 amino acids, about 5 to about 7 amino acids, about 6 to about 200 amino acids, about 6 to about 195 amino acids, about 6 to about 190 amino acids, about 6 to about 185 amino acids, about 6 to about 180 amino acids, about 6 to about 175 amino acids, about 6 to about 170 amino acids, about 6 to about 16 5 amino acids, about 6 amino acids to about 160 amino acids, about 6 amino acids to about 155 amino acids, about 6 amino acids to about 150 amino acids, about 6 amino acids to about 145 amino acids, about 6 amino acids to about 140 amino acids, about 6 amino acids to about 135 amino acids, about 6 amino acids to about 130 amino acids, about 6 amino acids to about 125 amino acids, about 6 amino acids to about 120 amino acids, about 6 amino acids to about 115 amino acids, about 6 amino acids to about 110 amino acids, about 6 amino acids to about 105 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 95 amino acids, about 6 amino acids to about 9 ... from about 6 amino acids to about 85 amino acids, from about 6 amino acids to about 80 amino acids, from about 6 amino acids to about 75 amino acids, from about 6 amino acids to about 70 amino acids, from about 6 amino acids to about 65 amino acids, from about 6 amino acids to about 60 amino acids, from about 6 amino acids to about 55 amino acids, from about 6 amino acids to about 50 amino acids, from about 6 amino acids to about 45 amino acids, from about 6 amino acids to about 40 amino acids, from about 6 amino acids to about 35 amino acids, from about 6 amino acids to about 30 amino acids, from about 6 amino acids to about 25 amino acids, from about 6 amino acids to about 20 amino acids, from about 6 amino acids to about 15 amino acids, from about 6 amino acids to about 10 amino acids, from about 6 amino acids to about 9 amino acids,About 6 amino acids to about 8 amino acids, about 7 amino acids to about 200 amino acids, about 7 amino acids to about 195 amino acids, about 7 amino acids to about 190 amino acids, about 7 amino acids to about 185 amino acids, about 7 amino acids to about 180 amino acids, about 7 amino acids to about 175 amino acids, about 7 amino acids to about 170 amino acids, about 7 amino acids to about 165 amino acids, about 7 amino acids to about 160 amino acids, about 7 amino acids to about 155 amino acids, about 7 amino acids to about 150 amino acids, about 7 amino acids to about 145 amino acids, about 7 amino acids to about 140 amino acids, about 7 amino acids to about 135 amino acids, about 7 amino acids to about 130 amino acids amino acids, about 7 amino acids to about 125 amino acids, about 7 amino acids to about 120 amino acids, about 7 amino acids to about 115 amino acids, about 7 amino acids to about 110 amino acids, about 7 amino acids to about 105 amino acids, about 7 amino acids to about 100 amino acids, about 7 amino acids to about 95 amino acids, about 7 amino acids to about 90 amino acids, about 7 amino acids to about 85 amino acids, about 7 amino acids to about 80 amino acids, about 7 amino acids to about 75 amino acids, about 7 amino acids to about 70 amino acids, about 7 amino acids to about 65 amino acids, about 7 amino acids to about 60 amino acids, about 7 amino acids to about 55 amino acids, about 7 amino acids to about 50 amino acids, About 7 to about 45 amino acids, about 7 to about 40 amino acids, about 7 to about 35 amino acids, about 7 to about 30 amino acids, about 7 to about 25 amino acids, about 7 to about 20 amino acids, about 7 to about 15 amino acids, about 7 to about 10 amino acids, about 7 to about 9 amino acids, about 8 to about 200 amino acids, about 8 to about 195 amino acids, about 8 to about 190 amino acids, about 8 to about 185 amino acids, about 8 to about 180 amino acids, about 8 to about 175 amino acids, about 8 to about 170 amino acids, about 8 to about 18 from about 8 amino acids to about 165 amino acids, from about 8 amino acids to about 160 amino acids, from about 8 amino acids to about 155 amino acids, from about 8 amino acids to about 150 amino acids, from about 8 amino acids to about 145 amino acids, from about 8 amino acids to about 140 amino acids, from about 8 amino acids to about 135 amino acids, from about 8 amino acids to about 130 amino acids, from about 8 amino acids to about 125 amino acids, from about 8 amino acids to about 120 amino acids, from about 8 amino acids to about 115 amino acids, from about 8 amino acids to about 110 amino acids, from about 8 amino acids to about 105 amino acids, from about 8 amino acids to about 100 amino acids, from about 8 amino acids to about 95 amino acids, from about 8 amino acids to about 90 amino acids,About 8 to about 85 amino acids, about 8 to about 80 amino acids, about 8 to about 75 amino acids, about 8 to about 70 amino acids, about 8 to about 65 amino acids, about 8 to about 60 amino acids, about 8 to about 55 amino acids, about 8 to about 50 amino acids, about 8 to about 45 amino acids, about 8 to about 40 amino acids, amino acids, about 8 to about 35 amino acids, about 8 to about 30 amino acids, about 8 to about 25 amino acids, about 8 to about 20 amino acids, about 8 to about 15 amino acids, about 8 to about 10 amino acids, about 10 to about 200 amino acids, about 10 to about 195 amino acids, about 10 to about 190 amino acids, about 10 to about 185 amino acids, about 10 to about 180 amino acids, about 10 to about 175 amino acids, about 10 to about 170 amino acids, about 10 to about 165 amino acids, about 10 to about 160 amino acids, about 10 Amino acids up to about 155 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 115 amino acids, about 10 amino acids to about 110 amino acids, about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 amino acids , about 10 amino acids to about 80 amino acids, about 10 amino acids to about 75 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 65 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 55 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 200 amino acids, amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to about 145 amino acids, about 15 amino acids to about 140 amino acids, about 15 amino acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15 amino acids to about 125 amino acids,about 15 amino acids to about 120 amino acids, about 15 amino acids to about 115 amino acids, about 15 amino acids to about 110 amino acids, about 15 amino acids to about 105 amino acids, about 15 amino acids to about 100 amino acids, about 15 amino acids to about 95 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 85 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 75 amino acids, about 15 amino acids to about 70 amino acids, about 15 amino acids to about 65 amino acids, about 15 amino acids to about 60 amino acids, about 15 amino acids to about 55 amino acids, about 15 amino acids to about 50 amino acids, about 1 5 amino acids to about 45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, About 20 to about 155 amino acids, about 20 to about 150 amino acids, about 20 to about 145 amino acids, about 20 to about 140 amino acids, about 20 to about 135 amino acids, about 20 to about 130 amino acids, about 20 to about 125 amino acids, about 20 to about 120 amino acids, about 20 to about 115 amino acids, about 20 to about 110 amino acids, about 20 to about 105 amino acids, about 20 to about 100 amino acids, about 20 to about 95 amino acids, about 20 to about 90 amino acids, about 20 to about 85 Amino acids, about 20 to about 80 amino acids, about 20 to about 75 amino acids, about 20 to about 70 amino acids, about 20 to about 65 amino acids, about 20 to about 60 amino acids, about 20 to about 55 amino acids, about 20 to about 50 amino acids, about 20 to about 45 amino acids, about 20 to about 40 amino acids, about 20 to about 35 amino acids, about 20 to about 30 amino acids, about 20 to about 25 amino acids, about 25 to about 200 amino acids, about 25 to about 195 amino acids, about 25 to about 190 amino acids,About 25 amino acids to about 185 amino acids, about 25 amino acids to about 180 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 11 5 amino acids, about 25 amino acids to about 110 amino acids, about 25 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 to about 35 amino acids, about 25 to about 30 amino acids, about 30 to about 200 amino acids, about 30 to about 195 amino acids, about 30 to about 190 amino acids, about 30 to about 185 amino acids, about 30 to about 180 amino acids, about 30 to about 175 amino acids, about 30 to about 170 amino acids, about 30 to about 165 amino acids, about 30 to about 160 amino acids, about 30 to about 155 amino acids, about 30 to about 150 amino acids, about 30 to about 145 amino acids, about 30 to about 140 amino acids amino acids, about 30 to about 135 amino acids, about 30 to about 130 amino acids, about 30 to about 125 amino acids, about 30 to about 120 amino acids, about 30 to about 115 amino acids, about 30 to about 110 amino acids, about 30 to about 105 amino acids, about 30 to about 100 amino acids, about 30 to about 95 amino acids, about 30 to about 90 amino acids, about 30 to about 85 amino acids, about 30 to about 80 amino acids, about 30 to about 75 amino acids, about 30 to about 70 amino acids, about 30 to about 65 amino acids,About 30 to about 60 amino acids, about 30 to about 55 amino acids, about 30 to about 50 amino acids, about 30 to about 45 amino acids, about 30 to about 40 amino acids, about 30 to about 35 amino acids, about 35 to about 200 amino acids, about 35 to about 195 amino acids, about 35 to about 190 amino acids, about 35 to about 185 amino acids, about 35 to about 180 amino acids, about 35 to about 175 amino acids, about 35 to about 170 amino acids, about 35 to about 165 amino acids, about 35 to about 160 amino acids amino acids, about 35 amino acids to about 155 amino acids, about 35 amino acids to about 150 amino acids, about 35 amino acids to about 145 amino acids, about 35 amino acids to about 140 amino acids, about 35 amino acids to about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino acids to about 125 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids, about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 18 ... about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids,About 40 to about 100 amino acids, about 40 to about 95 amino acids, about 40 to about 90 amino acids, about 40 to about 85 amino acids, about 40 to about 80 amino acids, about 40 to about 75 amino acids, about 40 to about 70 amino acids, about 40 to about 65 amino acids, about 40 to about 60 amino acids, about 40 to about 55 amino acids, about 40 to about 50 amino acids, about 40 to about 45 amino acids, about 45 to about 200 amino acids, about 45 to about 195 amino acids, about 45 to about 190 amino acids, about 45 amino acids to about 185 amino acids, about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165 amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to about 155 amino acids, about 45 amino acids to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids, about 45 amino acids to about 120 amino acids, about 45 amino acids to about 115 amino acids, About 45 amino acids to about 110 amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 75 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 65 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 55 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 20 ... to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids,About 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids or more, about 70 amino acids, about 50 to about 65 amino acids, about 50 to about 60 amino acids, about 50 to about 55 amino acids, about 55 to about 200 amino acids, about 55 to about 195 amino acids, about 55 to about 190 amino acids, about 55 to about 185 amino acids, about 55 to about 180 amino acids, about 55 to about 175 amino acids, about 55 to about 170 amino acids, about 55 to about 165 amino acids, about 55 to about 160 amino acids, about 55 to about 155 amino acids, about 55 to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 ... amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids,About 60 amino acids to about 65 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 1 35 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 7 0 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75 amino acids to about 200 amino acids, about 75 amino acids to about 195 amino acids, about 75 amino acids to about 190 amino acids, about 75 amino acids to about 185 amino acids, about 75 amino acids to about 180 amino acids,About 75 amino acids to about 175 amino acids, about 75 amino acids to about 170 amino acids, about 75 amino acids to about 165 amino acids, about 75 amino acids to about 160 amino acids, about 75 amino acids to about 155 amino acids, about 75 amino acids to about 150 amino acids, about 75 amino acids to about 145 amino acids, about 75 amino acids to about 140 amino acids, about 75 amino acids to about 135 amino acids, about 75 amino acids to about 130 amino acids, about 75 amino acids to about 125 amino acids, about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 10 5 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino acids to about 85 amino acids, about 85 amino acids amino acids to about 200 amino acids, about 85 amino acids to about 195 amino acids, about 85 amino acids to about 190 amino acids, about 85 amino acids to about 185 amino acids, about 85 amino acids to about 180 amino acids, about 85 amino acids to about 175 amino acids, about 85 amino acids to about 170 amino acids, about 85 amino acids to about 165 amino acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 155 amino acids, about 85 amino acids to about 150 amino acids, about 85 amino acids to about 145 amino acids, about 85 amino acids to about 140 amino acids, about 85 amino acids to about 135 amino acids, about 85 amino acids to about 130 amino acids,About 85 amino acids to about 125 amino acids, about 85 amino acids to about 120 amino acids, about 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100 amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to about 90 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 17 0 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids amino acids to about 95 amino acids, about 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185 amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95 amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids, about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino acids, about 95 amino acids to about 135 amino acids amino acids, about 95 amino acids to about 130 amino acids, about 95 amino acids to about 125 amino acids, about 95 amino acids to about 120 amino acids, about 95 amino acids to about 115 amino acids, about 95 amino acids to about 110 amino acids, about 95 amino acids to about 105 amino acids, about 95 amino acids to about 100 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids,About 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to about 200 Amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105 amino acids to about 180 amino acids, about 105 amino acids to about 175 amino acids, about 105 amino acids to about 170 amino acids, about 105 amino acids to about 165 amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105 amino acids to about 145 amino acids, about 105 amino acids to about 140 amino acids, about 105 amino acids to about 135 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids about 130 amino acids, about 105 amino acids to about 125 amino acids, about 105 amino acids to about 120 amino acids, about 105 amino acids to about 115 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 Amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids,About 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 15 0 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 Amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids,About 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 Amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 165 amino acids about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 Amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids,about 160 amino acids to about 165 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 1 The sequence may include a wild-type, full-length otoferlin protein (e.g., a wild-type, human, full-length otoferlin protein) having a deletion of 75 to about 190 amino acids, about 175 to about 185 amino acids, about 175 to about 180 amino acids, about 180 to about 200 amino acids, about 180 to about 195 amino acids, about 180 to about 190 amino acids, about 185 to about 200 amino acids, about 185 to about 195 amino acids, about 185 to about 190 amino acids, about 190 to about 200 amino acids, about 190 to about 195 amino acids, or about 195 to about 200 amino acids. In some embodiments where two or more amino acids are deleted from the sequence of the wild-type, full-length otoferlin protein, the two or more deleted amino acids may be contiguous in the sequence of the full-length protein. In other embodiments in which two or more amino acids are deleted from the sequence of the wild-type, full-length otoferlin protein, the two or more deleted amino acids are not contiguous in the sequence of the wild-type, full-length protein. One of skill in the art will understand that amino acids that are conserved between wild-type, full-length otoferlin proteins from different species are more likely to be involved in activity (than amino acids that are not conserved between different species) and should not be deleted, but that amino acids that are not conserved between wild-type, full-length otoferlin proteins from different species can be deleted without loss of activity. In some examples, the active otoferlin protein may be, for example, from 1 to about 100 amino acids, from 1 to about 95 amino acids, from 1 to about 90 amino acids, from 1 to about 85 amino acids, from 1 to about 80 amino acids, from 1 to about 75 amino acids, from 1 to about 70 amino acids, from 1 to about 65 amino acids, from 1 to about 60 amino acids, from 1 to about 55 amino acids, from 1 to about 50 amino acids, from 1 to about 45 amino acids, from 1 to about 40 amino acids, from 1 to about 35 amino acids, from 1 to about 30 amino acids, or from 1 to about 25 amino acids. amino acids, 1 to about 20 amino acids, 1 to about 15 amino acids, 1 to about 10 amino acids, 1 to about 9 amino acids, 1 to about 8 amino acids, 1 to about 7 amino acids, 1 to about 6 amino acids, 1 to about 5 amino acids, 1 to about 4 amino acids, 1 to about 3 amino acids, about 2 to about 100 amino acids, about 2 to about 95 amino acids, about 2 to about 90 amino acids, about 2 to about 85 amino acids, about 2 to about 80 amino acids, about 2 to about 75 amino acids, about 2 to about 70 amino acids, about 2 to about 10 About 65 amino acids, about 2 to about 60 amino acids, about 2 to about 55 amino acids, about 2 to about 50 amino acids, about 2 to about 45 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 to about 25 amino acids, about 2 to about 20 amino acids, about 2 to about 15 amino acids, about 2 to about 10 amino acids, about 2 to about 9 amino acids, about 2 to about 8 amino acids, about 2 to about 7 amino acids, about 2 to about 6 amino acids, about 2 to about 5 amino acids, about 2 amino acids about 4 amino acids, about 3 amino acids to about 100 amino acids, about 3 amino acids to about 95 amino acids, about 3 amino acids to about 90 amino acids, about 3 amino acids to about 85 amino acids, about 3 amino acids to about 80 amino acids, about 3 amino acids to about 75 amino acids, about 3 amino acids to about 70 amino acids, about 3 amino acids to about 65 amino acids, about 3 amino acids to about 60 amino acids, about 3 amino acids to about 55 amino acids, about 3 amino acids to about 50 amino acids, about 3 amino acids to about 45 amino acids, about 3 amino acids to about 40 amino acids, about 3 amino acids to about 35 amino acids, about 3 amino acids to about 30 amino acids, about 3 amino acids to about 25 amino acids,About 3 to about 20 amino acids, about 3 to about 15 amino acids, about 3 to about 10 amino acids, about 3 to about 9 amino acids, about 3 to about 8 amino acids, about 3 to about 7 amino acids, about 3 to about 6 amino acids, about 3 to about 5 amino acids, about 4 to about 100 amino acids, about 4 to about 95 amino acids, about 4 to about 90 amino acids, about 4 to about 85 amino acids, about 4 to about 80 amino acids, about 4 to about 75 amino acids, about 4 to about 70 amino acids, about 4 to about 65 amino acids, about 4 to about 60 amino acids acid, about 4 amino acids to about 55 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 9 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 7 amino acids, about 4 amino acids to about 6 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 95 amino acids, about 5 amino acids to about 90 Amino acids, about 5 to about 85 amino acids, about 5 to about 80 amino acids, about 5 to about 75 amino acids, about 5 to about 70 amino acids, about 5 to about 65 amino acids, about 5 to about 60 amino acids, about 5 to about 55 amino acids, about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 7 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 95 amino acids, about 6 amino acids to about 90 amino acids, about 6 amino acids to about 85 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids to about 75 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 65 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 55 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids,About 6 to about 30 amino acids, about 6 to about 25 amino acids, about 6 to about 20 amino acids, about 6 to about 15 amino acids, about 6 to about 10 amino acids, about 6 to about 9 amino acids, about 6 to about 8 amino acids, about 7 to about 100 amino acids, about 7 to about 95 amino acids, about 7 to about 90 amino acids, about 7 to about 85 amino acids, about 7 to about 80 amino acids, about 7 to about 75 amino acids, about 7 to about 70 amino acids, about 7 to about 65 amino acids, about 7 to about 60 amino acids, about 7 to about 5 5 amino acids, about 7 to about 50 amino acids, about 7 to about 45 amino acids, about 7 to about 40 amino acids, about 7 to about 35 amino acids, about 7 to about 30 amino acids, about 7 to about 25 amino acids, about 7 to about 20 amino acids, about 7 to about 15 amino acids, about 7 to about 10 amino acids, about 7 to about 9 amino acids, about 8 to about 100 amino acids, about 8 to about 95 amino acids, about 8 to about 90 amino acids, about 8 to about 85 amino acids, about 8 to about 80 amino acids, about 8 to about 75 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 65 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 55 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 150 amino acids about 85 amino acids, about 10 to about 80 amino acids, about 10 to about 75 amino acids, about 10 to about 70 amino acids, about 10 to about 65 amino acids, about 10 to about 60 amino acids, about 10 to about 55 amino acids, about 10 to about 50 amino acids, about 10 to about 45 amino acids, about 10 to about 40 amino acids, about 10 to about 35 amino acids, about 10 to about 30 amino acids, about 10 to about 25 amino acids, about 10 to about 20 amino acids, about 10 to about 15 amino acids, about 20 to about 100 amino acids,About 20 to about 95 amino acids, about 20 to about 90 amino acids, about 20 to about 85 amino acids, about 20 to about 80 amino acids, about 20 to about 75 amino acids, about 20 to about 70 amino acids, about 20 to about 65 amino acids, about 20 to about 60 amino acids, about 20 to about 55 amino acids, about 20 to about 50 amino acids, about 20 to about 45 amino acids, about 20 to about 40 amino acids, about 20 to about 35 amino acids, about 20 to about 30 amino acids, about 20 to about 25 amino acids, about 30 to about 100 amino acids, about 30 to about 95 amino acids, about 30 to about 90 amino acids, about 30 to about 85 amino acids, about 30 to about 80 amino acids, about 30 to about 75 amino acids, about 30 to about 70 amino acids, about 30 to about 65 amino acids, about 30 to about 60 amino acids, about 30 to about 55 amino acids, about 30 to about 50 amino acids, about 30 to about 45 amino acids, about 30 to about 40 amino acids, about 30 to about 35 amino acids, about 40 to about 100 amino acids, about 40 to about 95 amino acids, About 40 to about 90 amino acids, about 40 to about 85 amino acids, about 40 to about 80 amino acids, about 40 to about 75 amino acids, about 40 to about 70 amino acids, about 40 to about 65 amino acids, about 40 to about 60 amino acids, about 40 to about 55 amino acids, about 40 to about 50 amino acids, about 40 to about 45 amino acids, about 50 to about 100 amino acids, about 50 to about 95 amino acids, about 50 to about 90 amino acids, about 50 to about 85 amino acids, about 50 to about 80 amino acids, about 50 to about about 75 amino acids, about 50 to about 70 amino acids, about 50 to about 65 amino acids, about 50 to about 60 amino acids, about 50 to about 55 amino acids, about 60 to about 100 amino acids, about 60 to about 95 amino acids, about 60 to about 90 amino acids, about 60 to about 85 amino acids, about 60 to about 80 amino acids, about 60 to about 75 amino acids, about 60 to about 70 amino acids, about 60 to about 65 amino acids, about 70 to about 100 amino acids, about 70 to about 95 amino acids, about 70 to about 90 amino acids,It may comprise the sequence of a wild-type, full-length otoferlin protein with about 70 to about 85 amino acids, about 70 to about 80 amino acids, about 70 to about 75 amino acids, about 80 to about 100 amino acids, about 80 to about 95 amino acids, about 80 to about 90 amino acids, about 80 to about 85 amino acids, about 90 to about 100 amino acids, about 90 to about 95 amino acids, or about 95 to about 100 amino acids removed from its N-terminus and / or its C-terminus. In some embodiments, the active otoferlin protein may be, for example, from 1 to 50 amino acids, from 1 to 45 amino acids, from 1 to 40 amino acids, from 1 to 35 amino acids, from 1 to 30 amino acids, from 1 to 25 amino acids, from 1 to 20 amino acids, from 1 to 15 amino acids, from 1 to 10 amino acids, from 1 to 9 amino acids, from 1 to 8 amino acids, from 1 to 7 amino acids, from 1 to 6 amino acids, from 1 to 5 amino acids, from 1 to 4 amino acids, from 1 to 3 amino acids, from about 2 amino acids to 50 amino acids, from about 2 amino acids to 50 amino acids, or from about 2 amino acids to 50 amino acids. about 2 to 45 amino acids, about 2 to 40 amino acids, about 2 to 35 amino acids, about 2 to 30 amino acids, about 2 to 25 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, about 2 to 9 amino acids, about 2 to 8 amino acids, about 2 to 7 amino acids, about 2 to 6 amino acids, about 2 to 5 amino acids, about 2 to 4 amino acids, about 3 to 50 amino acids, about 3 to 45 amino acids, about 3 to 40 amino acids, about 3 to 35 amino acids, about 3 to 30 amino acids, about 3 to 25 amino acids, about 3 to 20 amino acids, about 3 to 15 amino acids, about 3 to 10 amino acids, about 3 to 9 amino acids, about 3 to 8 amino acids, about 3 to 7 amino acids, about 3 to 6 amino acids, about 3 to 5 amino acids, about 4 to 50 amino acids, about 4 to 45 amino acids, about 4 to 40 amino acids, about 4 to 35 amino acids, about 4 to 30 amino acids, about 4 to 25 amino acids, about 4 to 20 amino acids, about 4 to 15 amino acids, about 4 to 10 amino acids Amino acids, about 4 to 9 amino acids, about 4 to 8 amino acids, about 4 to 7 amino acids, about 4 to 6 amino acids, about 5 to 50 amino acids, about 5 to 45 amino acids, about 5 to 40 amino acids, about 5 to 35 amino acids, about 5 to 30 amino acids, about 5 to 25 amino acids, about 5 to 20 amino acids, about 5 to 15 amino acids, about 5 to 10 amino acids, about 5 to 9 amino acids, about 5 to 8 amino acids, about 5 to 7 amino acids, about 6 to 50 amino acids, about 6 to 45 amino acids,About 6 to 40 amino acids, about 6 to 35 amino acids, about 6 to 30 amino acids, about 6 to 25 amino acids, about 6 to 20 amino acids, about 6 to 15 amino acids, about 6 to 10 amino acids, about 6 to 9 amino acids, about 6 to 8 amino acids, about 7 to 50 amino acids, about 7 to 45 amino acids, about 7 to 40 amino acids, about 7 to 35 amino acids, about 7 to 30 amino acids, about 7 to 25 amino acids, about 7 to 20 amino acids, about 7 to 15 amino acids, about 7 amino acids about 7 to 9 amino acids, about 8 to 50 amino acids, about 8 to 45 amino acids, about 8 to 40 amino acids, about 8 to 35 amino acids, about 8 to 30 amino acids, about 8 to 25 amino acids, about 8 to 20 amino acids, about 8 to 15 amino acids, about 8 to 10 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 Amino acids to 20 amino acids, about 10 to 15 amino acids, about 15 to 50 amino acids, about 15 to 45 amino acids, about 15 to 40 amino acids, about 15 to 35 amino acids, about 15 to 30 amino acids, about 15 to 25 amino acids, about 15 to 20 amino acids, about 20 to 50 amino acids, about 20 to 45 amino acids, about 20 to 40 amino acids, about 20 to 35 amino acids, about 20 to 30 amino acids, about 20 to 25 amino acids, about 25 to 50 amino acids, about 25 amino acids The inserted amino acids may comprise a sequence of a wild-type, full-length otoferlin protein into which about 25 to 45 amino acids, about 25 to 40 amino acids, about 25 to 35 amino acids, about 25 to 30 amino acids, about 30 to 50 amino acids, about 30 to 45 amino acids, about 30 to 40 amino acids, about 30 to 35 amino acids, about 35 to 50 amino acids, about 35 to 45 amino acids, about 35 to 40 amino acids, about 40 to 50 amino acids, about 40 to 45 amino acids, or about 45 to 50 amino acids.The inserted amino acids may be inserted as a contiguous sequence into the sequence of the full-length protein. In some cases, the inserted amino acids are not inserted as a contiguous sequence into the sequence of the wild-type, full-length protein. As can be understood in the art, amino acids may be inserted into portions of the sequence of the wild-type, full-length protein that are not highly conserved between species.

[0231] Ranges: Throughout this disclosure, various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the range.

[0232] In one aspect, the present disclosure provides a recombinant AAV vector of SEQ ID NO: 96. In one aspect, the present disclosure provides a recombinant AAV vector of SEQ ID NO: 105.

[0233] In one aspect, the disclosure provides a recombinant AAV vector comprising, in 5' to 3' order, a CAG promoter comprising the 5' ITR sequence of SEQ ID NO: 97, a CMV early enhancer element of SEQ ID NO: 98, a chicken beta actin gene sequence of SEQ ID NO: 99, and a chimeric intron of SEQ ID NO: 100, a 5' OTOF coding region comprising exons 1 to 21 of the OTOF cDNA, an SD intron sequence of SEQ ID NO: 102, an AK recombination-inducing sequence of SEQ ID NO: 103, and a 3' ITR sequence of SEQ ID NO: 104. In some embodiments, the 5' OTOF coding region is SEQ ID NO: 101. In some embodiments, the 5'OTOF coding region is at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or 100% identical to SEQ ID NO:101 and encodes the same amino acid sequence as that encoded by SEQ ID NO:101.

[0234] In one aspect, the disclosure provides a recombinant AAV vector comprising, in 5' to 3' order, the 5' ITR sequence of SEQ ID NO: 97, an AK recombinogenic sequence of SEQ ID NO: 103, an SA intron sequence of SEQ ID NO: 106, a 3' OTOF coding region comprising exon 22 through exon 48 of the OTOF cDNA, a bgH polyA sequence of SEQ ID NO: 108, and a 3' ITR sequence of SEQ ID NO: 104. In some embodiments, the 3' OTOF coding region is SEQ ID NO: 107. In some embodiments, the 3' OTOF coding region is at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or 100% identical to SEQ ID NO: 107 and encodes the same amino acid sequence as that encoded by SEQ ID NO: 107.

[0235] In one aspect, the present disclosure provides an rAAV particle comprising one of the aforementioned recombinant AAV vectors encapsidated by an Anc80 capsid. In some embodiments, the Anc80 capsid comprises the polypeptide of SEQ ID NO: 109.

[0236] In one aspect, the present disclosure provides a composition comprising a first rAAV particle comprising a recombinant AAV vector of SEQ ID NO: 96 and a second rAAV particle comprising a recombinant AAV vector of SEQ ID NO: 105. In some embodiments, the recombinant AAV vector of the first rAAV particle is encapsidated by an Anc80 capsid. In some embodiments, the recombinant AAV vector of the second rAAV particle is encapsidated by an Anc80 capsid. In some embodiments, the recombinant AAV vectors of the first and second rAAV particles are each independently encapsidated by an Anc80 capsid. In some embodiments, the Anc80 capsid comprises a polypeptide of SEQ ID NO: 109.

[0237] In one aspect, the disclosure provides a method for producing an rAAV particle comprising: (a) a first rAAV particle comprising a recombinant AAV vector comprising, in 5' to 3' order: a CAG promoter comprising the 5' ITR sequence of SEQ ID NO: 97, a CMV early enhancer element of SEQ ID NO: 98, a chicken beta actin gene sequence of SEQ ID NO: 99, and a chimeric intron of SEQ ID NO: 100; a 5' OTOF coding region comprising exons 1 to 21 of the OTOF cDNA; an SD intron sequence of SEQ ID NO: 102; an AK recombinogenic sequence of SEQ ID NO: 103; and a 3' ITR sequence of SEQ ID NO: 104; and (b) a first rAAV particle comprising: a CAG promoter comprising, in 5' to 3' order: a 5' ITR sequence of SEQ ID NO: 97, a CMV early enhancer element of SEQ ID NO: 98, a chicken beta actin gene sequence of SEQ ID NO: 99, and a chimeric intron of SEQ ID NO: 100; a 5' OTOF coding region comprising exons 1 to 21 of the OTOF cDNA; an SD intron sequence of SEQ ID NO: 102; an AK recombinogenic sequence of SEQ ID NO: 103; an SA intron sequence of SEQ ID NO: 106; and a 3' ITR sequence of SEQ ID NO: 104. and a second rAAV particle comprising a recombinant AAV vector comprising a 3' OTOF coding region comprising exon 22 through exon 48 of the cDNA, the bgH polyA sequence of SEQ ID NO: 108, and the 3' ITR sequence of SEQ ID NO: 104. In some embodiments, the 5' OTOF coding region is SEQ ID NO: 101. In some embodiments, the 5' OTOF coding region is at least 70% identical to SEQ ID NO: 101 and encodes the same amino acid sequence as SEQ ID NO: 101. In some embodiments, the 3' OTOF coding region is SEQ ID NO: 107. In some embodiments, the 3' OTOF coding region is at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or 100% identical to SEQ ID NO: 107 and encodes the same amino acid sequence as that encoded by SEQ ID NO: 107. In some embodiments, the recombinant AAV vectors in the first and second rAAV particles are each independently encapsidated by an Anc80 capsid. In some embodiments, the Anc80 capsid comprises the polypeptide of SEQ ID NO: 109.

[0238] In one aspect, when the above-mentioned composition is introduced into a human cell, the recombinant AAV vectors of the first and second rAAV particles undergo concatemerization or homologous recombination with each other, thereby forming a recombinant nucleic acid encoding the full-length otoferlin protein within the cell.

[0239] In one aspect, the present disclosure provides a method comprising introducing a therapeutically effective amount of any of the aforementioned compositions into the cochlea of ​​a mammal (e.g., a human). In some embodiments, the mammal has been previously identified as having a defective otoferlin gene.

[0240] In one aspect, the present disclosure provides a method of increasing expression of full-length otoferlin protein in a mammalian cell, the method comprising introducing any of the aforementioned compositions into the mammalian cell, e.g., an inner hair cell, e.g., a human cell. In some embodiments, the mammalian cell has previously been determined to have a defective otoferlin gene.

[0241] In one aspect, the present disclosure provides a method of increasing expression of full-length otoferlin protein in inner hair cells of the cochlea of ​​a mammal, e.g., a human, comprising introducing a therapeutically effective amount of any of the aforementioned compositions into the cochlea of ​​the mammal. In some embodiments, the mammal has previously been identified as having a defective otoferlin gene.

[0242] In one aspect, the present disclosure provides a method for treating asymptomatic sensorineural hearing loss in a subject, e.g., a human, identified as having a defective otoferlin gene, comprising administering a therapeutically effective amount of any of the aforementioned compositions to the subject's cochlea. In some embodiments, the method further comprises determining that the subject has a defective otoferlin gene prior to the administering step. In some embodiments, the composition is administered to the cochlea using a microcatheter. In some embodiments, the microcatheter is shaped to enter the middle ear cavity via the ear canal and allow the end of the microcatheter to contact the RWM. In some embodiments, the distal end of the microcatheter comprises at least one microneedle having a diameter of 10 to 1,000 microns.

[0243] In one aspect, the present disclosure provides a kit comprising any of the aforementioned compositions. In some embodiments, the composition is preloaded into a device, such as a microcatheter. In some embodiments, the microcatheter is shaped to allow entry into the middle ear cavity via the ear canal and contact of the end of the microcatheter with the RWM. In some embodiments, the distal end of the microcatheter comprises at least one microneedle having a diameter of 10 to 1,000 microns.

[0244] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials for use in this disclosure are described herein, but other suitable methods and materials known in the art can also be used.Materials, methods, and examples are only illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In the event of any discrepancy, the present specification, including definitions, shall prevail. DETAILED DESCRIPTION OF THE INVENTION

[0245] Detailed Description Mutations in OTOF result in DFNB9, a recessively inherited, non-syndromic prelingual hearing loss. Deficiency of otoferlin, the protein encoded by OTOF, abolishes rapid exocytosis from auditory inner hair cells (IHCs). Due to impaired neurotransmission at the first auditory synapse, sound signals are not transmitted to the brain, resulting in profound hearing loss.

[0246] It has been discovered that the presently claimed methods result in expression of full-length otoferlin in inner auditory hair cells and successfully restore hearing in otoferlin knockout mice, with ABR thresholds of 30 dB to 70 dB for click stimuli and 50 dB to 90 dB for pure tones. In light of this discovery, compositions and methods are provided herein for treating asymptomatic sensorineural hearing loss in subjects (e.g., humans) identified as having a defective otoferlin gene.

[0247] Provided herein is a composition comprising at least two different nucleic acid vectors, each of which contains a coding sequence encoding a different portion of an otoferlin protein, each of which is at least 30 amino acid residues in length, and the amino acid sequence of each of the encoded portions optionally overlapping with the amino acid sequence of a different one of the encoded portions, wherein no single vector of the at least two different vectors encodes an active otoferlin protein (e.g., a full-length otoferlin protein), and wherein at least one of the coding sequences contains a nucleotide sequence spanning two adjacent exons of otoferlin genomic DNA and lacks intronic sequence between the two adjacent exons, and wherein, upon introduction into a mammalian cell, the at least two different vectors undergo homologous recombination with each other, thereby forming a recombinant nucleic acid, which encodes an active otoferlin protein (e.g., a full-length otoferlin protein). In some examples, the recombinant nucleic acid encoding the active otoferlin protein (e.g., a full-length otoferlin protein) is present as an episome in the mammalian cell (e.g., any of the types of mammalian cells described herein). Also provided are kits comprising any of the compositions described herein. Also provided herein are methods comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​a mammal.

[0248] Also provided herein is a method for increasing expression of active otoferlin protein (e.g., full-length otoferlin protein) in mammalian cells, the method comprising introducing any of the compositions described herein into the mammalian cells. Also provided herein is a method for increasing expression of active otoferlin protein (e.g., full-length otoferlin protein) in inner hair cells of a mammalian cochlea, the method comprising introducing a therapeutically effective amount of any of the compositions described herein into the mammalian cochlea. Also provided herein is a method for treating asymptomatic sensorineural hearing loss in a subject identified as having a defective otoferlin gene, the method comprising administering a therapeutically effective amount of any of the compositions described herein to the subject's cochlea.

[0249] Additional non-limiting aspects of the compositions, kits, and methods are described herein and may be used in any combination without limitation.

[0250] Otoferlin The human OTOF gene, in some embodiments, encodes otoferlin, a protein that plays a key role in priming, fusion, and / or replenishment of synaptic vesicles at inner hair cell synapses during sound coding. To date, hundreds of mutations in the human OTOF gene have been identified that cause severe prelingual hearing loss (DFNB9). Such mutations are responsible for hearing loss in 2–8% of individuals with autosomal recessive nonsyndromic hearing loss at birth across 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). Biallelic otoferlin gene mutations cause local synaptic defects between hair cells and the auditory nerve. Otoferlin allows sensory cells to release neurotransmitters in response to acoustic stimuli, activating auditory neurons, which then transmit electronically encoded acoustic information to the brain, resulting in "hearing." When biallelic mutations in OTOF are present, transmission is impaired, and as a result, the majority of subjects suffer from congenital severe to profound sensorineural hearing loss. For example, two substitutions at positions 490 and 515 of exon 15 in 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, in OTOF in a Spanish subject with prelingual nonsyndromic hearing loss (Migliosi et al. (2002) J. Med. Genet. 39(7): 502-506).

[0251] Further exemplary mutations in the otoferlin gene detected in subjects with hearing loss 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.

[0252] Otoacoustic emissions from DFNB9 subjects are normal up to at least 10 years of age, indicating the morphological integrity of the inner ear and proper function of the outer hair cells. Apart from the lack of synaptic transmission and subsequent synapse loss, the morphology and physiology of the inner ear remain preserved in DFNB9 up to at least 10 years of age in humans. Thus, in some embodiments, restoration of OTOF and / or otoferlin function may function to alleviate or prevent secondary degeneration of one or more cochlear structures.

[0253] Studies in mouse models have revealed that synapses are structurally normal and that IHCs preserve a normal number of synapses within the first postnatal week in mice. Approximately half of the synapses are lost between P6 and P15 (Roux et al. (2006) Cell 127 277-289). Animal models have made it possible to study the effects of mutations in otoferlin on synaptic transmission by recording changes in plasma membrane capacitance after vesicle fusion and activity in the auditory nerve. In otoferlin knockout (Otof- / -) mice, there was little exocytosis in IHCs that could be triggered by depolarization-induced Ca2+ influx through voltage-gated Ca2+ channels (Roux et al. (2006) Cell 127 277-289). In severely hearing-impaired pachanga (OtofPga / Pga) mice, which harbor a random point mutation in the C2F domain, brief (<10 ms) depolarization of IHCs induced vesicle fusion of similar size to wild-type mice, whereas sustained stimulation revealed a profound lack of vesicle replenishment relative 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 reduced speech comprehension and temperature-dependent hearing loss (Varga et al. (2006) J Med Genet 43 576-581), revealed an intermediate phenotype when studied in mouse models (Strenzke et al. (2016) EMBO J. 35:2519-2535). These OtofI515T / I515T mice showed moderately elevated hearing thresholds and a reduction in wave I amplitude as assessed by ABR, but behavioral testing and single auditory neuron recordings showed normal hearing thresholds. RRP exocytosis was again intact, although sustained exocytosis was reduced, albeit less severely than in OtofPga / Pga.At room temperature, wild-type mice can fuse 750 vesicles per second in each active field during sustained stimulation; this rate is reduced to 350 vesicles / second / active field in OtofI515T / I515T mice and 200 vesicles / second / active field in OtofPga / Pga IHCs (Pangrsic et al. (2010) Nat. Neurosci. 13 869-876, Strenzke et al. (2016) EMBO J. 35 2519-2535). This correlates with low otoferlin protein levels in the plasma membrane of IHCs, indicating that otoferlin levels fluctuate with exocytosis and hearing (Strenzke et al. (2016) EMBO J. 35 2519-2535).

[0254] Methods for detecting mutations in genes are well known in the art. Non-limiting examples of such techniques include real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.

[0255] The OTOF gene encodes otoferlin, a protein involved in synaptic vesicle exocytosis in cochlear hair cells (see, e.g., Johnson and Chapman (2010) J. Cell Biol. 191(1):187-198 and Heidrych et al. (2008) Hum. Mol. Genet. 17:3814-3821).

[0256] The human OTOF gene is located on chromosome 2p23.3. It contains 48 exons covering approximately 132 kilobases (kb) (NCBI accession number NG009937.1). The mRNA encoding the long form of otoferlin expressed in the brain contains 48 exons (Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000). Forward and reverse primers that can be used to amplify each of the 48 exons in the OTOF gene are listed in Table 2 of Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000. In some cases, the full-length OTOF protein is a full-length wild-type OTOF protein. The full-length wild-type OTOF protein expressed from the human OTOF gene is 1997 residues long.

[0257] An exemplary human wild-type otoferlin protein is or includes the sequence of any one of SEQ ID NOS: 1-5. Human otoferlin protein isoform e (SEQ ID NOS: 5) is encoded by an mRNA that includes exon 48 but not exon 47 of the otoferlin gene (Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000). In some embodiments, an active otoferlin protein has the sequence of SEQ ID NOS: 5 but lacks the 20 amino acids that comprise the RXR motif identified in Strenzke et al., EMBO J. 35(23):2499-2615, 2016. Non-limiting examples of nucleic acids encoding wild-type otoferlin proteins are or include any one of SEQ ID NOS: 7-11. As would be understood in the art, at least some or all of the codons in SEQ ID NOS: 7-11 can be codon-optimized to enable optimal expression in non-human mammals or humans. Orthologues of the human otoferlin protein are known in the art.

[0258] cDNA sequence of human otoferlin protein: Human canonical (long) isoform sequence (otoferlin protein) (SEQ ID NO: 1) (also called otoferlin isoform a) (NCBI accession number AAD26117.1) Human isoform 2 (short chain 1) (otoferlin protein) (SEQ ID NO: 2) (also called otoferlin isoform d) (NCBI accession number NP_919304.1) Human isoform 3 (short chain 2) (otoferlin protein) (SEQ ID NO: 3) (also called otoferlin isoform c) (NCBI accession number NP_919303.1) Human isoform 4 (short chain 3) (otoferlin protein) (SEQ ID NO: 4) (also called otoferlin isoform b) (NCBI accession number NP_004793.2) Human isoform 5 (short chain 4) (otoferlin protein) (SEQ ID NO: 5) (also called otoferlin isoform e) (NCBI accession number NP_001274418.1) Complete cd(otoferlin cDNA) (www.ncbi.nlm.nih.gov / nuccore / AF107403.1) (SEQ ID NO: 6) (encoding the protein of SEQ ID NO: 1) Human otoferlin transcript variant 1 (www.ncbi.nlm.nih.gov / nuccore / NM_194248.2) (SEQ ID NO: 7) (encoding the protein of SEQ ID NO: 1) Human otoferlin transcript variant 2 (www.ncbi.nlm.nih.gov / nuccore / NM_004802.3) (SEQ ID NO: 8) (encoding the protein of SEQ ID NO: 4) Human otoferlin transcript variant 3 (www.ncbi.nlm.nih.gov / nuccore / NM_194322.2) (SEQ ID NO: 9) (encoding the protein of SEQ ID NO: 3) Human otoferlin transcript variant 4 (www.ncbi.nlm.nih.gov / nuccore / NM_194323.2) (SEQ ID NO: 10) (encoding 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)

[0259] A non-limiting example of a human wild-type otoferlin genomic DNA sequence is SEQ ID NO: 12. The exons in SEQ ID NO: 12 are nucleotide positions 5001-5206 (exon 1), nucleotide positions 25925-25983 (exon 2), nucleotide positions 35779-35867 (exon 3), nucleotide positions 44590-44689 (exon 4), nucleotide positions 47100-47281 (exon 5), nucleotide positions 59854-59927 (exon 6), nucleotide positions 61273-61399 (exon 7), nucleotide positions 61891-61945 (exon 8), and nucleotide positions 61961-61968 (exon 9). 8626-68757 (exon 9), nucleotide positions 73959-74021 (exon 10), nucleotide positions 74404-74488 (exon 11), nucleotide positions 79066-79225 (exon 12), nucleotide positions 80051-80237 (exon 13), nucleotide positions 81107-81293 (exon 14), nucleotide positions 82690-82913 (exon 15), nucleotide positions 83388-83496 (exon 16), nucleotide positions 84046-84226 (exon 17). 7), nucleotide positions 84315-84435 (exon 18), nucleotide positions 85950-86050 (exon 19), nucleotide positions 86193-86283 (exon 20), nucleotide positions 86411-86527 (exon 21), nucleotide positions 86656-86808 (exon 22), nucleotide positions 87382-87571 (exon 23), nucleotide positions 87661-87785 (exon 24), nucleotide positions 88206-88340 (exon 25), nucleotide positions 8902-8903 5-89186 (exon 26), nucleotide positions 89589-89708 (exon 27), nucleotide positions 90132-90293 (exon 28), nucleotide positions 90405-90567 (exon 29), nucleotide positions 91050-91180 (exon 30), nucleotide positions 92549-92578 (exon 31), nucleotide positions 92978-93106 (exon 32), nucleotide positions 95225-95291 (exon 33), nucleotide positions 96198-96334 (exon 34),Nucleotide positions 96466-96600 (exon 35), nucleotide positions 96848-96985 (exon 36), nucleotide positions 97623-97750 (exon 37), nucleotide positions 97857-98027 (exon 38), nucleotide positions 98670-98830 (exon 39), nucleotide positions 99593-99735 (exon 40), nucleotide positions 100128-100216 (exon 41), nucleotide position 1 nucleotide positions 101518-101616 (exon 42), nucleotide positions 101762-102003 (exon 43), nucleotide positions 102669-102847 (exon 44), nucleotide positions 102952-103052 (exon 45), nucleotide positions 103494-103691 (exon 46), nucleotide positions 105479-106496 (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).

[0260] Introns are located between each consecutive pair of exons of SEQ ID NO:12, i.e., nucleotide positions 100-5001 (intron 1), nucleotides 5207-25924 (intron 2), nucleotide positions 25984-35778 (intron 3), nucleotide positions 35868-44589 (intron 4), nucleotide positions 44690-47099 (intron 5), nucleotide positions 47282-59853 (intron 6), nucleotide positions 59928-61272 (intron 7), nucleotide positions 61400-61890 (intron 8), and nucleotide positions 61400-61890 (intron 9). intron 8), nucleotide positions 61946-68625 (intron 9), nucleotide positions 68758-73958 (intron 10), nucleotide positions 74022-74403 (intron 11), nucleotide positions 74489-79065 (intron 12), nucleotide positions 79226-80050 (intron 13), nucleotide positions 80238-81106 (intron 14), nucleotide positions 81294-82689 (intron 15), nucleotide positions 82914-83387 (intron 16), nucleotide positions Positions 83497-84045 (intron 17), nucleotide positions 84227-84314 (intron 18), nucleotide positions 84436-85949 (intron 19), nucleotide positions 86051-86192 (intron 20), nucleotide positions 86284-86410 (intron 21), nucleotide positions 86528-86655 (intron 22), nucleotide positions 86809-87381 (intron 23), nucleotide positions 87572-87660 (intron 24), nucleotide positions 87786-882 05 (intron 25), nucleotide positions 88341-89024 (intron 26), nucleotide positions 89187-89588 (intron 27), nucleotide positions 89709-90131 (intron 28), nucleotide positions 90294-90404 (intron 29), nucleotide positions 90568-91049 (intron 30), nucleotide positions 91181-92548 (intron 31), nucleotide positions 92579-92977 (intron 32), nucleotide positions 93107-95224 (intron 33),Nucleotide positions 95292-96197 (intron 34), nucleotide positions 96335-96465 (intron 35), nucleotide positions 96601-96847 (intron 36), nucleotide positions 96986-97622 (intron 37), nucleotide positions 97751-97856 (intron 38), nucleotide positions 98028-98669 (intron 39), nucleotide positions 98831-99592 (intron 40), nucleotide positions 99736-100127 (intron 41), nucleotide positions 100217-101517 (intron 42), nucleotide positions 101617-101761 (intron 43), nucleotide positions 102004-102668 (intron 44), nucleotide positions 102848-102951 (intron 45), nucleotide positions 103053-103494 (intron 46), nucleotide positions 103692-105478 (intron 47), and nucleotide positions 106497-108496 (intron 48).

[0261] In some embodiments, the otoferlin gene may be split into two or more segments between or within any suitable exons and / or introns, with each segment being contained in a different vector of the present disclosure. In some embodiments, the otoferlin gene is split at exon 21, i.e., exons 1 through 21 in the first vector and exons 22 through 48 in 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, i.e., exons 1 through 21 in the first vector and exons 22 through 48 in the second vector, each vector lacking the otoferlin intron. In some embodiments, the otoferlin gene may be split by one or more other exons and / or introns, so long as the packaging capacity of the vector is not exceeded when combined with all other components 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 Canine 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.ncbi.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) Bovine topoferrin protein (SEQ ID NO: 22) Baboon otoferlin protein (SEQ ID NO: 23)

[0262] In some embodiments, the first vector comprises a 5' portion of the OTOF cDNA, e.g., as set forth in SEQ ID NO: 94. In some embodiments, the second vector comprises a 3' portion of the OTOF cDNA, e.g., as set forth in SEQ ID NO: 95. [ka] [ka] [ka] [ka]

[0263] Some embodiments of any of the compositions described herein may include a first vector that includes the coding sequence of SEQ ID NO: 94 (or includes a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, 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 that includes the coding sequence of SEQ ID NO: 95 (or includes a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 95).

[0264] Some embodiments of any of the compositions described herein may include a first vector having a 5' OTOF coding region comprising exons 1 through 21 of the OTOF cDNA. Some embodiments of any of the compositions described herein may include a first vector comprising the nucleotide sequence of SEQ ID NO:101 (or a sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:101). Some embodiments of any of the compositions described herein may include a first vector comprising a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 101 and encodes the same amino acid sequence as SEQ ID NO: 101. In some embodiments, the compositions of the present disclosure include a first vector comprising a codon-optimized version of SEQ ID NO: 101, i.e., a nucleotide sequence that encodes the same amino acid sequence as SEQ ID NO: 101 but with codons that are optimized for expression in a particular cell type, e.g., mammalian cells, e.g., human cells. In some embodiments, the first vector does not include any other portion of the OTOF gene. In some embodiments, the first vector does not include any other portion of the OTOF cDNA.

[0265] Some embodiments of any of the compositions described herein may include a second vector having a 3' OTOF coding region comprising exon 22 through exon 48 of the OTOF cDNA. Some embodiments of any of the compositions described herein may include a second vector comprising the nucleotide sequence of SEQ ID NO: 108 (or a sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 108). Some embodiments of any of the compositions described herein may include a second vector comprising a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 108 and encodes the same amino acid sequence as SEQ ID NO: 108. In some embodiments, the compositions of the present disclosure include a second vector comprising a codon-optimized version of SEQ ID NO: 108, i.e., a nucleotide sequence that encodes the same amino acid sequence as SEQ ID NO: 108 but with codons that are optimized for expression in a particular cell type, e.g., mammalian cells, e.g., human cells. In some embodiments, the second vector does not include any other portion of the OTOF gene. In some embodiments, the second vector does not include any other portion of the OTOF cDNA.

[0266] vector Provided herein are compositions and methods of use for treating diseases such as non-syndromic hearing loss using nucleic acid therapeutics, such as auditory polypeptide messenger RNA.Preferably, the auditory polypeptide nucleic acid is present in a viral vector, such as an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, and a retroviral vector.

[0267] The foregoing methods for packaging a recombinant vector into a desired AAV capsid to produce the rAAV (or rAAV particles) of the present disclosure are not meant to be limiting, and other suitable methods will be apparent to those of skill in the art.

[0268] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including promoters that are natural, constitutive, inducible, and / or tissue-specific, are known in the art and can be used.

[0269] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be "operably" linked when they are covalently linked in such a manner that the expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. When it is desired that the nucleic acid sequence be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to induce transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably linked to a nucleic acid sequence if it is capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into a desired protein or polypeptide. Similarly, two or more coding regions are operably linked if they are linked in such a manner that their transcription from a common promoter results in the expression of two or more proteins that are translated in frame. In some embodiments, the operably linked coding sequences result in a fusion protein. In some embodiments, the operably linked coding sequences result in a functional RNA (e.g., an shRNA, miRNA, or miRNA inhibitor).

[0270] For nucleic acids encoding proteins, a polyadenylation sequence is generally inserted following the transgene sequence and before the 3'AAV ITR sequence. rAAV constructs useful in the present disclosure may also contain an intron, desirably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40 T intron sequence. One possible intron sequence has the nucleotide sequence of SEQ ID NO: 100. Another vector element that can be used is an internal ribosome entry site (IRES). IRES sequences are used to produce more than one polypeptide from a single gene transcript. IRES sequences will be used to produce proteins containing more than one polypeptide chain. The selection of these and other common vector elements is routine, and many such sequences are available [see, e.g., Sambrook et al. "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989) and references cited therein, e.g., pages 3.18-3.26 and 16.17-16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, the foot-and-mouth disease virus 2A sequence is included in the polyprotein, which is a small peptide (approximately 18 amino acids in length) that has been shown to mediate polyprotein cleavage (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459).The cleavage activity of 2A sequences has previously been demonstrated in artificial systems, including plasmids and gene therapy vectors (AAV and retrovirus) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; pp. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931; and Klump, H et al., Gene Therapy, 2001;8:811-817).

[0271] The exact nature of the regulatory sequences required for gene expression in host cells may vary between species, tissues, or cell types, but will generally include, as necessary, 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, enhancer elements, and the like. In particular, such 5' non-transcribed regulatory sequences will include promoter regions, including promoter sequences for transcriptional control of operably linked genes. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired. Vectors of the present disclosure may also include 5' leader or signal sequences, as appropriate. The selection and design of appropriate vectors is within the ability and discretion of one of ordinary skill in the art.

[0272] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al. (1985) Cell, 41:521-530], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter (Gilham et al., J. Gene Med. 12(2):129-136, 2010), and the EF1α promoter [Invitrogen].

[0273] Inducible promoters allow gene expression to be regulated, and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological conditions, such as acute phase, specific differentiation state of cells, or only in replicating cells. Inducible promoters and induction systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionine (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. (1996) Proc. Natl. Acad. Sci. USA, 93:3346-3351), the tetracycline-repressible system (Gossen et al. Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al. Science, 268:1766-1769 (1995)), and the tetracycline-inducible system (Harvey et al. Curr. Opin. Chem. Biol., 2:512-518 (1995)). (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)). Still other types of inducible promoters that may be useful in this context include those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of the cell, or in replicating cells only.

[0274] In another embodiment, the native promoter of the transgene will be used. The native promoter may be preferred when it is desired that the expression of the transgene mimics the native expression. The native promoter can be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic the native expression.

[0275] In some embodiments, the regulatory sequence confers tissue-specific gene expression. In some cases, the tissue-specific regulatory sequence binds to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, 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; Hioki 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 (α-MHC) promoter, PDGF promoter (Patterna, Gene Ther. 7(15):1304-1311, 2000; Hioki et al., Gene Ther. 14:872-882, 2007), MecP2 promoter (Rastegar 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), nβ2 promoter (Xu et al., Human Gene Ther. 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. Other exemplary promoters include, among others, the beta-actin promoter, the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996), the alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)), the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998)), the immunoglobulin heavy chain promoter, the T cell receptor alpha chain promoter, and the neuronal, e.g., neuron-specific enolase (NSE) promoter (Andersen et al., J. Immunol., 161:1063-8 (1998)). 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. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), which will be apparent to those skilled in the art.

[0276] In some embodiments, one or more binding sites for one or more miRNAs are incorporated into a transgene in an rAAV vector to inhibit transgene expression in one or more tissues of a transgene-carrying subject. Those skilled in the art will understand that binding sites can be selected to control transgene expression in a tissue-specific manner. For example, a binding site for the liver-specific miR-122 can be incorporated into a transgene to inhibit its expression in the liver. Target sites in an mRNA can be located in the 5' UTR, 3' UTR, or coding region. Typically, target sites are located in the 3' UTR of an mRNA. Furthermore, transgenes can be designed so that multiple miRNAs regulate mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites can result in the cooperative action of multiple RISCs, providing highly efficient inhibition of expression. The target site sequence can contain a total of 5 to 100, 10 to 60, or more nucleotides. The target site sequence can include at least 5 nucleotides of the target gene binding site sequence.

[0277] Auditory Polypeptides and Auditory Polypeptide Transgene Coding Sequences The composition of the auditory polypeptide transgene sequence of the rAAV vector depends on the use for which the resulting vector is produced. Thus, the present disclosure encompasses the delivery of rAAV vectors encoding one or more auditory polypeptides, peptides, or proteins useful for treating or preventing disease states associated with hearing loss in mammalian subjects. Exemplary therapeutic proteins include otoferin, Ca v 1.3, bassoon, piccolo, ribeye, and harmonin; a scaffold protein selected from the group consisting of Vglut3, synaptotagmin, vesicle binding / docking protein, vesicle priming protein, vesicle fusion protein, GluA2 / 3, or GluA4.

[0278] Optionally, a polypeptide reporter protein is included in the AAV composition.Reporter sequences that can be provided in transgenes include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.When associated with the regulatory elements that drive their expression, reporter sequences provide signals that can be detected by conventional means, including enzymes, autoradiography, colorimetry, fluorescence or other spectrophotometric assays, fluorescence-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry.For example, if the marker sequence is the LacZ gene, the presence of a vector carrying a signal can be detected by assaying for β-galactosidase activity.If the transgene is green fluorescent protein or luciferase, a vector carrying a signal can be visually measured by color or light generation in a luminometer. Such reporters may be useful, for example, to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of rAAV.

[0279] In another example, the transgene encodes a protein or functional RNA that is intended to be used to generate an animal model of a disease. Suitable transgene coding sequences will be apparent to those skilled in the art.

[0280] The rAAV vector may contain a gene or portion of a gene encoding an auditory polypeptide that is to be transferred to a subject to treat a disease associated with reduced expression, lack of expression, or dysfunction of the auditory polypeptide gene or another gene in the functional pathway of the auditory polypeptide.

[0281] In a first aspect, a therapeutic composition is provided, comprising a plurality of adeno-associated virus (AAV) vectors, wherein the plurality of AAV vectors are capable of constructing auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition is administered. Preferably, the plurality of AAV vectors are capable of constructing full-length auditory polypeptide messenger RNA in target cells of the human subject to which the therapeutic composition is administered. The plurality of AAV vectors comprises a first AAV vector and a second AAV vector, wherein the first and second AAV vectors independently contain a packaging capacity of less than about 6 kb. The auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group consisting of otoferlin and its orthologs or homologs, as provided herein. The AAV vector contains at least one promoter sequence selected from the CBA, CMV, or CB7 promoter, or one or more cochlear-specific promoters. In some embodiments, the AAV vector may include a CMV enhancer and promoter sequence, for example, SEQ ID NO: 70. In some embodiments, an AAV vector may comprise a CMV enhancer and a chicken beta-actin (CBA) promoter, e.g., SEQ ID NO: 61. In some embodiments, an AAV vector may comprise a CMVd promoter sequence, e.g., SEQ ID NO: 86. In some embodiments, an AAV vector may comprise a CMV enhancer and a CBA promoter, e.g., a promoter comprising the nucleotide sequences of SEQ ID NOs: 98 and 99. In some embodiments, the nucleotide sequence of SEQ ID NO: 98 precedes the nucleotide sequence of SEQ ID NO: 99, optionally followed by a chimeric intron, e.g., the nucleotide sequence of SEQ ID NO: 100.

[0282] The compositions provided herein include at least two (e.g., two, three, four, five, or six) nucleic acid vectors, each of the at least two different vectors comprising a coding sequence encoding a different portion of an otoferlin protein, each of the encoded portions being at least 30 amino acids (e.g., from about 30 amino acids to about 1950 amino acids, from about 30 amino acids to about 1900 amino acids, from about 30 amino acids to about 1850 amino acids, from about 30 amino acids to about 1800 amino acids, from about 30 amino acids to about 1750 amino acids, from about 30 amino acids to about 1700 amino acids). amino acids, about 30 amino acids to about 1650 amino acids, about 30 amino acids to about 1600 amino acids, about 30 amino acids to about 1550 amino acids, about 30 amino acids to about 1500 amino acids, about 30 amino acids to about 1450 amino acids, about 30 amino acids to about 1400 amino acids, about 30 amino acids to about 1350 amino acids, about 30 amino acids to about 1300 amino acids, about 30 amino acids to about 1250 amino acids, about 30 amino acids to about 1200 amino acids, about 30 amino acids to about 1150 amino acids, about 30 amino acids to about 1100 amino acids, about 30 amino acids to about 1050 amino acids, about 30 amino acids to about 1000 amino acids, about 30 to about 950 amino acids, about 30 to about 900 amino acids, about 30 to about 850 amino acids, about 30 to about 800 amino acids, about 30 to about 750 amino acids, about 30 to about 700 amino acids, about 30 to about 650 amino acids, about 30 to about 600 amino acids, about 30 to about 550 amino acids, about 30 to about 500 amino acids, about 30 to about 450 amino acids, about 30 to about 400 amino acids, about 30 to about 350 amino acids, about 30 to about 300 amino acids , about 30 amino acids to about 250 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 1950 amino acids, about 50 amino acids to about 1900 amino acids, about 50 amino acids to about 1850 amino acids, about 50 amino acids to about 1800 amino acids, about 50 amino acids to about 1750 amino acids, about 50 amino acids to about 1700 amino acids, about 50 amino acids to about 1650 amino acids, about 50 amino acids to about 1600 amino acids, about 50 amino acids to about 1550 amino acids,About 50 amino acids to about 1500 amino acids, about 50 amino acids to about 1450 amino acids, about 50 amino acids to about 1400 amino acids, about 50 amino acids to about 1350 amino acids, about 50 amino acids to about 1300 amino acids, about 50 amino acids to about 1250 amino acids, about 50 amino acids to about 1200 amino acids, about 50 amino acids to about 1150 amino acids, about 50 amino acids to about 1100 amino acids, about 50 amino acids to about 1050 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids , about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 to about 1950 amino acids, about 100 to about 1900 amino acids, about 100 to about 1850 amino acids, about 100 to about 1800 amino acids, about 100 to about 1750 amino acids, about 100 to about 1700 amino acids, about 100 to about 1650 amino acids, about 100 to about 1600 amino acids, about 100 to about 1550 amino acids, about 100 to about 1500 amino acids, about 100 to about 1450 amino acids, about 100 to about 1400 amino acids, about 100 to about 13 50 amino acids, about 100 to about 1300 amino acids, about 100 to about 1250 amino acids, about 100 to about 1200 amino acids, about 100 to about 1150 amino acids, about 100 to about 1100 amino acids, about 100 to about 1050 amino acids, about 100 to about 1000 amino acids, about 100 to about 950 amino acids, about 100 to about 900 amino acids, about 100 to about 850 amino acids, about 100 to about 800 amino acids, about 100 to about 750 amino acids, about 100 to about 700 amino acids,About 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 1950 amino acids, about 150 amino acids to about 1900 amino acids, about 150 amino acids to about 185 0 amino acids, about 150 amino acids to about 1800 amino acids, about 150 amino acids to about 1750 amino acids, about 150 amino acids to about 1700 amino acids, about 150 amino acids to about 1650 amino acids, about 150 amino acids to about 1600 amino acids, about 150 amino acids to about 1550 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1450 amino acids, about 150 amino acids to about 1400 amino acids, about 150 amino acids to about 1350 amino acids, about 150 amino acids to about 1300 amino acids, about 150 amino acids to about 1250 amino acids, about 150 amino acids to about 1200 amino acids amino acids, about 150 amino acids to about 1150 amino acids, about 150 amino acids to about 1100 amino acids, about 150 amino acids to about 1050 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 5 ...650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 1950 amino acids, about 200 amino acids to about 1900 amino acids, about 200 amino acids to about 1850 amino acids, about 200 amino acids to about 1800 amino acids, about 200 amino acids to about 1750 amino acids, about 200 amino acids to about 1700 amino acids, about 200 amino acids to about 1650 amino acids,About 200 amino acids to about 1600 amino acids, about 200 amino acids to about 1550 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1450 amino acids, about 200 amino acids to about 1400 amino acids, about 200 amino acids to about 1350 amino acids, about 200 amino acids to about 1300 amino acids, about 200 amino acids to about 1250 amino acids, about 200 amino acids to about 1200 amino acids, about 200 amino acids to about 1150 amino acids, about 200 amino acids to about 1100 amino acids, about 200 amino acids to about 1050 amino acids, about 200 amino acids to about 1000 amino acids , about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 30 0 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 1950 amino acids, about 250 amino acids to about 1900 amino acids, about 250 amino acids to about 1850 amino acids, about 250 amino acids to about 1800 amino acids, about 250 amino acids to about 1750 amino acids, about 250 amino acids to about 1700 amino acids, about 250 amino acids to about 1650 amino acids, about 250 amino acids to about 1600 amino acids, about 250 amino acids to about 1550 amino acids, about 250 amino acids to about 1500 amino acids, about 250 amino acids to about 1450 amino acids, about 250 amino acids to about 1400 Amino acids, about 250 amino acids to about 1350 amino acids, about 250 amino acids to about 1300 amino acids, about 250 amino acids to about 1250 amino acids, about 250 amino acids to about 1200 amino acids, about 250 amino acids to about 1150 amino acids, about 250 amino acids to about 1100 amino acids, about 250 amino acids to about 1050 amino acids, about 250 amino acids to about 1000 amino acids, about 250 amino acids to about 950 amino acids, about 250 amino acids to about 900 amino acids, about 250 amino acids to about 850 amino acids, about 250 amino acids to about 800 amino acids, about 250 amino acids to about 750 amino acids,About 250 amino acids to about 700 amino acids, about 250 amino acids to about 650 amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 550 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 450 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 350 amino acids, about 250 amino acids to about 300 amino acids, about 300 amino acids to about 1950 amino acids, about 300 amino acids to about 1900 amino acids, about 300 amino acids to about 1850 amino acids, about 300 amino acids to about 1800 amino acids, about 300 amino acids about 1750 amino acids, about 300 amino acids to about 1700 amino acids, about 300 amino acids to about 1650 amino acids, about 300 amino acids to about 1600 amino acids, about 300 amino acids to about 1550 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 1450 amino acids, about 300 amino acids to about 1400 amino acids, about 300 amino acids to about 1350 amino acids, about 300 amino acids to about 1300 amino acids, about 300 amino acids to about 1250 amino acids, about 300 amino acids to about 1200 amino acids, about 300 amino acids to about 1150 amino acids, about 300 amino acids amino acids to about 1100 amino acids, about 300 amino acids to about 1050 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids amino acids, about 300 to about 400 amino acids, about 300 to about 350 amino acids, about 350 to about 1950 amino acids, about 350 to about 1900 amino acids, about 350 to about 1850 amino acids, about 350 to about 1800 amino acids, about 350 to about 1750 amino acids, about 350 to about 1700 amino acids, about 350 to about 1650 amino acids, about 350 to about 1600 amino acids, about 350 to about 1550 amino acids, about 350 to about 1500 amino acids, about 350 to about 1450 amino acids,About 350 amino acids to about 1400 amino acids, about 350 amino acids to about 1350 amino acids, about 350 amino acids to about 1300 amino acids, about 350 amino acids to about 1250 amino acids, about 350 amino acids to about 1200 amino acids, about 350 amino acids to about 1150 amino acids, about 350 amino acids to about 1100 amino acids, about 350 amino acids to about 1050 amino acids, about 350 amino acids to about 1000 amino acids, about 350 amino acids to about 950 amino acids, about 350 amino acids to about 900 amino acids, about 350 amino acids to about 850 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 1950 amino acids, about 400 amino acids to about 1 900 amino acids, about 400 amino acids to about 1850 amino acids, about 400 amino acids to about 1800 amino acids, about 400 amino acids to about 1750 amino acids, about 400 amino acids to about 1700 amino acids, about 400 amino acids to about 1650 amino acids, about 400 amino acids to about 1600 amino acids, about 400 amino acids to about 1550 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1450 amino acids, about 400 amino acids to about 1400 amino acids, about 400 amino acids to about 1350 amino acids, about 400 amino acids to about 1300 amino acids, about 400 amino acids to about 1250 amino acids, about 400 to about 1200 amino acids, about 400 to about 1150 amino acids, about 400 to about 1100 amino acids, about 400 to about 1050 amino acids, about 400 to about 1000 amino acids, about 400 to about 950 amino acids, about 400 to about 900 amino acids, about 400 to about 850 amino acids, about 400 to about 800 amino acids, about 400 to about 750 amino acids, about 400 to about 700 amino acids, about 400 to about 650 amino acids, about 400 to about 600 amino acids amino acids, about 400 to about 550 amino acids, about 400 to about 500 amino acids, about 400 to about 450 amino acids, about 450 to about 1950 amino acids, about 450 to about 1900 amino acids, about 450 to about 1850 amino acids, about 450 to about 1800 amino acids, about 450 to about 1750 amino acids, about 450 to about 1700 amino acids, about 450 to about 1650 amino acids, about 450 to about 1600 amino acids, about 450 to about 1550 amino acids, about 450 to about 1500 amino acids,About 450 amino acids to about 1450 amino acids, about 450 amino acids to about 1400 amino acids, about 450 amino acids to about 1350 amino acids, about 450 amino acids to about 1300 amino acids, about 450 amino acids to about 1250 amino acids, about 450 amino acids to about 1200 amino acids, about 450 amino acids to about 1150 amino acids, about 450 amino acids to about 1100 amino acids, about 450 amino acids to about 1050 amino acids, about 450 amino acids to about 1000 amino acids, about 450 amino acids to about 950 amino acids, about 450 amino acids to about 900 amino acids, about 450 amino acids to about 850 amino acids, about 45 0 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 1950 amino acids, about 500 amino acids to about 1900 amino acids, about 500 amino acids to about 1850 amino acids, about 500 amino acids to about 1800 amino acids, about 500 amino acids to about 1750 amino acids, about 500 amino acids to about 1700 amino acids, about 500 amino acids to about 1650 amino acids, about 500 amino acids to about 1600 amino acids, about 500 amino acids to about 1550 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1450 amino acids, about 500 amino acids to about 1400 amino acids, about 500 amino acids to about 1350 amino acids, about 500 amino acids to about 1300 amino acids, about 500 amino acids to about 1250 amino acids, about 500 amino acids to about 1200 amino acids, about 500 amino acids to about 1150 amino acids, about 500 amino acids to about 1100 amino acids, about 500 amino acids to about 1050 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 5...

Claims

1. Recombinant AAV vector of SEQ ID NO:

96.

2. Recombinant AAV vector of SEQ ID NO:

105.

3. In the order from 5' to 3', 5' ITR sequence of SEQ ID NO: 97; A CAG promoter comprising a CMV early enhancer element of SEQ ID NO: 98, a chicken beta actin gene sequence of SEQ ID NO: 99, and a chimeric intron of SEQ ID NO: 100; the 5' OTOF coding region, including exons 1 to 21 of the OTOF cDNA; SD intron sequence of SEQ ID NO: 102; an AK recombinogenic sequence of SEQ ID NO: 103; and the 3' ITR sequence of SEQ ID NO: 104; A recombinant AAV vector comprising:

4. The recombinant AAV vector of claim 3, wherein the 5'OTOF coding region is SEQ ID NO:

101.

5. 4. The recombinant AAV vector of claim 3, wherein the 5'OTOF coding region is at least 70%, 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%, at least 99.5%, or at least 99.9% identical to SEQ ID NO:101 and encodes the same amino acid sequence as that encoded by SEQ ID NO:

101.

6. In the order from 5' to 3', 5' ITR sequence of SEQ ID NO: 97; an AK recombinogenic sequence of SEQ ID NO: 103; and SA intron sequence of SEQ ID NO: 106; and the 3' OTOF coding region, including exon 22 through exon 48 of the OTOF cDNA; bgH polyA sequence of SEQ ID NO: 108; the 3' ITR sequence of SEQ ID NO: 104; A recombinant AAV vector comprising:

7. The recombinant AAV vector of claim 6, wherein the 3'OTOF coding region is SEQ ID NO:

107.

8. 7. The recombinant AAV vector of claim 6, wherein the 3'OTOF coding region is at least 70%, 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%, at least 99.5%, or at least 99.9% identical to SEQ ID NO:107 and encodes the same amino acid sequence as that encoded by SEQ ID NO:

107.

9. A recombinant AAV particle (rAAV particle) comprising the recombinant AAV vector of any one of claims 1 to 8 encapsidated by an Anc80 capsid.

10. The rAAV particle of claim 9, wherein the Anc80 capsid comprises a polypeptide of SEQ ID NO:

109.

11. A composition comprising a first rAAV particle comprising the recombinant AAV vector of claim 1 and a second rAAV particle comprising the recombinant AAV vector of claim 2.

12. A composition comprising a first rAAV particle comprising a recombinant AAV vector described in any one of claims 3 to 5, and a second rAAV particle comprising a recombinant AAV vector described in any one of claims 6 to 8.

13. 13. The composition of claim 11 or 12, wherein the first and second rAAV particles are each independently encapsidated by an Anc80 capsid.

14. The composition of claim 13, wherein the Anc80 capsid comprises the polypeptide of SEQ ID NO:

109.

15. The composition of any one of claims 11 to 14, wherein when the composition is introduced into a human cell, the recombinant AAV vector of SEQ ID NO: 96 and the recombinant vector of SEQ ID NO: 105 undergo concatemerization or homologous recombination with each other, thereby forming a recombinant nucleic acid encoding a full-length otoferlin protein in the cell.

16. The composition described in claim 12, wherein when the composition is introduced into a human cell, the recombinant AAV vector described in any one of claims 3 to 4 and the recombinant AAV vector described in any one of claims 6 to 7 undergo concatemer formation or homologous recombination with each other, thereby forming a recombinant nucleic acid encoding a full-length otoferlin protein in the cell.

17. The composition of any one of claims 11 to 16, further comprising at least one pharmaceutically acceptable excipient.

18. The composition of any one of claims 11 to 17, wherein the composition is for introducing a therapeutically effective amount into the cochlea of ​​a mammal.

19. 19. The composition of claim 18, wherein the mammal is a human.

20. the mammal has previously been identified as having a defective otoferlin gene; 20. The composition of claim 18 or 19.

21. The composition according to any one of claims 11 to 20, which is a composition for increasing the expression of full-length otoferlin protein in mammalian cells.

22. 22. The composition of claim 21, wherein the mammalian cell is an inner hair cell.

23. 23. The composition of claim 21 or 22, wherein the mammalian cell is a human cell.

24. 24. The composition of claim 23, wherein the mammalian cell has previously been determined to have a defective otoferlin gene.

25. The composition of any one of claims 11 to 17, for use in increasing expression of full-length otoferlin protein in inner hair cells of the cochlea of ​​a mammal, wherein a therapeutically effective amount of the composition is introduced into the cochlea of ​​the mammal.

26. 26. The composition of claim 25, wherein the mammal has previously been determined to have a defective otoferlin gene.

27. 27. The composition of claim 25 or 26, wherein the mammal is a human.

28. 18. The composition of any one of claims 11 to 17, for use in treating asymptomatic sensorineural hearing loss in a subject identified as having a defective otoferlin gene, wherein the composition is administered to the cochlea of ​​the subject.

29. 29. The composition of claim 28, wherein the subject is a human.

30. 30. The composition of claim 28 or 29, comprising determining that the subject has a defective otoferlin gene prior to the administering step.

31. The composition of any one of claims 28 to 30, wherein the composition is administered to the cochlea using a microcatheter.

32. 32. The composition of claim 31, wherein the microcatheter is shaped to allow entry into the middle ear cavity via the ear canal and contact of the end of the microcatheter with the RWM.

33. 33. The composition of claim 32, wherein the distal end of the microcatheter is comprised of at least one microneedle having a diameter of 10 to 1,000 microns.

34. The composition of any one of claims 28 to 30, wherein the composition is administered to the cochlea using a device comprising a needle.

35. 35. The composition of claim 34, wherein the needle comprises a curved portion and an angled tip.

36. A kit comprising the composition of any one of claims 11 to 33.

37. 37. The kit of claim 36, wherein the composition is pre-loaded into a device.

38. 38. The kit of claim 37, wherein the device is a microcatheter.

39. 39. The kit of claim 38, wherein the microcatheter is shaped to allow entry into the middle ear cavity via the ear canal and contact of the end of the microcatheter with the RWM.

40. 40. The kit of claim 38 or 39, wherein the distal end of the microcatheter comprises at least one microneedle having a diameter of 10 to 1,000 microns.

41. 37. The kit of claim 36, further comprising a device.

42. 42. The kit of claim 41, wherein the device comprises a needle comprising a curved portion and an angled tip.

43. below: a) a first rAAV vector comprising the nucleic acid sequence of SEQ ID NO: 96; and b) a second rAAV vector comprising the nucleic acid sequence of SEQ ID NO: 105 A plurality of recombinant adeno-associated virus (rAAV) vectors, including:

44. 44. The plurality of rAAV vectors of claim 43, wherein the first and second rAAV vectors are each encapsulated by an AAV capsid.

45. 45. The multiple rAAV vectors of claim 44, wherein the AAV capsid encapsulating the first rAAV vector is of a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh39, AAVrh43, or Anc80.

46. 44. The multiple rAAV vectors of claim 43, wherein the AAV capsid encapsulating the second rAAV vector is of a serotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh39, AAVrh43, or Anc80.

47. 44. The plurality of rAAV vectors of claim 43, wherein a first rAAV vector is encapsulated by an Anc80 capsid and a second rAAV vector is encapsulated by an Anc80 capsid.

48. 48. The rAAV vectors of claim 47, wherein the Anc80 capsid comprises the polypeptide sequence of SEQ ID NO:

109.

49. 49. A composition comprising a plurality of the rAAV vectors of any one of claims 43-48, wherein the composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

50. 50. The composition of claim 49, wherein the composition is formulated for intracochlear administration.

51. 51. The composition of claim 49 or 50, wherein the composition comprises a synthetic perilymphatic solution.

52. 1. A composition for expressing a recombinant full-length otoferlin protein in a mammalian cell, comprising a plurality of recombinant adeno-associated virus (rAAV) vectors, the plurality of rAAV vectors comprising: a) a first rAAV vector comprising the nucleic acid sequence of SEQ ID NO: 96; and b) a second rAAV vector comprising the nucleic acid sequence of SEQ ID NO: 105; wherein the mammalian cell has reduced expression, lack of expression or impaired function of otoferlin.

53. 53. The composition of claim 52, wherein the mammalian cell is an inner hair cell.

54. 54. The composition of claim 52 or 53, wherein the mammalian cell is a human cell.

55. The composition of any one of claims 52 to 54, wherein the mammalian cell comprises a defective otoferlin gene.

56. 1. A composition for treating hearing loss in a subject with otoferlin-associated hearing loss, comprising a plurality of recombinant adeno-associated virus (rAAV) vectors, wherein the plurality of rAAV vectors comprises: a) a first rAAV vector comprising the nucleic acid sequence of SEQ ID NO: 96; and b) a second rAAV vector comprising the nucleic acid sequence of SEQ ID NO: 105; wherein the subject has reduced expression, lack of expression, or dysfunction of otoferlin.

57. 57. The composition of claim 56, wherein the subject is a human.

58. 58. The composition of any one of claims 52 to 57, wherein the first and second rAAV vectors are each encapsulated by an AAV capsid.

59. 59. The composition of claim 58, wherein the AAV capsid encapsulating the first rAAV vector is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh39, AAVrh43, or Anc80.

60. 60. The composition of claim 58 or 59, wherein the AAV capsid encapsulating the second rAAV vector is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh39, AAVrh43, or Anc80.

61. 58. The composition of any one of claims 52 to 57, wherein the first rAAV vector is encapsulated by an Anc80 capsid and the second rAAV vector is encapsulated by an Anc80 capsid.

62. 62. The composition of claim 61, wherein the Anc80 capsid comprises the polypeptide sequence of SEQ ID NO:

109.

63. 63. The composition of any one of claims 52-62, wherein the multiple rAAV vectors are administered as a single dose, or the multiple rAAV vectors are administered as two or more doses.

64. below: a) a first rAAV vector comprising, in 5' to 3' order: (i) the 5′ ITR sequence of SEQ ID NO: 97; (ii) CAG promoter; (iii) the 5′ OTOF coding region, including exons 1 to 21 of the OTOF cDNA; (iv) the SD intron sequence of SEQ ID NO: 102; (v) the AK recombinogenic sequence of SEQ ID NO: 103; and (vi) the 3' ITR sequence of SEQ ID NO: 104; and b) a second rAAV vector comprising, in 5' to 3' order: (i) the 5' ITR sequence of SEQ ID NO: 97 (ii) the AK recombinogenic sequence of SEQ ID NO: 103 (iii) SA intron sequence of SEQ ID NO: 106 (iv) the 3' OTOF coding region including exon 22 to exon 48 of the OTOF cDNA; (v) a polyA sequence; and (vi) the 3′ ITR sequence of SEQ ID NO:

104. A plurality of recombinant adeno-associated (rAAV) vectors, including:

65. 65. A composition comprising a plurality of rAAV vectors of claim 64 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

66. A composition comprising a first rAAV vector encapsulated by an Anc80 capsid and a second rAAV vector encapsulated by an Anc80 capsid, the composition comprising: a) The first rAAV vector comprises, in 5' to 3' order: (i) the 5′ ITR sequence of SEQ ID NO: 97; (ii) the CAG promoter, (iii) the 5' OTOF coding region, including exon 1 to exon 21 of the OTOF cDNA; (iv) SD intron sequence of SEQ ID NO: 102 (v) an AK recombinogenic sequence of SEQ ID NO: 103, and (vi) the 3′ ITR sequence of SEQ ID NO: 104; and b) The second rAAV vector comprises, in 5' to 3' order: (i) the 5′ ITR sequence of SEQ ID NO: 97; (ii) the AK recombinogenic sequence of SEQ ID NO: 103; (iii) the SA intron sequence of SEQ ID NO: 106; (iv) the 3' OTOF coding region, including exon 22 to exon 48 of the OTOF cDNA; (v) a polyA sequence, and (vi) the 3′ ITR sequence of SEQ ID NO: 104; wherein the composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

67. below: (i) the CAG promoter comprises a CMV early enhancer element of SEQ ID NO: 98, a chicken β-actin gene sequence of SEQ ID NO: 99, and a chimeric intron of SEQ ID NO: 100; and / or (ii) the polyA is a bGH polyA having the sequence of SEQ ID NO: 108; A plurality of rAAVs according to claim 64 or a composition according to claim 65 or 66.

68. 67. A plurality of rAAVs as described in claim 64 or a composition as described in claim 65 or 66, wherein the 5'OTOF coding region is at least 80% identical to SEQ ID NO: 101 and encodes the same amino acid sequence as encoded by SEQ ID NO:

101.

69. 67. The plurality of rAAVs of claim 64 or the composition of claim 65 or 66, wherein the 5'OTOF coding region comprises SEQ ID NO:

101.

70. 67. A plurality of rAAVs as described in claim 64 or a composition as described in claim 65 or 66, wherein the 3'OTOF coding region is at least 80% identical to SEQ ID NO: 107 and encodes the same amino acid sequence as encoded by SEQ ID NO:

107.

71. 67. The plurality of rAAVs of claim 64 or the composition of claim 65 or 66, wherein the 3'OTOF coding region comprises SEQ ID NO:

107.

72. A plurality of rAAVs as described in claim 64 or a composition as described in claim 65 or 66, wherein the first rAAV vector comprises the nucleic acid sequence of SEQ ID NO: 96; and the second rAAV vector comprises the nucleic acid sequence of SEQ ID NO:

105.

73. 67. The composition of claim 66, wherein an Anc80 capsid encapsulating the first rAAV vector comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 109; and an Anc80 capsid encapsulating the second rAAV vector comprises a polypeptide having at least 90% sequence identity to SEQ ID NO:

109.

74. 67. The composition of claim 65 or 66, wherein the composition is formulated for intracochlear administration.

75. 75. The composition of claim 74, wherein the composition comprises a synthetic perilymphatic solution.

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