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

Abstract

Compositions and methods for treating non-age-related hearing loss in human subjects are provided. [Solution] 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 invention generally relates to the use of nucleic acids to treat hearing loss in a human subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 494,866, filed August 23, 2016. No. 6,239,999, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present invention generally relates to the use of nucleic acids to treat hearing loss in a human subject. Regarding use. [Background technology]

[0003] Background of the Invention The ear is a complex organ, classically consisting of the outer ear, middle ear, inner ear, auditory nerve, and auditory system (the The ear is explained as a system that processes sound as it travels from the brain to the brain. In addition, it helps maintain balance. Therefore, disorders of the inner ear can lead to hearing loss, tinnitus, and dizziness. Vertigo is a hallucination of movement and is a major symptom of vestibular disorders. Dizziness can be caused by problems in the inner ear or central nervous system. Common inner ear causes of dizziness include: vestibular neuritis (sudden unilateral vestibular loss) , Meniere's disease (episodic vertigo), benign paroxysmal positional vertigo (BPPV), and bilateral vestibular Common central nervous system causes of dizziness include: post-concussion syndrome, cervical dizziness, vestibular migraine, cerebrovascular disease, and acoustic neuroma.

[0004] Hearing loss is one of the most common human sensory deficits and can occur for many reasons. Some people may have a congenital hearing loss, while others may lose their hearing slowly over time. Presbycusis is age-related hearing loss. Approximately 36 million American adults have reported some degree of hearing loss, with one in three people over 60 and half of people over 85 However, he has experienced hearing loss.

[0005] Hearing loss can be the result of environmental factors or a combination of genetic and environmental factors. Ringing, or the illusory noise in the auditory system (ringing, buzzing) Buzzing, chirping, humming, About half of all people with hearing loss or beating sounds also have a condition known as hyperacusis. They also have a hypersensitivity / intolerance to certain sound frequencies and volume ranges. Liam's syndrome (also known as Williams-Beuren syndrome) is a LIM-Kinner syndrome. A 1.6 Mb region of 7q11.23 encompassing approximately 26 genes, including the gene encoding LIMK1 (LIMK1). It is a multisystem disorder caused by a hemizygous deletion. The body manifests hyperacusis and progressive hearing loss, with early onset of hyperacusis resulting in its absence It is suggested that this may be related to one of the genes.

[0006] Environmental causes of hearing loss include certain medications, certain infections before or after birth, and long-term exposure to loud noise. Hearing loss can be caused by noise, ototoxic agents, It can be due to presbycusis, disease, infection, or cancer affecting certain parts of the ear. Ischemic injury can cause hearing loss through pathophysiological mechanisms initiated by it Another example is autoimmune inner ear disease (AIED), which occurs when the body's immune system fights off viruses or bacteria. Rapidly progressive bilateral sensorineural hearing loss that occurs when the brain attacks cells in the inner ear that are misplaced It is characterized by:

[0007] Approximately 1.5 in 1,000 children are born with profound hearing loss, and 1.5 in 1,000 children Another 2-3 people per 100,000 people are born with partial hearing loss (Smith et al., 2005, Lancet 3 65:879-890. More than half of these cases are attributable to a genetic basis. (DiDomenico, et al., 2011, J. Cell. Physiol. 226:2494-2499 (Non-Patent Document 2)).

[0008] Nonsyndromic 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. Most cases of hereditary hearing loss (70-80%) are nonsyndromic; the remaining cases are due to a specific genetic cause. It is caused by a genetic syndrome.

[0009] Hearing loss can be conductive (originating from the ear canal or middle ear), sensorineural (originating from the inner ear or auditory nerve), or Most forms of nonsyndromic hearing loss are due to structures in the inner ear. It is associated with permanent hearing loss caused by damage to the human sensorineural The majority of hearing loss is caused by abnormalities in the hair cells of the organ of Corti in the cochlea. Also, a very rare sensorineural hearing disorder involving the 8th cranial nerve (vestibulocochlear nerve) or the auditory part of the brain. In the rarest of these types of hearing loss, only the hearing centers of the brain are affected. In this condition, cortical deafness, sounds can be heard at normal thresholds but are not perceived. The quality of the sounds produced is so poor that the language cannot be understood. Sensorineural hearing loss in children is due to insufficient hair cell function. Hair cells are abnormal at birth and Noise trauma and External causes of damage, such as infection, as well as factors that play an important role in the anatomy or physiology of the cochlea There are both intrinsic abnormalities, such as congenital mutations in genes that play a role.

[0010] Hearing loss caused by changes in the middle ear is called conductive hearing loss. The form involves changes in both the inner and middle ear, called mixed hearing loss. Hearing loss that exists before language is learned is classified as prelingual or congenital. Hearing loss that occurs later in development is classified as postlingual. Most autosomal recessive loci are , which causes severe to profound hearing loss before language acquisition.

[0011] Non-syndromic hearing loss can have a variety of inheritance patterns and can occur at any age. The types of symptomatic hearing loss are named according to their inheritance pattern: DF, which is autosomal dominant; The autosomal recessive form is called DFNB, and the X-linked form is called DFN. Each type also has its own For example, DFNA1 is the first autosomal dominant gene described. The hearing loss was nonsyndromic, of the female type.

[0012] 75% to 80% of cases are inherited in an autosomal recessive pattern, meaning that the gene in each cell This means that both copies of the gene have the mutation. Usually, each copy of the gene in an individual with autosomal recessive hearing loss The parents carry one copy of the mutated gene but are not affected by this form of hearing loss. not present.

[0013] Another 20% to 25% of cases of nonsyndromic hearing loss are autosomal dominant, meaning that the gene This means that one copy of the altered gene in a gene is sufficient to cause hearing loss. People with chromosomal dominant hearing loss most often inherit a genetic mutation from a parent with hearing loss. Inherit the copy.

[0014] 1% to 2% of cases show an X-linked pattern of inheritance, which means that the mutated gene responsible for the condition This means that the gene is located on the X chromosome (one of the two sex chromosomes). Men with syndromic hearing loss are more likely to develop hearing loss in life than women who inherit two copies of the same gene mutation. They tend to develop hearing loss earlier and more severely. X-linked inheritance is characterized by the father having the X-linked trait. It is something that cannot be passed on to his son.

[0015] Mitochondrial nonsyndromic hearing loss, caused by changes to mitochondrial DNA, is a common condition in the United States. occurs in less than 1% of cases. The altered mitochondrial DNA is passed from the mother to the This type of hearing loss is not inherited from the father.

[0016] Hearing impairment characterized by normal outer hair cell function and abnormal or absent auditory brainstem responses Auditory neuropathy spectrum disorders (ANSDs), which are harmful to infants and young children, One of the most common conditions that results in hearing and speech communication barriers in children Approximately 10% of children with permanent hearing loss may have ANSD. This is the first gene identified for an autosomal recessive non-syndromic ANSD, and is the first gene identified in OTOF. Mutations account for approximately all cases of autosomal recessive nonsyndromic hearing loss in some populations. It has been found that it accounts for 5% of the total (Rodriguez-Ballesterose et al. 2008 Human Mut 2 9(6):823-831 (Non-Patent Document 3).

[0017] The causes of nonsyndromic hearing loss are complex. Researchers have identified several conditions associated with nonsyndromic hearing loss that may be altered. have identified more than 30 genes that contribute to the The mechanisms by which different mutations in the same gene cause different types of may be associated with hearing loss, and several genes are involved in syndromic and nonsyndromic hearing loss. is related to both.

[0018] For example, genes associated with non-syndromic hearing loss include, but are not limited to, ATP2B2, ACTG 1, CDH23, CLDN14, COCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB3, KCNQ4 , LHFPL5, MYO1A, MYO15A, MYO6, MYO7A, OTOF, PCDH15, SLC26A4, STRC, TECTA, TMC1, These include TMIE, TMPRSS3, TRIOBP, USH1C, and WFS1.

[0019] The most common cause of hearing loss is nonsyndromic hearing loss (Nonsyndromic Hearing Loss and Deafness). ) DFNB1 (GJB2-related DFNB1 nonsyndromic hearing loss; autosomal recessive hearing loss 1; neurosensory nonsyndromic recessive hearing loss 1) Non-syndromic hearing loss (DFNB1) is a congenital, non-progressive, mild to profound hearing loss. It is characterized by the loss of sensorineural hearing due to the expression of GJB2 (which encodes the protein connexin 26) and It is caused by mutations in DFNB1 and GJB6 (encoding connexin 30). The diagnosis is based on alterations in the gap junction beta-2 protein (connexin 26), GJB2 and Molecular genetic testing to identify deafness-causing mutations in the cis-regulatory elements and upstream Molecular genetic testing of GJB2 has demonstrated the presence of deafness-inducing genes in these genes. Detects more than 99% of mutations that cause hearing loss. Unlike some other forms of hearing loss, DFNB1 non-syndromic hearing loss does not affect balance or movement. The degree of hearing loss varies depending on how well a person It is difficult to predict the incidence of HIV based on the genetic mutations present in the individual members of the same family. Even though members are affected by DFNB1 non-syndromic hearing loss, the degree of hearing loss varies among members. may vary.

[0020] Mutations in the genes encoding connexin 26 (Cx26) and / or Cx30 are common in humans. Cx26 is associated with nearly half of all cases of chromosomal nonsyndromic prelingual hearing loss. Cx30 and Cx40 are the two major Cx isoforms found in the cochlea and coassemble with each other. to form hybrid (heteromeric and heterotypic) gap junctions (GJs) (Ahmad, et al., Proc. Natl. Acad. Sci., 2007, 104(4):1337-1341 (Non-Patent Document 4) )) Nonsyndromic hearing loss DFNA3 binds to the protein connexin 26 (Cx26) or connexin 26, respectively. Dominant negative mutations in the GJB2 or GJB6 genes that alter either Cx30 (Cx30) or Cx40 (Cx41) It is caused by a pathogenic variant in the brain and can range from mild to severe in prelingual or postlingual It is characterized by progressive high-frequency sensorineural hearing loss.

[0021] OTOF-related hearing loss (DFNB9 nonsyndromic hearing loss) has two phenotypes: prelingual nonsyndromic hearing loss and and less frequently, temperature-sensitive nonsyndromic auditory neuropathy (TS-NSAN) ) Mutations in the otoferlin gene (e.g., I1573T mutation or P1987R Another form of progressive hearing loss associated with the E1700Q mutation (and / or the E1700Q mutation) is temperature sensitivity. isn't it.

[0022] Pendred syndrome / DFNB4 (deafness with goiter) is an autosomal recessive genetic disorder Pendred syndrome accounts for 7.5% of all cases of congenital hearing loss. The PDS gene (DFNB4, EVA, PDS, TDH2B) on the long arm of chromosome 7 (7q31) encodes a protein. , and mutations in solute carrier family 26 member 4, also known as SLC26A4 Mutations in this gene are also linked to vestibular aqueduct dysfunction, another congenital cause of hearing loss. It also causes ectasia (EVA or EVAS); certain mutations are more likely to cause EVAS. Others are more likely to be linked to Pendred syndrome (Azaiez, et al. (December er 2007), Hum.Genet. 122 (5): 451-7 (Non-Patent Document 5)).

[0023] The transmembrane protease, serine 3, is expressed in the TMPRSS3 gene (DFNB10, DFNB8, ECHOS1, and TAD The gene is encoded by the mitochondrial ATPase inhibitor G12 (also known as G12). It was identified by its association with childhood-onset autosomal recessive hearing loss. Mutations in the TMPRSS3 gene are associated with postlingual and rapidly progressive hearing loss. The encoded protein contains a serine protease domain, a transmembrane domain, and an LDL receptor. It contains a scavenger receptor-like domain, and a cysteine-rich domain. Proteases are known to be involved in various biological processes, and their dysfunction This gene is responsible for the development of fetal cochleae and multiple myeloma. It is expressed in many other tissues, including the inner ear or the development of the perilymphatic and endolymphatic contents. This gene is also overexpressed in ovarian tumors. Four alternative splicins were also identified as tumor-associated genes that are expressed in the nucleus. Two variants have been described, two of which encode identical products.

[0024] DFN3 hearing loss is caused by mutations in the POU3F4 gene, which is located on the X chromosome. In people with this condition, one of the small bones in the middle ear (the stapes) The characteristic symptom of DFN3 is the inability to move normally, which can lead to hearing problems. At least four other regions of the X chromosome have been implicated in hearing loss. DFNB59, also known as Pejvakin or PJVK, is a rare but rare form of DFNB59. (Autosomal recessive deafness 59) is a genetic disorder that affects vertebrates. DFNB59 is a 352 amino acid protein belonging to the gasdermin family. , human chromosome 2q31, essential for proper function of auditory pathway neurons and outer hair cell function The mutation in DFNB59 is absent. A condition characterized by a mild or severely abnormal auditory brainstem response but normal otoacoustic emissions. Forms of auditory impairment (auditory neuropathy or auditory dyssynchrony) It is thought to cause an autosomal recessive form of nonsyndromic sensorineural hearing loss59, which is a synchronous disorder. DFNB59 shares significant similarity with DFNA5, suggesting that these genes share a common origin. Indicates that.

[0025] Alport syndrome is a condition characterized by the deletion of COL4A3, COL4A4, and COL4A5, which are involved in collagen biosynthesis. It is caused by a mutation in one of these genes. Mutations result in the loss of type IV collagen, a key structural component of the basement membrane in the kidney, inner ear, and eye. The proper production or assembly of the collagen network is prevented. One of the criteria used for diagnosis is bilateral sensorineural loss in the 2000 Hz to 8000 Hz range. Hearing loss develops gradually, is not present in early infancy, and generally appears before age 30. can be.

[0026] Defects in ion channels are associated with hearing loss: DFNA2 nonsyndromic hearing loss is a potential The potassium voltage-gated channel subunit Kv7.4 is also known as Kv7.4. Autosomal dominant in the KCNQ4 gene, encoding channel subfamily KQT member 4 Inherited as a mutation. DFNA2 nonsyndromic hearing loss is progressive across all frequencies. It is characterized by symmetric, primarily high-frequency sensorineural hearing loss (SNHL). At younger ages, hearing loss is primarily caused by low-frequency in older adults, hearing loss tends to be mild in the low frequencies and moderate in the high frequencies; Hearing loss is usually moderate at high frequencies and severe to profound at high frequencies. Hearing, particularly at high frequencies, is impaired from birth as detected during routine hearing evaluation. Those most affected are between the ages of 10 and 40, and are more likely to have hearing aids to help amplify sounds. By age 70, all people with DFNA2 hearing loss will have severe-to-profound hearing loss. Has a physical disability.

[0027] Mutations in the KCNE1 and KCNQ1 genes are associated with severe bilateral hearing loss and long QT syndrome. This causes a type of syndrome called Jervell-Lange-Nielsen syndrome (JLNS). The condition is an autosomal recessive disorder that affects an estimated 1.6 to 6 in 1 million children, and It accounts for less than 10% of all cases of long T syndrome. It has a significantly higher incidence in Sweden, up to 1:200,000. The proteins produced by the KCNQ1 and KCNQ2 genes work together to transport positively charged potassium These channels form potassium channels that transport potassium ions out of the cell. The movement of potassium ions through the urea is important for maintaining normal function of the inner ear and heart muscle. .

[0028] It is characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt wasting. EAST / SeSAME syndrome is caused by mutations in KCNJ10, which inwardly rectifies potassium channels. It is believed to be caused by this.

[0029] Bartter syndrome type 4 with sensorineural hearing loss (also known as Bartter syndrome IV or BSND) ) have a defect in the Cl- channel accessory subunit.

[0030] Mutations in the ATP6V1B1 gene, which is expressed in both the kidney and the cochlea, cause distal tubular amyloidosis. A significant proportion of children with autosomal recessive DRTA have been associated with dyslipidemia (DRTA). A percentage was also found to experience progressive bilateral sensorineural hearing loss.

[0031] Usher syndrome (Hallgren syndrome, Asher-Hallgren syndrome, retinitis pigmentosa) -Hearing Deficiency Syndrome, and Retinal Dystrophy Hearing Deficiency Syndrome) Any of at least 10 genes that result in a combination of deafness and progressive visual impairment It is a rare disorder caused by mutations in one of these genes and is the leading cause of deafness and blindness. Hearing loss is caused by a defective inner ear, while vision loss is due to the degeneration of retinal cells. Caused by retinitis pigmentosa (RP), Usher syndrome is designated as I, II, and III There are three clinical subtypes: Subjects with Usher I are born profoundly deaf; Instead, they begin to lose their vision in the first decade of life and develop as children due to problems in their vestibular system. They are slow to learn to walk and exhibit balance problems. Subjects who are not born deaf but have hearing loss, but who have balance problems, They also do not appear to have any noticeable problems with their vision later in life (in their teens). They may begin to lose their vision (at least in the early stages of life) and may still retain some vision even in middle age. Subjects with Sher Syndrome III are not born deaf, but their hearing is impaired. They experience a gradual loss of strength and vision. They may have balance problems, or There may be cases where it does not have one.

[0032] A mouse model of congenital hearing loss lacks the gene encoding vesicular glutamate transporter-3 (VGLUT3). Recently, virally mediated gene Treatment was used to restore hearing in VGLUT3 knockout mice (Akil, et al., 20 12, Neuron75:283-293 (Non-Patent Document 6)).

[0033] Math1 (the mouse homolog of ATH1; HATH1, or ATOH1) is a Drosophila (Drosophila phila homolog), is essential for hair cell development in the inner ear; Therefore, Math1 acts as a "pro-haircell" gene in the development of sensory epithelia. It has been proposed that the ATP acts as a ATP-dependent ATP synthase (Bermingham et al., 1999, Science 284:1837-1841 (Non-Patent Document 7)). Several studies have now shown that viral delivery of Math1 has a negative effect on Hair cell regeneration and improvement of both hearing and balance demonstrated in injured mouse cochleae (Baker et al., 2009, Adv. Otorhinolaryngol. 66:52-63 (Non-Patent Document 8) Husseman andRaphael, 2009, Adv. Otorhinolaryngol. 66:37-51 (Non-patent document 9);Izum Ikawa et al., 2008, Hear. Res. 240:52-56 (Non-Patent Document 10); Kawamoto et al., 2003, J. Neurosci.23:4395-4400 (Non-patent document 11); Praetorius et al., 2010, Acta Otolar yngol.130:215-222 (Non-patent Document 12); Staecker et al., 2007, Otol. Neurotol. 28:22 3-231 (Non-Patent Document 13).

[0034] Mutations in the WFS1 gene cause more than 90% of Wolfram syndrome type 1 cases Wolfram syndrome is a condition that causes high blood sugar levels due to a deficiency of the hormone insulin. (diabetes mellitus), and progressive vision loss due to degeneration of the nerves that carry information from the eye to the brain (optic nerve It is a condition that affects many of the body's systems and is most often characterized by atrophy (atrophy of the body's organs). However, people with Wolfram syndrome also often experience excessive urine production (diabetes insipidus). ), hearing loss caused by changes in the inner ear (sensorineural hearing loss), urinary tract problems, and in men Low levels of the sex hormone testosterone (hypogonadism), or neuropathy They also have pituitary dysfunction, which can lead to mental disorders. Approximately 65% ​​have hearing loss ranging in severity from birth to mild hearing loss that begins in adolescence and worsens over time. have sensorineural hearing loss, which may range from deafness to hearing loss. Additionally, people with Wolfram syndrome Approximately 60% of individuals develop neurological or psychiatric disorders, most commonly idiopathic encephalopathy, that typically begin in early adulthood. develop problems with balance and coordination (ataxia).

[0035] The WFS1 gene encodes wolframin ( Wolfram syndrome is thought to be caused by a gene called WFS1. When caused by mutations in , it is inherited in an autosomal recessive pattern, Wolframin protein has reduced or absent function. As a result, Calcium levels in the cell are not regulated and the endoplasmic reticulum does not function properly. Without the specific wolframin, cells induce their own cell death (apoptosis). The death of cells in the pancreas, specifically the cells that produce insulin (beta cells), is called walloping. Causes diabetes mellitus in people with Flamm syndrome. Cellular progression along the optic nerve. The loss of vision ultimately leads to visual loss in affected individuals. The death of cells that cause the various signs and symptoms of Wolfram syndrome type 1 may be due to the is high.

[0036] Mutations in the mitochondrial genes MT-TS1 and MT-RNR1 cause nonsyndromic hearing loss. Non-syndromic mitochondrial hearing loss has been found to increase the risk of developing Characterized by moderate to severe hearing loss. Pathogenic variants in MT-TS1 usually cause sensorineural hearing loss. Pathogenic variants in MT-RNR1 are associated with childhood onset of the disorder. Hearing loss (ototoxicity) when exposed to certain antibiotic medications called lycosides, and / or associated with a predisposition to late-onset sensorineural hearing loss; however, Some people with mutations that cause this disease may have difficulty with these antibiotics even without exposure. Hearing loss associated with aminoglycoside ototoxicity is bilateral and severe to profound. Yes, gentamicin, tobramycin, amikacin, kanamycin, or streptomycin after administration of any amount (even a single dose) of an aminoglycoside antibiotic such as mycobacterium thrombin It occurs within days to weeks.

[0037] Treatment for hearing loss currently consists of hearing amplification for mild to severe loss and hearing aids for severe to profound loss. The cochlear implant procedure for hearing loss (Kral and O'Donoghue, 2010, N. Engl. J. Med. 363:1438-1450 (Non-Patent Document 14). To date, the majority of research in this area has focused on elderly Applicable to the most common forms of hearing loss, including inflammatory hearing loss, noise injury, infection, and ototoxicity One, focusing on cochlear hair cell regeneration.

[0038] In animal models of cochlear ischemia, ischemic injury was associated with the upregulation of insulin-like growth factor (IGF- 1), AM-111 (apoptosis inhibitor), edaravone (free radical scavenger), Ginsenoside RB1 (Kappo), glial cell line-derived neurotrophic factor (GDNF), BDNF, CNTF, SOD1 It can be blocked by compounds such as SOD2, necrostatin-1, DFNA5, and MSRB3. However, it appears that combinations of substances may be more effective than single compounds (e.g. oxidative stress, exotoxicity, blood flow, calcium and stimuli overload, Complementary therapies for modulating apoptotic pathways, neurotrophic or hormonal control mechanisms .

[0039] Inhibition of JNK-1-induced apoptosis (mitochondrial-induced) was observed by a dominant-negative J It can be blocked by compounds such as NK-1 and d-stereoisomer JNK-1 (Mol. Pharmacol. 200 7 March;71(3):654-66 (Non-Patent Document 15); the contents of which are incorporated herein by reference in their entirety. incorporated).

[0040] Agents and methods for preventing or reversing hearing loss have been studied for many years. There remains a desperate need. [Prior art documents] [Non-patent literature]

[0041] [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 [Non-patent document 4] Ahmad, et al., Proc. Natl. Acad. Sci., 2007, 104(4):1337-1341 [Non-patent document 5] Azaiez, et al. (December 2007), Hum. Genet. 122 (5): 451-7 [Non-patent document 6] Akil, et al., 2012, Neuron 75:283-293 [Non-Patent Document 7] Bermingham et al., 1999, Science 284:1837-1841 [Non-patent document 8] Baker et al., 2009, Adv. Otorhinolaryngol. 66:52-63 [Non-Patent Document 9] Husseman and Raphael, 2009, Adv. Otorhinolaryngol. 66:37-51 [Non-Patent Document 10] Izumikawa et al., 2008, Hear. Res. 240:52-56 [Non-Patent Document 11] Kawamoto et al., 2003, J. Neurosci. 23:4395-4400 [Non-Patent Document 12] Praetorius et al., 2010, Acta Otolaryngol. 130:215-222 [Non-Patent Document 13] Staecker et al., 2007, Otol. Neurotol. 28:223-231 [Non-Patent Document 14] Kral and O'Donoghue, 2010, N. Engl. J. Med. 363:1438-1450 [Non-Patent Document 15] Mol. Pharmacol. 2007 March; 71(3):654-66 [Brief explanation of the drawings]

[0042] [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 human β-actin promoter (hbA) and CMV enhancer (CMVe) 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 6194-211. 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 stabilization sequence, a woodchuck hepatitis virus post-translational regulatory element (WPRE), and a polyadenylation signal (pA). [Figure 2] Figure 1 shows an exemplary schematic 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 (AK) from F1 phage were included at the 3' end of the 5' vector and the 5' end of the 3' vector to allow for precise vector assembly (Trapani et al. (2014) EMBO Mol Med 6 194-211). [Figure 3] Graph showing auditory brainstem response (ABR) amplitude over time in non-transduced Otof− / − mice (n=25; thin black line) and Otof− / − mice after dual AAV-mediated expression of otoferlin (n=14; gray lines represent individual animals, thick black line is the average response across all animals). [Figure 4]Graph showing ABR amplitude over time in untransduced Otof− / − mice (n=25; long dashed line), Otof− / − mice after dual AAV-mediated expression of otoferlin (n=14; short dashed line), and wild-type Otofwt / wt mice after dual AAV transduction of enhanced green fluorescent protein (eGFP) (n=5; thin black lines represent individual animals, thick black line is the average amplitude across all animals). [Figure 5] 1 is an exemplary graph showing ABR thresholds (in dB SPL) across frequencies (in kHz) in response to pure tones or click stimuli. The minimum sound pressure level (SPL) at which waves can be detected is presented for individual Otof− / − animals with dual AAV-mediated expression of otoferlin (open circles represent individual animals, and hatched circles are the average thresholds across all animals), for non-transduced Otof− / − animals (closed circles), and for Otofwt / wt animals after dual AAV transduction of eGFP (checked circles). [Figure 6] Graph showing the transduction rate of IHCs along the cochlea (e.g., entire organ of Corti, apical turn, and middle / basal turn) as determined by immunohistochemistry using two different antibodies, one that binds to the N-terminal otoferlin fragment and the other that binds to the very C-terminal portion of otoferlin. [Figure 7]This is a set of immunohistochemistry images of one organ of Corti from an Otof- / - mouse after dual AAV-mediated expression of otoferlin. 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) in the N-terminal half of otoferlin. The right panel shows C-terminal anti-otoferlin antibody staining (Synaptic Systems). Together, all three panels demonstrate full-length otoferlin expression in IHC. Importantly, even though AAV transduces 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. [Figure 8] Graph showing Ca2+ currents over depolarizing stimuli for Otof- / - IHCs (white; n=10), Otof- / - IHCs after dual AAV-mediated expression of otoferlin (hatched; n=8), and wild-type IHCs from background-matched control animals (black; n=6). [Figure 9] Graph showing plasma membrane capacitance (ΔCm) over the duration of depolarization in dual AAV-transduced IHCs of Otof- / - mice (black line, individual IHCs; hatched diamonds: mean ± sem; n = 8), two individual dual AAV-transduced IHCs from injected Otof- / - mice that exhibited only small 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. [Figure 10] Representative graph showing Qreal over depolarization duration (ms) in Otof− / − IHC (open diamonds), dual AAV-transduced IHC of Otof− / − mice (filled circles), and background-matched wild-type controls (filled diamonds). [Figure 11]A representative plasmid map of pAAV-AK-SA-3'mOTOF-EWB. [Figure 12] A representative plasmid map of pAAV-SA-3'mOTOF-EWB. [Figure 13] A representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD. [Figure 14] A representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD-AK. [Figure 15] 3 is a graph showing plasma membrane capacitance (ΔCm) over the duration of depolarization in IHCs of Otof− / − mice transduced with the two vectors shown in FIG. 2 (medium shading; thick line), in IHCs in wild-type mice (dark shading; medium thickness line), and in IHCs in Otof− / − mice. [Figure 16] Representative graph showing Qreal over depolarization duration (ms) in Otof − / − IHCs (open diamonds), IHCs from Otof − / − mice transduced with the two vectors shown in Figure 2 (gray diamonds), and IHCs from background-matched wild-type controls (black diamonds). Summary of the Invention

[0043] overview The present invention provides a method for producing an active otoferlin protein (e.g., a full-length otoferlin protein). The coding sequence is produced in mammalian cells, thereby preventing the development of a non-human rhesus monkey in a subject in need thereof. A composition comprising at least two different nucleic acid vectors for treating symptomatic sensorineural hearing loss. wherein each of the at least two different vectors encodes a different gene for the otoferlin protein. The invention relates to a method for producing a composition comprising: Based on the view.

[0044] As used herein, a composition comprising at least two different nucleic acid vectors, At least two different vectors each encoding a different portion of the otoferlin protein. Each encoded portion contains a coding sequence that is at least 30 amino acids long. residues, and the amino acid sequence of each of the encoded portions is optionally the amino acid sequences of the at least two portions may overlap with the amino acid sequences of different portions; Any single vector among the different vectors encodes the full-length otoferlin protein. at least one of the coding sequences is located in the two otoferlin genomic DNA sequences; A nucleotide sequence spanning adjacent exons and between the two adjacent exons lacking intron sequences; and wherein the at least two different vectors are When introduced into cells, they undergo concatenation or homologous recombination with each other, thereby The composition is provided in which a recombinant nucleic acid encoding a full-length otoferlin protein is formed. In some embodiments of any of the compositions described herein, Each of at least two different vectors may be a plasmid, transposon, cosmid, The compositions described herein are artificial chromosomes, or viral vectors. In some embodiments of any one of the at least two different vectors, each of the at least two different vectors comprises a human Human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived Some of the compositions described herein are proteases, proteases, and proteases. In some embodiments, each of the at least two different vectors is an adeno-associated virus. (AAV) vector, adenoviral vector, lentiviral vector, or retroviral vector The composition is a viral vector selected from the group consisting of viral vectors. In some embodiments of any of the above, the vectors are at least two different vectors. Each is an AAV vector.

[0045] In some embodiments of any of the compositions described herein, the code The amino acid sequence of one of the encoded portions is the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, In this manner, the amino acid sequence of each of the encoded portions is The amino acid sequence of any of the compositions described herein overlaps with that of the In some embodiments, the length of the overlapping amino acid sequences ranges from about 30 amino acid residues to about 1000 amino acids. It is an amino acid residue.

[0046] In some embodiments of any of the compositions described herein, a vector The vector comprises two different vectors, each of which contains a different intron. The intron comprises a segment of an intron present in otoferlin genomic DNA. The sequences of the two different segments comprise a nucleotide sequence of at least 100 nucleotides. In some embodiments of any of the compositions described herein, In this case, the sequences of the two different segments overlap by about 100 to about 800 nucleotides. In some embodiments of any of the compositions described herein, The length of each nucleotide sequence of at least two different vectors is about 500 nucleotides. Any of the compositions described herein may be used in a nucleic acid sequence of from about 10,000 to about 10,000 nucleotides. In some embodiments, the nucleotide sequences of each of at least two different vectors are The length of the string ranges from 500 nucleotides to 5,000 nucleotides.

[0047] In some embodiments of any of the compositions described herein, the composition The number of different vectors in any of the compositions described herein is two. In some embodiments, the first of the two different vectors is an otofena The coding sequence encoding the N-terminal portion of the phosphoprotein is described herein. In some embodiments of any of the compositions, the N-terminal portion of the otoferlin protein The length of the fragment is between 30 amino acids and 1600 amino acids. In some embodiments of any of the above, the length of the N-terminal portion of the otoferlin protein is 2 00 to 1500 amino acids. In some embodiments, the first vector comprises one of a promoter and a Kozak sequence. or both. In this embodiment, the first vector comprises an inducible promoter, a constitutive promoter, or Includes promoters that are tissue-specific promoters.

[0048] In some embodiments of any of the compositions described herein, two The second of the different vectors encodes the C-terminal portion of the otoferlin protein. Some of the compositions described herein include a coding sequence for In one embodiment, the length of the C-terminal portion of the otoferlin protein is between 30 amino acids and 1600 amino acids. In some embodiments of any of the compositions described herein, The length of the C-terminal portion of the otoferlin protein is 200 to 1500 amino acids. In some embodiments of any of the compositions described herein, a second vector The target further comprises a poly(dA) signal sequence. In some embodiments of either, the second vector further comprises a sequence for mRNA stabilization. Some embodiments of any of the compositions described herein include pharmaceutical The composition further comprises an excipient acceptable to the pharmaceutical industry.

[0049] Also provided herein are compositions comprising any of the compositions described herein. Also provided are kits. Some embodiments of any of the kits described herein further comprises a pre-filled syringe containing the composition.

[0050] Also provided herein is a therapeutically effective amount of any of the compositions described herein. Also provided are methods for administering the method described herein to a mammalian subject, comprising the step of introducing any of the methods described herein into the cochlea of ​​the 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 comprises: It has previously been identified as having a defective otoferlin gene.

[0051] Also provided herein are methods for administering any of the compositions described herein to mammals. A method for expressing a full-length otoferlin protein in a mammalian cell, comprising introducing the protein into the mammalian cell. Also provided are methods for increasing the expression of any of the methods described herein. In some embodiments, the mammalian cell is a cochlear inner hair cell. In some embodiments of any of the methods, 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.

[0052] Also provided herein is a therapeutically effective amount of any of the compositions described herein. In the inner hair cells of the mammalian cochlea, the method comprises introducing either Methods for increasing expression of full-length otoferlin protein are also provided. In some embodiments of any of the methods, the mammal comprises a defective otoferlin The gene has been previously identified as having any of the methods described herein. In some embodiments, the mammal is a human.

[0053] Also provided herein is a therapeutically effective amount of any of the compositions described herein. and administering any one of the compositions to the cochlea of ​​a subject having a defective otoferlin gene. Also provided is a method for treating non-syndromic sensorineural hearing loss in an identified subject. In some embodiments of any of the methods described in, the subject is a human. Some embodiments of any of the methods described herein may further comprise administering to the subject a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, ... The method further comprises determining that the subject has a defective otoferlin gene.

[0054] Also provided herein is a composition comprising two different nucleic acid vectors, wherein the two different nucleic acid vectors a first nucleic acid vector among the nucleic acid vectors, comprising a promoter, a 3'-terminal sequence of the promoter, a first coding sequence encoding the N-terminal portion of the otoferlin protein located at and a splicing donor signal sequence located at the 3' end of the first coding sequence. and a second nucleic acid vector of the two different nucleic acid vectors comprises a splicing aid. a splicing acceptor signal sequence located at the 3' end of the splicing acceptor signal sequence; a second coding sequence encoding the C-terminal portion of the otoferlin protein, and 2 coding sequences, including a polyadenylation sequence at the 3' end of each of the coding sequences; the length of the encoded portion is at least 30 amino acid residues, and the amino acid sequences of the encoded portions do not overlap; Any single vector of the two different vectors expresses the full-length otoferlin protein. does not encode a protein, and the coding sequence is transcribed in mammalian cells to produce an RNA transcript. When the product is produced, the splicing donor signal sequence on one transcript and the splicing donor signal sequence on the other transcript are fused. Splicing occurs between the splicing acceptor signal sequence on the transcript of the target gene. thereby forming a recombinant RNA molecule encoding the full-length otoferlin protein. Also provided are compositions comprising any of the compositions described herein. In some embodiments, the coding sequence of at least one of the vectors is A nucleotide sequence spanning two adjacent exons of the feline genomic DNA, It lacks intronic sequences between adjacent exons.

[0055] In the present specification, a promoter, an otoconia located on the 3' side of the promoter, a first coding sequence encoding the N-terminal portion of a ferrin protein; A splicing donor signal sequence located at the end, and the splicing donor - a first nucleic acid sequence containing a first detectable marker gene located 3' to the signal sequence; an acid vector; and a second detectable marker gene, A splicing acceptor signal sequence located on the 3' side of a gene, The C-terminus of the otoferlin protein is located at the 3' end of the acceptor signal sequence. a second coding sequence encoding a portion of the a second nucleic acid vector different from the first nucleic acid vector, the second nucleic acid vector comprising a polyadenylation sequence; 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 with each other, and the encoded portions are encoded by two different vectors. None of the vectors encodes the full-length otoferlin protein, and when the coding sequence is transcribed in mammalian cells to produce an RNA transcript, A splicing donor signal on one transcript and a splicing donor signal on the other transcript Splicing occurs between the nucleotide acceptor signal and the full-length otoferase. Also provided is the composition, wherein a recombinant RNA molecule encoding the phosphoprotein is formed. In some embodiments of any of the compositions described herein, the vector The coding sequence of at least one of the vectors is located between two adjacent loci of otoferlin genomic DNA. a nucleotide sequence spanning two adjacent exons and an interspace between the two adjacent exons; Some of the compositions described herein lack a nucleotide sequence. In embodiments, 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.

[0056] In the present specification, a promoter, an otoconia located on the 3' side of the promoter, a first coding sequence encoding the N-terminal portion of a ferrin protein; A splicing donor signal sequence located at the end, and the splicing donor - A highly recombinogenic sequence located 3' of the signal sequence (e.g., F1 phage a first nucleic acid vector comprising a highly recombinogenic region; and a second highly recombinogenic region. (e.g., the recombinogenic region of F1 phage), said second highly recombinogenic sequence splicing accessions located 3' to the recombinogenic region of the F1 phage an acceptor signal sequence, located at the 3' end of the splicing acceptor signal sequence; a second coding sequence encoding the C-terminal portion of the otoferlin protein, and the first nucleic acid vector comprising a polyadenylation sequence located at the 3' end of the coding sequence of a composition comprising a second nucleic acid vector different from the first nucleic acid vector; The length of each of the encoded portions is at least 30 amino acid residues. The vectors do not overlap with each other, and no single vector of the two different vectors contains the full-length otoconia. It does not encode a ferrin protein, and the coding sequence is transduced in mammalian cells. When transcribed to produce RNA transcripts, splicing donor signals on one transcript splicing between the splicing acceptor signal on the other transcript and the This results in the production of a recombinant RNA molecule encoding the full-length otoferlin protein. Also provided is the composition, wherein

[0057] In some embodiments of any of the compositions described herein, a vector The coding sequence of at least one of the vectors is located between two flanking loci of otoferlin genomic DNA. A nucleotide sequence spanning two adjacent exons and an interleukin-1 between the two adjacent exons. It lacks intronic sequences.

[0058] Also provided herein are compositions comprising any of the compositions described herein. Also provided are kits. Some embodiments of any of the kits described herein further comprises a pre-filled syringe containing the composition.

[0059] Also provided herein is a therapeutically effective amount of any of the compositions described herein. Also provided are methods for administering the method described herein to a mammalian subject, comprising the step of introducing any of the methods described herein into the cochlea of ​​the mammal. In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal is It has previously been identified as carrying the otoferlin gene.

[0060] Also provided herein are methods for administering any of the compositions described herein to mammals. A method for expressing a full-length otoferlin protein in a mammalian cell, comprising introducing the protein into the mammalian cell. Also provided are methods for increasing the expression of any of the methods described herein. In some embodiments, the mammalian cell is a cochlear inner hair cell. In some embodiments of any of the methods, 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.

[0061] Also provided herein is a therapeutically effective amount of any of the compositions described herein. A method for producing a full-length otofilament in inner hair cells of a mammalian cochlea, comprising the step of introducing either one of the otofilament or the otofilament into the cochlea. Also provided are methods for increasing expression of a telomerase protein. In some embodiments of any of the above, the mammal has a defective otoferlin gene. It has been previously identified that any of the methods described herein In some embodiments, the mammal is a human.

[0062] Also provided herein is a therapeutically effective amount of any of the compositions described herein. The method includes administering either one of the following into the cochlea of ​​a subject identified as having a defective otoferlin gene: Also provided are methods of treating non-syndromic sensorineural hearing loss in a subject. In some embodiments of any of the methods described herein, the subject is a human. Some embodiments of any of the described methods further comprise administering to the subject a deficiency The method further includes the step of determining whether the mutant strain has the otoferlin gene.

[0063] As used herein, a therapeutic composition comprising a plurality of adeno-associated virus (AAV) vectors wherein the plurality of AAV vectors are transfected into target cells of a human subject to which the therapeutic composition is administered. The ability to organize messenger RNA for auditory polypeptides in the The therapeutic composition is provided, comprising:

[0064] In some embodiments of any of the therapeutic compositions described herein, The multiple AAV vectors produce full-length auditory receptors in target cells of a human subject to which the therapeutic composition is administered. It has the ability to construct messenger RNA for the system polypeptides.

[0065] Some embodiments of any of the therapeutic compositions described herein include a first A and a second AAV vector, wherein the first and second AAV vectors are independently and has a packaging capacity of less than about 6 kb.

[0066] In some embodiments of any of the therapeutic compositions described herein, The messenger RNAs for auditory system polypeptides are encoded by otoferlin and its orthologues or The polypeptide encodes an auditory system polypeptide selected from the group of homologs.

[0067] Some embodiments of any of the therapeutic compositions described herein may comprise Ca v 1.3 bassoon, piccolo, ribeye, and harmonium a scaffolding protein selected from (harmonin); Vglut3; synaptotagmin; and vesicle tetherin Docking / docking proteins; vesicle priming proteins; vesicle fusion proteins; GluA2 / 3 and an auditory system polypeptide encoding an auditory system polypeptide selected from the group consisting of GluA4; A nucleic acid (e.g., a vector) comprising a nucleic acid sequence encoding the messenger RNA of the peptide It may further include:

[0068] In some embodiments of any of the therapeutic compositions described herein, The first AAV vector comprises at least one promoter selected from a CBA, CMV, or CB7 promoter. It further comprises a promoter sequence.

[0069] In some embodiments of any of the therapeutic compositions described herein, The first AAV vector contains at least one promoter selected from cochlear-specific promoters. It further comprises a target sequence.

[0070] In some embodiments of any of the therapeutic compositions described herein, The therapeutic compositions are formulated for intracochlear administration. In some embodiments of any of the compositions, the therapeutic composition comprises lipid nanoparticles. Any of the therapeutic compositions described herein are formulated as follows: In some embodiments, the therapeutic composition is formulated to include polymeric nanoparticles. In some embodiments of any of the therapeutic compositions described herein In some cases, therapeutic compositions are formulated to contain small circular DNA. In some embodiments of any of the therapeutic compositions, the therapeutic composition comprises CELiD DN A. Any of the therapeutic compositions described herein is formulated to contain A. In some embodiments, the therapeutic composition is formulated to include synthetic perilymph. In some embodiments of any of the therapeutic compositions described herein, wherein the therapeutic composition comprises 20-200 mM NaCl; 1-5 mM KCl; 0.1-10 mM CaCl; 1-10 mM glucose; and 2 to 50 mM HEPES, including a synthetic perilymph solution having a pH of about 6 to about 9. It is formulated as follows.

[0071] Also provided herein are therapeutic compositions comprising messenger RNA for auditory system polypeptides. Goods are also provided.

[0072] Also provided herein are therapeutics comprising one or more adenoviral (AV) vectors. a therapeutic composition, wherein the one or more AV vectors are Capable of assembling messenger RNA for auditory system polypeptides in target cells of interest The therapeutic composition is also provided.

[0073] In some embodiments of any of the therapeutic compositions described herein, The one or more AV vectors are capable of transfecting target cells of a human subject to which the therapeutic composition is administered. It has the ability to construct messenger RNA for full-length auditory system polypeptides.

[0074] Also provided herein are therapeutic compositions comprising one or more lentiviral vectors. wherein the one or more lentiviral vectors are administered to a subject in need thereof. Ability to organize messenger RNA for auditory system polypeptides in target cells of human subjects Also provided is the therapeutic composition, comprising:

[0075] In some embodiments of any of the therapeutic compositions described herein, The one or more lentiviral vectors are targeted to a human subject to which the therapeutic composition is administered. It has the ability to synthesize messenger RNA for full-length auditory system polypeptides in cells.

[0076] Also provided herein is a method of: i) introducing a first incision into the cochlea of ​​a human subject at a first incision point; ii) administering an effective dose of a therapeutic composition (e.g., a therapeutic composition described herein) to the patient; Also provided is a surgical method comprising intracochlearly administering a therapeutic agent (any of

[0077] In some embodiments of any of the methods described herein, the therapeutic composition The composition is administered to the subject at the first incision point. In some embodiments, the therapeutic composition is administered into or through the first incision. is administered to the subject.

[0078] In some embodiments of any of the methods described herein, the therapeutic composition The composition is administered to the subject into or through the cochlear oval window membrane. In some embodiments of any of the methods, the therapeutic composition is administered to a subject in the cochlear round window membrane. It is administered to or through a subject.

[0079] In some embodiments of any of the methods described herein, the therapeutic composition The composition is administered using a medical device capable of creating multiple incisions in the round window membrane. can be.

[0080] In some embodiments of any of the methods described herein, a medical device The device includes a plurality of microneedles. In some embodiments, the medical device comprises a plurality of masks including a generally circular first surface. and microneedles, wherein each microneedle has a diameter of at least about 10 microns. It has.

[0081] In some embodiments of any of the methods described herein, a medical device The device includes a base and / or reservoir capable of holding a therapeutic composition. In some embodiments of any of the methods described herein, a medical device The chair comprises a plurality of hollow microneedles, each containing a lumen capable of transferring a therapeutic composition. Includes

[0082] In some embodiments of any of the methods described herein, a medical device The vise includes means for generating at least a partial vacuum.

[0083] Also provided herein is a method for treating a human subject in need thereof, comprising: i) making multiple incisions in the round window membrane of the inner ear; and ii) a medical device capable of producing multiple adeno-associated viruses (AAVs). a therapeutic composition comprising a plurality of AAV vectors, wherein the plurality of AAV vectors are capable of expressing AAV in target cells of the inner ear. The therapeutic composition has the ability to synthesize messenger RNA of a full-length auditory system polypeptide. A therapeutic delivery system containing an effective dose of the composition is also provided.

[0084] Also referred to herein are: i) an effective dose of a therapeutic composition (e.g., a composition comprising a compound as described herein); and administering intracochlearly to a human subject in need thereof a therapeutic composition comprising: 1. A method of treating a wound, comprising: a) administering to a patient a therapeutic composition comprising: a) a means for creating multiple incisions in the round window membrane; and and b) administered by using a medical device containing an effective dose of the therapeutic composition. Also provided is a means for performing a surgical method comprising the steps described above, which may be performed.

[0085] Any of the means for performing the surgical methods described herein In some embodiments, the medical device comprises a plurality of microneedles.

[0086] Also provided herein are therapeutic compositions comprising a single adeno-associated virus (AAV) vector. wherein the AAV vector is capable of transfecting target cells of a human subject to which the therapeutic composition is administered. said therapeutic composition having the ability to synthesize messenger RNA of auditory system polypeptides in Goods are also provided.

[0087] In some embodiments of any of the therapeutic compositions described herein, A single AAV vector produces full-length auditory receptors in target cells of a human subject to which the therapeutic composition is administered. The ability to construct messenger RNA for the system polypeptides described herein. In some embodiments of any of the methods, an active otoferlin protein (e.g., For example, any of the truncated active otoferlin proteins described herein. ) a single vector (e.g., a vector described herein) containing sequences encoding (any of the above) can be administered to the subject.

[0088] In some embodiments of any of the therapeutic compositions described herein, The therapeutic composition further comprises a second vector other than an AAV vector, wherein the single AAV vector The vector and the second vector independently have a packaging capacity of less than about 6 kb.

[0089] In some embodiments of any of the therapeutic compositions described herein, The messenger RNAs for auditory system polypeptides are encoded by otoferlin and its orthologues or The polypeptide encodes an auditory system polypeptide selected from the group of homologs.

[0090] In some embodiments of any of the therapeutic compositions described herein, Messenger RNAs for auditory polypeptides are derived from otoferin and its truncated variants. The polypeptide encodes an auditory system polypeptide selected from the group consisting of:

[0091] In some embodiments of any of the therapeutic compositions described herein, The otoferlin truncation mutant comprises at least one C2 domain of: JPEG0007814280000001.jpg53127

[0092] In some embodiments of any of the therapeutic compositions described herein, The otoferlin truncation mutant does not contain the C-terminal region of the endogenous otoferlin polypeptide.

[0093] In some embodiments of any of the therapeutic compositions described herein, Messenger RNA for auditory system polypeptides is expressed in Ca v 1.3; Bassoon, Piccolo, Ribeye, and harmonin; Vglut3; synaptotagmin; vesicle tethering proteins RING / docking proteins; vesicle priming proteins; vesicle fusion proteins; Glu A2 / 3; and GluA4.

[0094] In some embodiments of any of the therapeutic compositions described herein, The single AAV vector comprises at least one promoter selected from the group consisting of a CBA, CMV, or CB7 promoter. The promoter sequence of

[0095] In some embodiments of any of the therapeutic compositions described herein, The single AAV vector contains at least one promoter selected from the cochlear-specific promoters. It further comprises a target sequence.

[0096] In some embodiments of any of the therapeutic compositions described herein, The therapeutic composition is formulated for intracochlear administration.

[0097] In some embodiments of any of the therapeutic compositions described herein, The therapeutic composition is formulated to include lipid nanoparticles.

[0098] In some embodiments of any of the therapeutic compositions described herein, The therapeutic composition is formulated to include polymeric nanoparticles.

[0099] In some embodiments of any of the therapeutic compositions described herein, The therapeutic composition is formulated to include small circular DNA.

[0100] In some embodiments of any of the therapeutic compositions described herein, Therapeutic compositions are formulated to contain CELiD DNA.

[0101] In some embodiments of any of the therapeutic compositions described herein, The therapeutic composition is formulated to include synthetic perilymph fluid.

[0102] In some embodiments of any of the therapeutic compositions described herein, The therapeutic composition contains 20-200 mM NaCl; 1-5 mM KCl; 0.1-10 mM CaCl; 1-10 mM glucose. Formulated to contain synthetic perilymph containing 2 to 50 mM HEPES and having a pH of about 6 to about 9 It has been done.

[0103] Also herein, the present invention relates to an auditory system polypeptide encoding an otoferlin truncated mutant. Therapeutic compositions comprising messenger RNA are also provided.

[0104] Unless otherwise defined, all technical and scientific terms used herein has the same meaning as commonly understood by a person skilled in the art in the technical field to which the present invention pertains. It has.

[0105] The terms "a" and "an" refer to one or more of the grammatical objects of the article in question. refers to a plurality (i.e., at least one). For example, "an element" refers to one It includes elements and elements of.

[0106] The term "about" when referring to a measurable value such as an amount, time duration, etc. These variations are meant to encompass variations of up to ±20% from the specified value. For example, up to ±10%, or in some cases ±5%, or in some cases ±1%, or in some instances, ±0.1%, it being understood that such variations are within the scope of the disclosure. This is because it is suitable for carrying out the method described.

[0107] The term "signaling domain" refers to a domain that generates a second messenger and by functioning as an effector in response to such messengers, Transmitting intracellular information to regulate cellular activity through defined signaling pathways Otoferlin is a functional part of a protein that acts by activating the endothelial cells (ECCs) of the inner hair cells. for exocytosis in the retina and neurotransmitter release in immature outer hair cells It is believed that the otoferlin protein is a calcium sensor required for Five of the six C2 domains (C2B–F) bind calcium with moderate and low affinity constants in solution. Binds to ammonium ions (Kd = 25-95 μM and 400-700 μM, respectively) (Padmanarayana et al. 2014Biochem 53:5023-5033). In the presence of phosphatidylserine (PS), 10 μM The calcium concentration of otoferlin C2C-C2E domains significantly increased the C2-liposome phase. Thus, otoferlin operates using an "electrostatic switch" mechanism. It has a domain that appears to bind calcium and a domain that binds calcium independently. PI(4,5)P2, a major signaling molecule at synapses, acts in a calcium-independent manner. , which has been shown to interact with the C2C and C2F domains of otoferlin (Pa dmanarayana et al. 2014 Biochem 53:5023-5033).

[0108] As used herein, the term "antibody" refers to an immunoglobulin that specifically binds to an antigen. Antibodies refer to proteins or polypeptide sequences derived from a globulin molecule. Clonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins Antibodies can be any antibody and may be derived from natural or recombinant sources. The term "antibody fragment" refers to an intact antibody. or a recombinant variant thereof, and directed against a target such as an antigen. An antigen-binding domain (e.g., an antibody fragment) sufficient to confer recognition and specific binding to the antibody fragment. Examples of antibody fragments include, but are not limited to, , Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, sdAb (VL or VH fragments) single domain antibodies, such as camelid VHH domains, and antibodies formed from antibody fragments. The term "scFv" refers to a multispecific antibody that contains at least one variable region of the light chain. and a fusion protein comprising at least one antibody fragment containing a variable region of a heavy chain. The term "protein" refers to a protein in which the light and heavy chain variable regions are separated by a short, flexible polypeptide linker. and are capable of being expressed as a single polypeptide chain; , scFv retains the specificity of the intact antibody from which it is derived. As used herein, an scFv refers to a polypeptide that has, for example, an N-terminal and a C-terminal end thereof. For example, scFvs may have the VL and VH variable regions in either order, with the VL-linker-VH or may comprise VH-linker-VL.

[0109] The term "recombinant polypeptide" refers to a polypeptide produced using recombinant DNA technology. This term refers to a polypeptide expressed by a viral vector expression system. It also refers to polypeptides (such as those produced by synthesis of DNA molecules encoding the polypeptides) The DNA molecule expresses a protein, or an amino acid sequence that identifies the polypeptide. The term "DNA or amino acid sequence" should also be construed to mean a sequence of amino acids, where the DNA or amino acid sequence is , recombinant DNA or amino acid sequence techniques available and well known in the art This was obtained using

[0110] The term "mutation in the otoferlin gene" refers to a mutation in the wild-type otoferlin protein. In comparison, deletion of one or more amino acids, substitution of one or more amino acids, and and otoferlin proteins having one or more of one or more amino acid insertions. and / or the encoded protein in mammalian cells without the mutation. The expression level of the encoded otoferlin protein in mammalian cells was compared with that of the otoferlin protein The wild-type otoferlin gene, resulting in a decrease in the expression level of the otoferlin protein In some embodiments, the mutation refers to a change in one or more amino acids (e.g., For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 This can result in the production of otoferlin proteins with deletions of several amino acids. In embodiments, the mutation may result in a frameshift in the otoferlin gene. The term "frameshift" refers to a sequence that results in a shift in the reading frame of a coding sequence. It is known in the art to encompass any mutation in the coding sequence. In some embodiments, the frameshift can result in a non-functional protein. In some embodiments, the point mutation may be a nonsense mutation (i.e., a point mutation in an exon of a gene). Nonsense mutations result in a stop codon in the middle of the sequence (compared to the corresponding wild-type protein). of truncated proteins that may or may not be functional (compared to In some embodiments, the mutation may result in the production of otoferlin mRNA or otoferlin. Loss of expression (or reduced levels) of telomerase protein or both mRNA and protein In some embodiments, the mutation may result in a wild-type otoferlin protein. Loss, reduction, or alteration of one or more biological activities (functions) compared to This may result in the production of the otoferlin protein.

[0111] In some embodiments, the mutation is at one or more nucleotides of the otoferlin gene. In some embodiments, the mutation is an insertion into the regulatory sequence of the otoferlin gene. In some embodiments, the regulatory sequence is in the portion of the gene that is not the coding sequence. The mutations in the sequence may be in the promoter or enhancer region, and The proper transcription of the gene may be prevented or reduced.

[0112] Modifications can be made by methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. The nucleotide sequence can be introduced by standard techniques.

[0113] The term "conservative sequence modifications" refers to the binding of an antibody or antibody fragment containing an amino acid sequence Refers to amino acid modifications that do not significantly affect or significantly alter a property. Such conservative modifications include amino acid substitutions, additions, and deletions. Standard techniques known in the art, such as directed mutagenesis and PCR-mediated mutagenesis Conservative amino acid substitutions can be introduced into the antibody or antibody fragment of the present invention by is one 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, amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), non Amino acids with polar side chains (e.g., alanine, valine, leucine, isoleucine, proline) phosphorus, phenylalanine, and methionine), amino acids with β-branched side chains (e.g. , threonine, valine, and isoleucine), and amino acids with aromatic side chains ( (e.g., tyrosine, phenylalanine, tryptophan, and histidine) .

[0114] The term "encoding" refers to a polynucleotide such as a gene, cDNA, or mRNA. A specific sequence of nucleotides in a molecule, followed by a defined sequence of amino acids according to the genetic code. It refers to the inherent property of serving as a template for the synthesis of a gene, cDNA, or RNA. A is produced as a protein by transcription and translation of the mRNA corresponding to that gene, cDNA, or RNA. If the nucleotide sequence is the mRNA sequence, it encodes a protein. the coding strand, which is identical to the sequence shown and is generally provided in the sequence listing, and Both the non-coding strands used herein may be referred to as encoding a protein product.

[0115] The terms "homology" or "identity" refer to the relationship between two polymer molecules, e.g., two DNA molecules. Subunits between two nucleic acid molecules, such as two RNA molecules or two polypeptide molecules Refers to unit sequence identity. The positions of the subunits in both of the two molecules are the same. When occupied by two subunits; for example, in each of two DNA molecules If a position is occupied by an 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, half of the positions in the two sequences (e.g., a polymer 10 subunits long) If 90% of the positions (e.g., 5 positions in For example, if 9 out of 10 (9 of 10) of the sequences match or are homologous, then the two sequences are 90% homologous. do.

[0116] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" are abbreviations of each other. All nucleotide sequences that are duplicate versions and therefore code for the same amino acid sequence Nucleotide sequences encoding proteins may also contain introns. .

[0117] The term "endogenous" refers to a substance that is native to or located within an organism, cell, tissue, or system. refers to any substance produced by

[0118] The term "exogenous" refers to something that is foreign to or originates from an organism, cell, tissue, or system. refers to any substance produced outside of the

[0119] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally occurring in a living animal is considered "isolated." The same substance that is not "contained" but is partially or completely separated from the coexisting substances in its natural state The same nucleic acid or peptide is "isolated." An isolated nucleic acid or protein is one that has been It can be present in a qualitatively purified form or can be present in a native form, e.g., a host cell. It can exist in a non-industrial environment.

[0120] The term "substantially purified cells" refers to cells that are essentially free of other cell types. A substantially purified cell also refers to a cell that is free from other cells with which it is normally associated in its naturally occurring state. It also refers to cells that have been separated from a cell type. In some instances, substantially purified cells A population of cells refers to a homogenous population of cells. In other instances, the term simply refers to a population of cells in their natural state. refers to cells that are separated from the cells with which they are naturally associated. The cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0121] "Transfected" or "transformed" or "transduced" The term refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. "Transfected" or "transformed" or "transduced" "Cells" are cells that have been transfected, transformed, or modified with exogenous nucleic acid. A transduced cell includes the primary subject cell and its progeny.

[0122] The term "expression" refers to the transcription of a particular nucleotide sequence driven by a promoter. Refers to transcription and / or translation.

[0123] As used herein, "transient" means that the The expression of a non-integrated transgene occurs over a period of time in a host cell. integrated into the genome or contained within a stable plasmid replicon The duration of gene expression is less than that of the case where

[0124] The term "subject" refers to a living organism in which an immune response can be elicited (e.g., In some embodiments, the subject is a rodent. (e.g., rats or mice), rabbits, sheep, dogs, cats, horses, non-human primates In some embodiments, the subject has non-syndromic hearing loss or In some embodiments, the subject has or is at risk of developing otoferlin In some embodiments, the subject has previously been identified as having a mutation in an offspring. Patients who have been identified as having a mutation in the phosphodiesterase gene and have been diagnosed with nonsyndromic sensorineural hearing loss In some embodiments, the subject is identified as having non-syndromic sensorineural hearing loss. .

[0125] As used herein, the term "therapeutic" means treatment. Therapeutic benefit is achieved by the reduction, suppression, remission, or eradication of the disease state. do.

[0126] As used herein, the term "prophylaxis" refers to the prevention of a disease or disorder. Prevention of a disease state or protective treatment for a disease or disease state "Prevention" in this context means the prevention of a disease. This includes reducing the likelihood of experiencing

[0127] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein. , the compounds, formulations described herein effective to achieve a particular biological outcome Refers to the quantity of a substance or composition.

[0128] In some embodiments, a therapeutically effective amount of the composition comprises an active otoferlin protein (e.g., For example, wild-type full-length otoferlin protein, or an otoferlin protein having the desired activity. an increase in the expression level of a protein variant (e.g., an increase in the expression level compared to the expression level before treatment with the composition) In some embodiments, a therapeutically effective amount of the composition may result in target cell proliferation. Active otoferlin protein (e.g., wild-type whole hair cells) in the cochlear cavity (e.g., cochlear inner hair cells) This may result in increased expression levels of the long otoferlin protein or an active variant. In some embodiments, a therapeutically effective amount of the composition is administered to target cells (e.g., cochlear inner hair cells). ) in the presence of an active otoferlin protein (e.g., a wild-type full-length otoferlin protein or In some embodiments, the therapeutic A therapeutically effective amount of the composition is an active otoferlin protein (e.g., a wild-type full-length otoferlin protein). Increased expression levels of the target protein or its active variant, and / or Increase in one or more activities of otoferlin protein (e.g., in a subject prior to treatment) levels, levels in subjects with mutations in the otoferlin gene, or nonsyndromic sensorineural loss (compared to a reference level, such as the level in a hearing subject or population of subjects) It is possible.

[0129] The term "parenteral" administration of a composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (IM) These include intrameatal (im) or intrasternal injection or infusion techniques.

[0130] The term "nucleic acid" or "polynucleotide" refers to a nucleic acid in either single-stranded or double-stranded form. Either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and its polymers Unless specifically limited, the term refers to a nucleic acid that has similar binding properties to the reference nucleic acid, or and known forms of natural nucleotides that are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also includes a conservative its variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated, are implicitly encompassed. indicates that degenerate codon substitutions occur at the third position of one or more selected (or all) codons. positions are substituted with mixed base and / or deoxyinosine residues. This may be achieved by (Batzer et al., Nucleic Acid Res. 19:5081 (1991) ;Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., M ol. Cell. Probes 8:91-98 (1994)).

[0131] In some embodiments of any of the nucleic acids described herein, the nucleic acid is a DNA A. In some embodiments of any of the nucleic acids described herein, The nucleic acid is RNA.

[0132] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, and "T" refers to "U" refers to uridine.

[0133] As used herein, "in vitro transcribed RNA" refers to RNA that has been transcribed in vitro. Generally, in vitro transcribed RNA refers to RNA synthesized in vitro, preferably mRNA. In vitro transcription vectors are prepared from in vitro transcription vectors. The template used to generate the RNA.

[0134] The terms "peptide," "polypeptide," and "protein" are used interchangeably. A compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids. There is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. A polypeptide is a molecule that contains two or more amino acids linked together by peptide bonds. It includes any peptide or protein that contains amino acids. When used herein, the term is used interchangeably with other terms used in the art, e.g., peptides, oligopeptides, and Short chains, also commonly referred to as oligomers, and those generally known in the art as proteins, are also known as nucleotides. There are many types of proteins, both long chains called "polypeptides." "Peptides" include, inter alia, for example, biologically active fragments, substantially homologous polypeptides, Peptides, oligopeptides, homodimers, heterodimers, variants and modifications of polypeptides Polypeptides include polypeptides, derivatives, analogs, and fusion proteins. The peptides include recombinant peptides, recombinant peptides, or combinations thereof.

[0135] The term "signaling pathway" refers to the process by which a signal is transmitted from one part of a cell to another part of the cell. It refers to the biochemical relationships between the various signaling molecules that play a role in signal transmission. The phrase "cell surface receptor" refers to a molecule that receives a signal and transmits the signal across the cell membrane. This includes molecules and complexes of molecules that have the ability to

[0136] The term "active otoferlin protein" refers to an otherwise wild-type mammalian protein. The full-length otoferlin protein is expressed in auditory hair cells (e.g., auditory inner hair cells). If both wild-type alleles encoding the When expressed in cells of a mammal, the mammal is similar to a mammal that is entirely wild type. DNA coding results in animals having a level of hearing close to normal. A non-limiting example of an active otoferlin protein is a full-length otoferlin protein. a toferlin protein (e.g., a full-length toferlin protein described herein) (either of the above).

[0137] For example, active otoferlin proteins may contain from 1 amino acid substitution to about 240 amino acid substitutions, 1 amino acid substitution to approximately 235 amino acid substitutions, 1 amino acid substitution to approximately 230 amino acid substitutions, 1 amino acid substitution to approximately 225 amino acid substitutions, 1 amino acid substitution to approximately 220 amino acid substitutions, 1 amino acid substitution to approximately 215 amino acids substitution, 1 amino acid substitution to approximately 210 amino acid substitutions, 1 amino acid substitution to approximately 205 amino acid substitutions, 1 amino acid substitution to approximately 200 amino acid substitutions, 1 amino acid substitution to approximately 195 amino acid substitutions, 1 amino acid substitution to approximately 190 amino acid substitutions, 1 amino acid substitution to approximately 185 amino acid substitutions, 1 amino acid substitution to approximately 180 amino acids substitution, 1 amino acid substitution to about 175 amino acid substitution, 1 amino acid substitution to about 170 amino acid substitution, 1 amino acid substitution From about 165 amino acid substitutions, 1 amino acid substitution to about 160 amino acid substitutions, 1 amino acid substitution 1 to about 155 amino acid substitutions, 1 to about 150 amino acid substitutions, 1 to about 145 amino acid substitutions amino acid substitution, 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 approximately 130 amino acid substitutions, 1 amino acid substitution to approximately 125 amino acid substitutions, 1 amino acid substitution 1 to about 120 amino acid substitutions, 1 to about 115 amino acid substitutions, 1 to about 110 amino acid substitutions amino acid substitution, 1 amino acid substitution to about 105 amino acid substitutions, 1 amino acid substitution to about 100 amino acid substitutions, 1 to approximately 95 amino acid substitutions, 1 to approximately 90 amino acid substitutions, 1 amino acid substitution ~approximately 85 amino acid substitutions, 1 amino acid substitution to approximately 80 amino acid substitutions, 1 amino acid substitution to approximately 75 amino acid substitutions Substitution, 1 amino acid substitution to about 70 amino acid substitutions, 1 amino acid substitution to about 65 amino acid substitutions, 1 amino acid substitution 1 amino acid substitution to approximately 60 amino acid substitutions, 1 amino acid substitution to approximately 55 amino acid substitutions, 1 amino acid substitution to approximately 50 amino acid substitutions amino acid substitution, 1 amino acid substitution to about 45 amino acid substitutions, 1 amino acid substitution to about 40 amino acid substitutions, 1 Amino acid substitutions: Approximately 35 amino acid substitutions, 1 amino acid substitution: Approximately 30 amino acid substitutions, 1 amino acid substitution Approximately 25 amino acid substitutions, 1 to approximately 20 amino acid substitutions, 1 to approximately 15 amino acid substitutions substitution, 1 to about 10 amino acid substitutions, 1 to about 9 amino acid substitutions, 1 amino acid substitution 1 to about 8 amino acid substitutions, 1 to about 7 amino acid substitutions, 1 to about 6 amino acid substitutions Substitution, 1 to about 5 amino acid substitutions, 1 to about 4 amino acid substitutions, 1 amino acid Substitutions up to about 3 amino acid substitutions, about 2 amino acid substitutions to about 240 amino acid substitutions, about 2 amino acid substitutions to about 23 5 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 amino acid substitution, about 2 amino acid substitutions to about 220 amino acid substitutions, about 2 amino acid substitutions to about 215 amino acid substitutions, Approximately 2 amino acid substitutions to approximately 210 amino acid substitutions, approximately 2 amino acid substitutions to approximately 205 amino acid substitutions, approximately ... Approximately 200 amino acid substitutions, approximately 2 amino acid substitutions, approximately 195 amino acid substitutions, approximately 2 amino acid substitutions ~ about 190 amino acid substitutions, ~ about 2 amino acid substitutions ~ about 185 amino acid substitutions, ~ about 2 amino acid substitutions ~ about 180 Amino acid substitution, about 2 amino acid substitutions to about 175 amino acid substitutions, about 2 amino acid substitutions to about 170 amino acids substitution, about 2 amino acid substitutions to about 165 amino acid substitutions, about 2 amino acid substitutions to about 160 amino acid substitutions, about 2 amino acid substitutions to about 155 amino acid substitutions, about 2 amino acid substitutions to about 150 amino acid substitutions, about 2 amino acid substitutions Approximately 145 amino acid substitutions, approximately 2 amino acid substitutions, approximately 140 amino acid substitutions, approximately 2 amino acid substitutions Approximately 135 amino acid substitutions, approximately 2 amino acid substitutions to approximately 130 amino acid substitutions, approximately 2 amino acid substitutions to approximately 125 amino acid substitutions amino acid substitution, about 2 amino acid substitutions to about 120 amino acid substitutions, about 2 amino acid substitutions to about 115 amino acid substitutions substitutions, about 2 to about 110 amino acid substitutions, about 2 to about 105 amino acid substitutions, about 2 Amino acid substitutions up to about 100 amino acid substitutions, about 2 amino acid substitutions to about 95 amino acid substitutions, about 2 amino acid substitutions substitutions up 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 substitution, about 2 amino acid substitutions to about 75 amino acid substitutions, about 2 amino acid substitutions to about 70 amino acid substitutions substitution, about 2 amino acid substitution to about 65 amino acid substitution, about 2 amino acid substitution to about 60 amino acid substitution, about 2 amino acid substitution to about 65 amino acid substitution Amino acid substitutions - approximately 55 amino acid substitutions, approximately 2 amino acid substitutions - approximately 50 amino acid substitutions, approximately 2 amino acid substitutions ~ about 45 amino acid substitutions, ~ about 2 amino acid substitutions ~ about 40 amino acid substitutions, ~ about 2 amino acid substitutions ~ about 35 amino acid substitutions amino acid substitution, 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 amino acid substitutions Approximately 10 amino acid substitutions, approximately 2 amino acid substitutions to approximately 9 amino acid substitutions, approximately 2 amino acid substitutions to approximately 8 Amino acid substitution, 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 amino acid substitutions Approximately 240 amino acid substitutions, approximately 3 amino acid substitutions, approximately 235 amino acid substitutions, approximately 3 amino acid substitutions Approximately 230 amino acid substitutions, approximately 3 amino acid substitutions to approximately 225 amino acid substitutions, approximately 3 amino acid substitutions to approximately 220 amino acid substitutions amino acid substitution, about 3 amino acid substitutions to about 215 amino acid substitutions, about 3 amino acid substitutions to about 210 amino acid substitutions substitution, about 3 amino acid substitution to about 205 amino acid substitution, about 3 amino acid substitution to about 200 amino acid substitution, about 3 Amino acid substitutions - approximately 195 amino acid substitutions, approximately 3 amino acid substitutions - approximately 190 amino acid substitutions, approximately 3 amino acid substitutions Substitutions up 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, approximately 3 amino acid substitutions to approximately 170 amino acid substitutions, approximately 3 amino acid substitutions to approximately 165 amino acid substitutions amino acid substitution, about 3 amino acid substitutions to about 160 amino acid substitutions, about 3 amino acid substitutions to about 155 amino acid substitutions , about 3 to about 150 amino acid substitutions, about 3 to about 145 amino acid substitutions, 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 about 3 amino acid substitutions to about 130 amino acid substitutions, about 3 amino acid substitutions to about 125 amino acid substitutions, about 3 amino acid substitutions to about 12 0 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 amino acid substitution, about 3 amino acid substitutions to about 105 amino acid substitutions, about 3 amino acid substitutions to about 100 amino acid substitutions, About 3 to about 95 amino acid substitutions, about 3 to about 90 amino acid substitutions, about 3 amino acid substitutions Approximately 85 amino acid substitutions, approximately 3 amino acid substitutions to approximately 80 amino acid substitutions, approximately 3 amino acid substitutions to approximately 75 amino acid substitutions, approximately 3 to approximately 70 amino acid substitutions, approximately 3 to approximately 65 amino acid substitutions substitution, 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 up to about 50 amino acid substitutions, about 3 amino acid substitutions to about 45 amino acid substitutions, about 3 amino acid substitutions about 3 to about 40 amino acid substitutions, about 3 to about 35 amino acid substitutions, about 3 to about 30 amino acid substitutions Amino acid substitution, about 3 to about 25 amino acid substitutions, about 3 to about 20 amino acid substitutions , about 3 to about 15 amino acid substitutions, about 3 to about 10 amino acid substitutions, about 3 amino acid substitutions 1 amino acid substitution to about 9 amino acid substitutions, about 3 amino acid substitutions to about 8 amino acid substitutions, about 3 amino acid substitutions to about 7 amino acid substitutions, about 3 to about 6 amino acid substitutions, about 3 to about 5 amino acid substitutions substitutions, about 4 to about 240 amino acid substitutions, about 4 to about 235 amino acid substitutions, about 4 Amino acid substitutions - approximately 230 amino acid substitutions, approximately 4 amino acid substitutions - approximately 225 amino acid substitutions, approximately 4 amino acid substitutions Substitutions up to about 220 amino acid substitutions, about 4 amino acid substitutions to about 215 amino acid substitutions, about 4 amino acid substitutions to about 210 amino acid substitutions, approximately 4 amino acid substitutions to approximately 205 amino acid substitutions, approximately 4 amino acid substitutions to approximately 200 amino acid substitutions amino acid substitution, about 4 amino acid substitution to about 195 amino acid substitution, about 4 amino acid substitution to about 190 amino acid substitution , 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 185 amino acid substitutions, Approximately 175 amino acid substitutions, approximately 4 amino acid substitutions, approximately 170 amino acid substitutions, approximately 4 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 15 5 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 amino acid substitution, about 4 amino acid substitutions to about 140 amino acid substitutions, about 4 amino acid substitutions to about 135 amino acid substitutions, Approximately 4 amino acid substitutions to approximately 130 amino acid substitutions, approximately 4 amino acid substitutions to approximately 125 amino acid substitutions, approximately ... Approximately 120 amino acid substitutions, approximately 4 amino acid substitutions, approximately 115 amino acid substitutions, approximately 4 amino acid substitutions ~ 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 substitution, about 4 amino acid substitutions to about 95 amino acid substitutions, about 4 amino acid substitutions to about 90 amino acid substitutions substitution, about 4 amino acid substitution to about 85 amino acid substitution, about 4 amino acid substitution to about 80 amino acid substitution, about 4 amino acid substitution to about 85 amino acid substitution Amino acid substitutions to approximately 75 amino acid substitutions, approximately 4 amino acid substitutions to approximately 70 amino acid substitutions, approximately 4 amino acid substitutions ~ about 65 amino acid substitutions, ~ about 4 amino acid substitutions ~ about 60 amino acid substitutions, ~ about 4 amino acid substitutions ~ about 55 amino acid substitutions amino acid substitution, about 4 amino acid substitution to about 50 amino acid substitution, about 4 amino acid substitution to about 45 amino acid substitution, About 4 amino acid substitutions to about 40 amino acid substitutions, about 4 amino acid substitutions to about 35 amino acid substitutions, about ... Approximately 4 amino acid substitutions to approximately 30 amino acid substitutions, approximately 4 amino acid substitutions to approximately 25 amino acid substitutions, approximately 4 amino acid substitutions to approximately 20 amino acid substitutions, about 4 to about 15 amino acid substitutions, about 4 to about 10 amino acid substitutions substitution, about 4 amino acid substitutions to about 9 amino acid substitutions, about 4 amino acid substitutions to about 8 amino acid substitutions, about 4 amino acid substitutions to about 9 amino acid substitutions Amino acid substitutions: about 7 amino acid substitutions, about 4 amino acid substitutions: about 6 amino acid substitutions, about 5 amino acid substitutions Approximately 240 amino acid substitutions, approximately 5 amino acid substitutions to approximately 235 amino acid substitutions, approximately 5 amino acid substitutions to approximately 230 amino acid substitutions amino acid substitution, about 5 amino acid substitutions to about 225 amino acid substitutions, about 5 amino acid substitutions to about 220 amino acid substitutions substitutions, about 5 to about 215 amino acid substitutions, about 5 to about 210 amino acid substitutions, about 5 Amino acid substitutions - approximately 205 amino acid substitutions, approximately 5 amino acid substitutions - approximately 200 amino acid substitutions, approximately 5 amino acid substitutions substitutions up to about 195 amino acid substitutions, about 5 amino acid substitutions up to about 190 amino acid substitutions, about 5 amino acid substitutions up to about 185 amino acid substitutions, approximately 5 amino acid substitutions to approximately 180 amino acid substitutions, approximately 5 amino acid substitutions to approximately 175 amino acid substitutions amino acid substitution, about 5 to about 170 amino acid substitution, about 5 to about 165 amino acid substitution , about 5 amino acid substitutions to about 160 amino acid substitutions, about 5 amino acid substitutions to about 155 amino acid substitutions, amino acid substitutions to about 150 amino acid substitutions, about 5 amino acid substitutions to about 145 amino acid substitutions, about 5 amino acid substitutions about 5 amino acid substitutions to about 140 amino acid substitutions, about 5 amino acid substitutions to about 135 amino acid substitutions, about 5 amino acid substitutions to about 13 0 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 amino acid substitution, about 5 amino acid substitutions to about 115 amino acid substitutions, about 5 amino acid substitutions to about 110 amino acid substitutions, Approximately 5 amino acid substitutions to approximately 105 amino acid substitutions, approximately 5 amino acid substitutions to approximately 100 amino acid substitutions, approximately ... Approximately 95 amino acid substitutions, approximately 5 amino acid substitutions, approximately 90 amino acid substitutions, approximately 5 amino acid substitutions Approximately 85 amino acid substitutions, approximately 5 to approximately 80 amino acid substitutions, approximately 5 to approximately 75 amino acid substitutions amino acid substitutions, about 5 to about 70 amino acid substitutions, about 5 to about 65 amino acid substitutions, about 5 amino acid substitutions to about 60 amino acid substitutions, about 5 amino acid substitutions to about 55 amino acid substitutions, about 5 amino acid substitutions substitutions up to about 50 amino acid substitutions, about 5 amino acid substitutions to about 45 amino acid substitutions, about 5 amino acid substitutions to about 40 Amino acid substitution, about 5 to about 35 amino acid substitutions, about 5 to about 30 amino acid substitutions substitution, about 5 to about 25 amino acid substitutions, about 5 to about 20 amino acid substitutions, about 5 to about 25 amino acid substitutions Amino acid substitutions up to about 15 amino acid substitutions, about 5 amino acid substitutions up to about 10 amino acid substitutions, about 5 amino acid substitutions up to about 9 amino acid substitutions, about 5 amino acid substitutions to about 8 amino acid substitutions, about 5 amino acid substitutions to about 7 amino acid substitutions amino acid substitution, about 6 amino acid substitutions to about 240 amino acid substitutions, about 6 amino acid substitutions to about 235 amino acid substitutions, Approximately 6 amino acid substitutions to approximately 230 amino acid substitutions, approximately 6 amino acid substitutions to approximately 225 amino acid substitutions, approximately ... Approximately 220 amino acid substitutions, approximately 6 amino acid substitutions, approximately 215 amino acid substitutions, approximately 6 amino acid substitutions ~ 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 substitution, about 6 amino acid substitutions to about 195 amino acid substitutions, about 6 amino acid substitutions to about 190 amino acids substitution, 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 Approximately 165 amino acid substitutions, approximately 6 amino acid substitutions, approximately 160 amino acid substitutions, approximately 6 amino acid substitutions Approximately 155 amino acid substitutions, approximately 6 amino acid substitutions to approximately 150 amino acid substitutions, approximately 6 amino acid substitutions to approximately 145 amino acid substitutions amino acid substitution, about 6 amino acid substitutions to about 140 amino acid substitutions, about 6 amino acid substitutions to about 135 amino acid substitutions substitutions, about 6 to about 130 amino acid substitutions, about 6 to about 125 amino acid substitutions, about 6 Amino acid substitutions - approximately 120 amino acid substitutions, approximately 6 amino acid substitutions - approximately 115 amino acid substitutions, approximately 6 amino acid substitutions Substitutions up 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, approximately 6 amino acid substitutions to approximately 95 amino acid substitutions, approximately 6 amino acid substitutions Amino acid substitutions - approximately 90 amino acid substitutions, approximately 6 amino acid substitutions - approximately 85 amino acid substitutions, approximately 6 amino acid substitutions ~ about 80 amino acid substitutions, ~ about 6 amino acid substitutions ~ about 75 amino acid substitutions, ~ about 6 amino acid substitutions ~ about 70 amino acid substitutions amino acid substitution, about 6 amino acid substitution to about 65 amino acid substitution, about 6 amino acid substitution to about 60 amino acid substitution, Approximately 6 to 55 amino acid substitutions, approximately 6 to 50 amino acid substitutions, approximately 6 amino acid substitutions Approximately 45 amino acid substitutions, approximately 6 amino acid substitutions to approximately 40 amino acid substitutions, approximately 6 amino acid substitutions to approximately 35 amino acid substitutions, about 6 to about 30 amino acid substitutions, about 6 to about 25 amino acid substitutions substitution, 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 up to about 10 amino acid substitutions, about 6 amino acid substitutions to about 9 amino acid substitutions, about 6 amino acid substitutions about 7 amino acid substitutions to about 240 amino acid substitutions; about 7 amino acid substitutions to about 235 Amino acid substitution, about 7 amino acid substitutions to about 230 amino acid substitutions, about 7 amino acid substitutions to about 225 amino acids substitution, 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 Approximately 200 amino acid substitutions, approximately 7 amino acid substitutions, approximately 195 amino acid substitutions, approximately 7 amino acid substitutions Approximately 190 amino acid substitutions, approximately 7 amino acid substitutions to approximately 185 amino acid substitutions, approximately 7 amino acid substitutions to approximately 180 amino acid substitutions amino acid substitution, about 7 amino acid substitution to about 175 amino acid substitution, about 7 amino acid substitution to about 170 amino acid substitution 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 - approximately 155 amino acid substitutions, approximately 7 amino acid substitutions - approximately 150 amino acid substitutions, approximately 7 amino acid substitutions Substitutions up 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, approximately 7 amino acid substitutions to approximately 130 amino acid substitutions, approximately 7 amino acid substitutions to approximately 125 amino acid substitutions amino acid substitution, about 7 amino acid substitution to about 120 amino acid substitution, about 7 amino acid substitution to about 115 amino acid substitution , about 7 amino acid substitutions to about 110 amino acid substitutions, about 7 amino acid substitutions to about 105 amino acid substitutions, 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 about 7 amino acid substitutions to about 90 amino acid substitutions, about 7 amino acid substitutions to about 85 amino acid substitutions, about 7 amino acid substitutions to about 80 amino acid substitutions amino acid substitution, about 7 to about 75 amino acid substitution, about 7 to about 70 amino acid substitution , about 7 amino acid substitutions to about 65 amino acid substitutions, about 7 amino acid substitutions to about 60 amino acid substitutions, about 7 amino acid substitutions to about 65 amino acid substitutions, Approximately 55 amino acid substitutions, approximately 7 amino acid substitutions, approximately 50 amino acid substitutions, approximately 7 amino acid substitutions Approximately 45 amino acid substitutions, approximately 7 to approximately 40 amino acid substitutions, approximately 7 to approximately 35 amino acid substitutions amino acid substitution, about 7 to about 30 amino acid substitution, about 7 to about 25 amino acid substitution, about 7 to about 20 amino acid substitutions, about 7 to about 15 amino acid substitutions, about 7 amino acid substitutions Substitutions up to about 10 amino acid substitutions, about 7 amino acid substitutions to about 9 amino acid substitutions, about 8 amino acid substitutions to about 240 Amino acid substitution, about 8 amino acid substitutions to about 235 amino acid substitutions, about 8 amino acid substitutions to about 230 amino acids substitution, about 8 amino acid substitutions to about 225 amino acid substitutions, about 8 amino acid substitutions to about 220 amino acid substitutions, about 8 amino acid substitutions to about 215 amino acid substitutions, about 8 amino acid substitutions to about 210 amino acid substitutions, about 8 amino acid substitutions Approximately 205 amino acid substitutions, approximately 8 amino acid substitutions, approximately 200 amino acid substitutions, approximately 8 amino acid substitutions Approximately 195 amino acid substitutions, approximately 8 amino acid substitutions to approximately 190 amino acid substitutions, approximately 8 amino acid substitutions to approximately 185 amino acid substitutions amino acid substitution, about 8 amino acid substitutions to about 180 amino acid substitutions, about 8 amino acid substitutions to about 175 amino acid substitutions substitutions, about 8 to about 170 amino acid substitutions, about 8 to about 165 amino acid substitutions, about 8 Amino acid substitutions - approximately 160 amino acid substitutions, approximately 8 amino acid substitutions - approximately 155 amino acid substitutions, approximately 8 amino acid substitutions Substitutions up 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, approximately 8 amino acid substitutions to approximately 135 amino acid substitutions, approximately 8 amino acid substitutions to approximately 130 amino acid substitutions amino acid substitution, about 8 amino acid substitution to about 125 amino acid substitution, about 8 amino acid substitution to about 120 amino acid substitution , 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 115 amino acid substitutions, 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 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 amino acid substitution, about 8 to about 80 amino acid substitution, about 8 to about 75 amino acid substitution , about 8 amino acid substitutions to about 70 amino acid substitutions, about 8 amino acid substitutions to about 65 amino acid substitutions, about 8 amino acid substitutions to about 70 amino acid substitutions, Approximately 60 amino acid substitutions, approximately 8 amino acid substitutions, approximately 55 amino acid substitutions, approximately 8 amino acid substitutions Approximately 50 amino acid substitutions, approximately 8 to approximately 45 amino acid substitutions, approximately 8 to approximately 40 amino acid substitutions amino acid substitution, about 8 to about 35 amino acid substitution, about 8 to about 30 amino acid substitution, about 8 amino acid substitutions to about 25 amino acid substitutions, about 8 amino acid substitutions to about 20 amino acid substitutions, about 8 amino acid substitutions Substitutions up to about 15 amino acid substitutions, about 8 amino acid substitutions to about 10 amino acid substitutions, about 10 amino acid substitutions to about 2 40 amino acid substitutions, approximately 10 amino acid substitutions to approximately 235 amino acid substitutions, approximately 10 amino acid substitutions to approximately 230 amino acid substitutions amino acid substitution, about 10 amino acid substitutions to about 225 amino acid substitutions, about 10 amino acid substitutions to about 220 amino acid substitutions substitution, 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 approximately 205 amino acid substitutions, approximately 10 amino acid substitutions to approximately 200 amino acid substitutions, approximately 10 amino acid substitutions 10 amino acid substitutions to about 195 amino acid substitutions, about 10 amino acid substitutions to about 190 amino acid substitutions, about 10 amino acid substitutions about 10 amino acid substitutions to about 185 amino acid substitutions, about 10 amino acid substitutions to about 180 amino acid substitutions, about 10 amino acid substitutions to about 175 amino acid substitutions, approximately 10 amino acid substitutions to approximately 170 amino acid substitutions, approximately 10 amino acid substitutions to approximately 165 amino acid substitutions amino acid substitution, about 10 amino acid substitutions to about 160 amino acid substitutions, about 10 amino acid substitutions to about 155 amino acids substitution, about 10 amino acid substitutions to about 150 amino acid substitutions, about 10 amino acid substitutions to about 145 amino acid substitutions, Approximately 10 to 140 amino acid substitutions, approximately 10 to 135 amino acid substitutions, approximately 10 to 140 amino acid substitutions amino acid substitutions to about 130 amino acid substitutions, about 10 amino acid substitutions to about 125 amino acid substitutions, about 10 amino acid substitutions Substitutions: Approximately 120 amino acid substitutions, approximately 10 amino acid substitutions, approximately 115 amino acid substitutions, approximately 10 amino acid substitutions Approximately 110 amino acid substitutions, approximately 10 to approximately 105 amino acid substitutions, approximately 10 to approximately 100 Amino acid substitution, about 10 amino acid substitutions to about 95 amino acid substitutions, about 10 amino acid substitutions to about 90 amino acids substitution, about 10 amino acid substitutions to about 85 amino acid substitutions, about 10 amino acid substitutions to about 80 amino acid substitutions, about 10 amino acid substitutions to about 75 amino acid substitutions, about 10 amino acid substitutions to about 70 amino acid substitutions, about 10 amino acid substitutions Approximately 65 amino acid substitutions, approximately 10 amino acid substitutions, approximately 60 amino acid substitutions, approximately 10 amino acid substitutions Approximately 55 amino acid substitutions, approximately 10 to approximately 50 amino acid substitutions, approximately 10 to approximately 45 amino acid substitutions amino acid substitution, about 10 to about 40 amino acid substitution, about 10 to about 35 amino acid substitution , about 10 to about 30 amino acid substitutions, about 10 to about 25 amino acid substitutions, amino acid substitution to about 20 amino acid substitutions, about 10 amino acid substitutions to about 15 amino acid substitutions, about 15 amino acid substitutions about 15 amino acid substitutions 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, approximately 15 amino acid substitutions to approximately 225 amino acid substitutions, approximately 15 amino acid substitutions to approximately 220 amino acid substitutions amino acid substitution, about 15 amino acid substitutions to about 215 amino acid substitutions, about 15 amino acid substitutions to about 210 amino acids substitution, about 15 amino acid substitutions to about 205 amino acid substitutions, about 15 amino acid substitutions to about 200 amino acid substitutions, Approximately 15 to 195 amino acid substitutions, approximately 15 to 190 amino acid substitutions, approximately 15 to 195 amino acid substitutions Amino acid substitutions - approximately 185 amino acid substitutions, approximately 15 amino acid substitutions - approximately 180 amino acid substitutions, approximately 15 amino acid substitutions Substitutions - approximately 175 amino acid substitutions, approximately 15 amino acid substitutions - approximately 170 amino acid substitutions, approximately 15 amino acid substitutions Approximately 165 amino acid substitutions, approximately 15 to approximately 160 amino acid substitutions, approximately 15 to approximately 155 Amino acid substitution, about 15 amino acid substitutions to about 150 amino acid substitutions, about 15 amino acid substitutions to about 145 amino acid substitutions amino acid substitution, about 15 to about 140 amino acid substitution, about 15 to about 135 amino acid substitution , about 15 to about 130 amino acid substitutions, about 15 to about 125 amino acid substitutions, about 15 Amino acid substitutions up to about 120 amino acid substitutions, about 15 amino acid substitutions to about 115 amino acid substitutions, about 15 amino acid substitutions Approximately 110 amino acid substitutions, approximately 15 amino acid substitutions to approximately 105 amino acid substitutions, approximately 15 amino acid substitutions ~ about 100 amino acid substitutions, ~ about 15 amino acid substitutions to about 95 amino acid substitutions, ~ about 15 amino acid substitutions to about 90 Amino acid substitution, about 15 to about 85 amino acid substitutions, about 15 to about 80 amino acid substitutions substitution, about 15 to about 75 amino acid substitutions, about 15 to about 70 amino acid substitutions, about 15 amino acid substitutions to about 65 amino acid substitutions, about 15 amino acid substitutions to about 60 amino acid substitutions, about 15 amino acid substitutions Approximately 55 amino acid substitutions, approximately 15 amino acid substitutions, approximately 50 amino acid substitutions, approximately 15 amino acid substitutions Approximately 45 amino acid substitutions, approximately 15 to approximately 40 amino acid substitutions, approximately 15 to approximately 35 amino acid substitutions amino acid substitution, about 15 to about 30 amino acid substitution, about 15 to about 25 amino acid substitution , about 15 to about 20 amino acid substitutions, about 20 to about 240 amino acid substitutions, about 20 Amino acid substitutions - approximately 235 amino acid substitutions, approximately 20 amino acid substitutions - approximately 230 amino acid substitutions, approximately 20 amino acid substitutions Approximately 225 amino acid substitutions, approximately 20 amino acid substitutions, approximately 220 amino acid substitutions, approximately 20 amino acid substitutions ~ about 215 amino acid substitutions, ~ about 20 amino acid substitutions ~ about 210 amino acid substitutions, ~ about 20 amino acid substitutions ~ about 20 5 amino acid substitutions, approximately 20 to approximately 200 amino acid substitutions, approximately 20 to approximately 195 amino acid substitutions amino acid substitution, about 20 amino acid substitutions to about 190 amino acid substitutions, about 20 amino acid substitutions to about 185 amino acid substitutions substitution, about 20 to about 180 amino acid substitutions, about 20 to about 175 amino acid substitutions, 20 amino acid substitutions to approximately 170 amino acid substitutions, approximately 20 amino acid substitutions to approximately 165 amino acid substitutions, approximately 20 amino acid substitutions Approximately 160 amino acid substitutions, approximately 20 amino acid substitutions, approximately 155 amino acid substitutions, approximately 20 amino acid substitutions 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, approximately 20 amino acid substitutions to approximately 135 amino acid substitutions, approximately 20 amino acid substitutions to approximately 130 amino acid substitutions amino acid substitution, about 20 amino acid substitutions to about 125 amino acid substitutions, about 20 amino acid substitutions to about 120 amino acids substitution, about 20 amino acid substitutions to about 115 amino acid substitutions, about 20 amino acid substitutions to about 110 amino acid substitutions, Approximately 20 to 105 amino acid substitutions, approximately 20 to 100 amino acid substitutions, approximately 20 amino acid substitutions to about 95 amino acid substitutions, about 20 amino acid substitutions to about 90 amino acid substitutions, about 20 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 substitution, about 20 to about 70 amino acid substitutions, about 20 to about 65 amino acid substitutions 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 amino acid substitutions Approximately 40 amino acid substitutions, approximately 20 amino acid substitutions, approximately 35 amino acid substitutions, approximately 20 amino acid substitutions Approximately 30 amino acid substitutions, approximately 20 to approximately 25 amino acid substitutions, approximately 25 to approximately 240 amino acid substitutions amino acid substitution, about 25 amino acid substitutions to about 235 amino acid substitutions, about 25 amino acid substitutions to about 230 amino acids substitution, about 25 amino acid substitutions to about 225 amino acid substitutions, about 25 amino acid substitutions to about 220 amino acid substitutions, Approximately 25 to 215 amino acid substitutions, approximately 25 to 210 amino acid substitutions, approximately 25 Amino acid substitutions - approximately 205 amino acid substitutions, approximately 25 amino acid substitutions - approximately 200 amino acid substitutions, approximately 10 amino acid substitutions Substitutions: Approximately 195 amino acid substitutions, approximately 25 amino acid substitutions, approximately 190 amino acid substitutions, approximately 25 amino acid substitutions Approximately 185 amino acid substitutions, approximately 25 to approximately 180 amino acid substitutions, approximately 25 to approximately 175 Amino acid substitutions, about 25 amino acid substitutions to about 170 amino acid substitutions, about 25 amino acid substitutions to about 165 amino acid substitutions amino acid substitution, about 25 to about 160 amino acid substitution, about 25 to about 155 amino acid substitution , about 25 to about 150 amino acid substitutions, about 25 to about 145 amino acid substitutions, about 25 Amino acid substitutions up to about 140 amino acid substitutions, about 25 amino acid substitutions to about 135 amino acid substitutions, about 25 amino acid substitutions Approximately 130 amino acid substitutions, approximately 25 amino acid substitutions, approximately 125 amino acid substitutions, approximately 25 amino acid substitutions ~ about 120 amino acid substitutions, ~ about 25 amino acid substitutions to about 115 amino acid substitutions, ~ about 25 amino acid substitutions to about 11 0 amino acid substitution, about 25 to about 105 amino acid substitutions, about 25 to about 100 amino acid substitutions amino acid substitution, about 25 to about 95 amino acid substitution, about 25 to about 90 amino acid substitution , about 25 to about 85 amino acid substitutions, about 25 to about 80 amino acid substitutions, amino acid substitutions ~ approximately 75 amino acid substitutions, approximately 25 amino acid substitutions to about 70 amino acid substitutions, about 25 amino acid substitutions to about 65 amino acid substitutions, about 25 amino acid substitutions Approximately 60 amino acid substitutions, approximately 25 amino acid substitutions, approximately 55 amino acid substitutions, approximately 25 amino acid substitutions Approximately 50 amino acid substitutions, approximately 25 to approximately 45 amino acid substitutions, approximately 25 to approximately 40 amino acid substitutions amino acid substitution, about 25 to about 35 amino acid substitution, about 25 to about 30 amino acid substitution , about 30 to about 240 amino acid substitutions, about 30 to about 235 amino acid substitutions, about 30 Amino acid substitutions up to about 230 amino acid substitutions, about 30 amino acid substitutions to about 225 amino acid substitutions, about 30 amino acid substitutions Approximately 220 amino acid substitutions, approximately 30 amino acid substitutions, approximately 215 amino acid substitutions, approximately 30 amino acid substitutions ~ about 210 amino acid substitutions, ~ about 30 amino acid substitutions ~ about 205 amino acid substitutions, ~ about 30 amino acid substitutions ~ about 20 0 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 amino acid substitution, about 30 amino acid substitution to about 185 amino acid substitution, about 30 amino acid substitution to about 180 amino acid substitution substitution, 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 approximately 165 amino acid substitutions, approximately 30 amino acid substitutions to approximately 160 amino acid substitutions, approximately 30 amino acid substitutions Approximately 155 amino acid substitutions, approximately 30 amino acid substitutions, approximately 150 amino acid substitutions, approximately 30 amino acid substitutions 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, approximately 30 amino acid substitutions to approximately 130 amino acid substitutions, approximately 30 amino acid substitutions to approximately 125 amino acid substitutions amino acid substitution, about 30 amino acid substitutions to about 120 amino acid substitutions, about 30 amino acid substitutions to about 115 amino acids substitution, about 30 amino acid substitutions to about 110 amino acid substitutions, about 30 amino acid substitutions to about 105 amino acid substitutions, Approximately 30 to 100 amino acid substitutions, approximately 30 to 95 amino acid substitutions, approximately 30 amino acid substitutions to about 90 amino acid substitutions, about 30 amino acid substitutions to about 85 amino acid substitutions, about 30 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 substitution, about 30 to about 65 amino acid substitutions, about 30 to about 60 amino acid substitutions substitution, 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 amino acid substitutions Approximately 35 amino acid substitutions, approximately 35 amino acid substitutions to approximately 240 amino acid substitutions, approximately 35 amino acid substitutions ~ about 235 amino acid substitutions, ~ about 35 amino acid substitutions ~ about 230 amino acid substitutions, ~ about 35 amino acid substitutions ~ about 22 5 amino acid substitutions, approximately 35 amino acid substitutions to approximately 220 amino acid substitutions, approximately 35 amino acid substitutions to approximately 215 amino acid substitutions amino acid substitution, about 35 amino acid substitution to about 210 amino acid substitution, about 35 amino acid substitution to about 205 amino acid substitution substitutions, about 35 to about 200 amino acid substitutions, about 35 to about 195 amino acid substitutions, 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 Approximately 180 amino acid substitutions, approximately 35 amino acid substitutions, approximately 175 amino acid substitutions, approximately 35 amino acid substitutions 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, approximately 35 amino acid substitutions to approximately 155 amino acid substitutions, approximately 35 amino acid substitutions to approximately 150 amino acid substitutions amino acid substitution, about 35 amino acid substitutions to about 145 amino acid substitutions, about 35 amino acid substitutions to about 140 amino acids substitution, about 35 amino acid substitutions to about 135 amino acid substitutions, about 35 amino acid substitutions to about 130 amino acid substitutions, Approximately 35 to 125 amino acid substitutions, approximately 35 to 120 amino acid substitutions, approximately 35 Amino acid substitutions - approximately 115 amino acid substitutions, approximately 35 amino acid substitutions - approximately 110 amino acid substitutions, approximately 35 amino acid substitutions Substitutions: Approximately 105 amino acid substitutions, approximately 35 amino acid substitutions, approximately 100 amino acid substitutions, approximately 35 amino acid substitutions Approximately 95 amino acid substitutions, approximately 35 to approximately 90 amino acid substitutions, approximately 35 to approximately 85 amino acid substitutions amino acid substitution, about 35 to about 80 amino acid substitution, about 35 to about 75 amino acid substitution , about 35 to about 70 amino acid substitutions, about 35 to about 65 amino acid substitutions, amino acid substitutions to about 60 amino acid substitutions, about 35 amino acid substitutions to about 55 amino acid substitutions, about 35 amino acid substitutions about 50 to about 50 amino acid substitutions, about 35 to about 45 amino acid substitutions, about 35 to about 40 Amino acid substitution, about 40 amino acid substitutions to about 240 amino acid substitutions, about 40 amino acid substitutions to about 235 amino acid substitutions amino acid substitution, about 40 to about 230 amino acid substitution, about 40 to about 225 amino acid substitution , about 40 to about 220 amino acid substitutions, about 40 to about 215 amino acid substitutions, about 40 Amino acid substitutions - approximately 210 amino acid substitutions, approximately 40 amino acid substitutions - approximately 205 amino acid substitutions, approximately 40 amino acid substitutions Approximately 200 amino acid substitutions, approximately 40 amino acid substitutions to approximately 195 amino acid substitutions, approximately 40 amino acid substitutions ~ about 190 amino acid substitutions, ~ about 40 amino acid substitutions ~ about 185 amino acid substitutions, ~ about 40 amino acid substitutions ~ about 18 0 amino acid substitution, about 40 amino acid substitutions to about 175 amino acid substitutions, about 40 amino acid substitutions to about 170 amino acid substitutions amino acid substitution, about 40 amino acid substitution to about 165 amino acid substitution, about 40 amino acid substitution to about 160 amino acid substitution substitution, about 40 to about 155 amino acid substitutions, about 40 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, about 40 amino acid substitutions Approximately 135 amino acid substitutions, approximately 40 amino acid substitutions, approximately 130 amino acid substitutions, approximately 40 amino acid substitutions substitutions to about 125 amino acid substitutions, about 40 amino acid substitutions to about 120 amino acid substitutions, about 40 amino acid substitutions to about 115 amino acid substitutions, approximately 40 amino acid substitutions to approximately 110 amino acid substitutions, approximately 40 amino acid substitutions to approximately 105 amino acid substitutions amino acid substitution, about 40 to about 100 amino acid substitution, about 40 to about 95 amino acid substitution substitution, about 40 to about 90 amino acid substitutions, about 40 to about 85 amino acid substitutions, about 40 amino acid substitutions to about 80 amino acid substitutions, about 40 amino acid substitutions to about 75 amino acid substitutions, about 40 amino acid substitutions Approximately 70 amino acid substitutions, approximately 40 amino acid substitutions, approximately 65 amino acid substitutions, approximately 40 amino acid substitutions Approximately 60 amino acid substitutions, approximately 40 to approximately 55 amino acid substitutions, approximately 40 to approximately 50 amino acid substitutions amino acid substitution, about 40 amino acid substitution to about 45 amino acid substitution, about 45 amino acid substitution to about 240 amino acid substitution substitution, 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, about 45 amino acid substitutions Approximately 215 amino acid substitutions, approximately 45 amino acid substitutions, approximately 210 amino acid substitutions, approximately 45 amino acid substitutions substitutions to about 205 amino acids, substitutions to about 200 amino acids, substitutions to about 45 amino acids 195 amino acid substitutions, approximately 45 amino acid substitutions to approximately 190 amino acid substitutions, approximately 45 amino acid substitutions to approximately 185 amino acid substitutions amino acid substitution, about 45 amino acid substitutions to about 180 amino acid substitutions, about 45 amino acid substitutions to about 175 amino acids substitution, about 45 amino acid substitutions to about 170 amino acid substitutions, about 45 amino acid substitutions to about 165 amino acid substitutions, Approximately 45 to 160 amino acid substitutions, approximately 45 to 155 amino acid substitutions, approximately 45 to 160 amino acid substitutions 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 Substitutions: Approximately 140 amino acid substitutions, approximately 45 amino acid substitutions: Approximately 135 amino acid substitutions, approximately 45 amino acid substitutions Approximately 130 amino acid substitutions, approximately 45 to approximately 125 amino acid substitutions, approximately 45 to approximately 120 Amino acid substitution, about 45 amino acid substitutions to about 115 amino acid substitutions, about 45 amino acid substitutions to about 110 amino acid substitutions amino acid substitution, about 45 to about 105 amino acid substitution, about 45 to about 100 amino acid substitution , about 45 to about 95 amino acid substitutions, about 45 to about 90 amino acid substitutions, amino acid substitutions to about 85 amino acid substitutions, about 45 amino acid substitutions to about 80 amino acid substitutions, about 45 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 substitution, about 45 to about 60 amino acid substitutions, about 45 to about 55 amino acid substitutions substitution, about 45 amino acid substitutions to about 50 amino acid substitutions, about 50 amino acid substitutions to about 240 amino acid substitutions, Approximately 50 to 235 amino acid substitutions, approximately 50 to 230 amino acid substitutions, approximately 50 Amino acid substitutions - approximately 225 amino acid substitutions, approximately 50 amino acid substitutions - approximately 220 amino acid substitutions, approximately 50 amino acid substitutions Substitutions ~ Approximately 215 amino acid substitutions, approximately 50 amino acid substitutions ~ Approximately 210 amino acid substitutions, approximately 50 amino acid substitutions Approximately 205 amino acid substitutions, approximately 50 to approximately 200 amino acid substitutions, approximately 50 to approximately 195 Amino acid substitution, about 50 amino acid substitutions to about 190 amino acid substitutions, about 50 amino acid substitutions to about 185 amino acid substitutions amino acid substitution, about 50 to about 180 amino acid substitution, about 50 to about 175 amino acid substitution , about 50 to about 170 amino acid substitutions, about 50 to about 165 amino acid substitutions, about 50 Amino acid substitutions up to about 160 amino acid substitutions, about 50 amino acid substitutions to about 155 amino acid substitutions, about 50 amino acid substitutions Approximately 150 amino acid substitutions, approximately 50 amino acid substitutions, approximately 145 amino acid substitutions, approximately 50 amino acid substitutions ~ about 140 amino acid substitutions, ~ about 50 amino acid substitutions ~ about 135 amino acid substitutions, ~ about 50 amino acid substitutions ~ about 13 0 amino acid substitution, about 50 to about 125 amino acid substitution, about 50 to about 120 amino acid substitution amino acid substitution, about 50 amino acid substitution to about 115 amino acid substitution, about 50 amino acid substitution to about 110 amino acid substitution substitution, about 50 to about 105 amino acid substitutions, about 50 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 Approximately 85 amino acid substitutions, approximately 50 amino acid substitutions, approximately 80 amino acid substitutions, approximately 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 amino acid substitution, about 50 to about 60 amino acid substitution, about 50 to about 55 amino acid substitution , about 60 to about 240 amino acid substitutions, about 60 to about 235 amino acid substitutions, about 60 Amino acid substitutions up to about 230 amino acid substitutions, about 60 amino acid substitutions to about 225 amino acid substitutions, about 60 amino acid substitutions Approximately 220 amino acid substitutions, approximately 60 amino acid substitutions, approximately 215 amino acid substitutions, approximately 60 amino acid substitutions ~ about 210 amino acid substitutions, ~ about 60 amino acid substitutions ~ about 205 amino acid substitutions, ~ about 60 amino acid substitutions ~ about 20 0 amino acid substitutions, about 60 to about 195 amino acid substitutions, about 60 to about 190 amino acid substitutions amino acid substitution, about 60 amino acid substitution to about 185 amino acid substitution, about 60 amino acid substitution to about 180 amino acid substitution substitution, about 60 to about 175 amino acid substitutions, about 60 to about 170 amino acid substitutions, about 60 amino acid substitutions to approximately 165 amino acid substitutions, approximately 60 amino acid substitutions to approximately 160 amino acid substitutions, approximately 60 amino acid substitutions Approximately 155 amino acid substitutions, approximately 60 amino acid substitutions, approximately 150 amino acid substitutions, approximately 60 amino acid substitutions 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, approximately 60 amino acid substitutions to approximately 130 amino acid substitutions, approximately 60 amino acid substitutions to approximately 125 amino acid substitutions amino acid substitution, about 60 amino acid substitutions to about 120 amino acid substitutions, about 60 amino acid substitutions to about 115 amino acids substitution, about 60 amino acid substitutions to about 110 amino acid substitutions, about 60 amino acid substitutions to about 105 amino acid substitutions, Approximately 60 to 100 amino acid substitutions, approximately 60 to 95 amino acid substitutions, approximately 60 amino acid substitutions to about 90 amino acid substitutions, about 60 amino acid substitutions to about 85 amino acid substitutions, about 60 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 substitution, about 60 amino acid substitutions to about 65 amino acid substitutions, about 70 amino acid substitutions to about 240 amino acid substitutions amino acid substitution, about 70 to about 235 amino acid substitution, about 70 to about 230 amino acid substitution , about 70 amino acid substitutions to about 225 amino acid substitutions, about 70 amino acid substitutions to about 220 amino acid substitutions, about 70 Amino acid substitutions - about 215 amino acid substitutions, about 70 amino acid substitutions - about 210 amino acid substitutions, about 10 amino acid substitutions Approximately 205 amino acid substitutions, approximately 70 amino acid substitutions, approximately 200 amino acid substitutions, approximately 70 amino acid substitutions ~ about 195 amino acid substitutions, ~ about 70 amino acid substitutions ~ about 190 amino acid substitutions, ~ about 70 amino acid substitutions ~ about 18 5 amino acid substitutions, approximately 70 to approximately 180 amino acid substitutions, approximately 70 to approximately 175 amino acid substitutions amino acid substitution, about 70 amino acid substitution to about 170 amino acid substitution, about 70 amino acid substitution to about 165 amino acid substitution substitution, about 70 to about 160 amino acid substitutions, about 70 to about 155 amino acid substitutions, 70 amino acid substitutions to approximately 150 amino acid substitutions, approximately 70 amino acid substitutions to approximately 145 amino acid substitutions, approximately 70 amino acid substitutions Approximately 140 amino acid substitutions, approximately 70 amino acid substitutions, approximately 135 amino acid substitutions, approximately 70 amino acid substitutions substitutions to about 130 amino acid substitutions, about 70 amino acid substitutions to about 125 amino acid substitutions, about 70 amino acid substitutions to about 120 amino acid substitutions, approximately 70 to approximately 115 amino acid substitutions, approximately 70 to approximately 110 amino acid substitutions amino acid substitution, about 70 amino acid substitutions to about 105 amino acid substitutions, about 70 amino acid substitutions to about 100 amino acids substitution, about 70 to about 95 amino acid substitutions, about 70 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 Approximately 75 amino acid substitutions substitution, about 80 to about 240 amino acid substitutions, about 80 to about 235 amino acid substitutions, 80 amino acid substitutions to approximately 230 amino acid substitutions, approximately 80 amino acid substitutions to approximately 225 amino acid substitutions, approximately 80 amino acid substitutions Approximately 220 amino acid substitutions, approximately 80 amino acid substitutions, approximately 215 amino acid substitutions, approximately 80 amino acid substitutions Approximately 210 amino acid substitutions, approximately 80 amino acid substitutions to approximately 205 amino acid substitutions, approximately 80 amino acid substitutions to approximately 200 amino acid substitutions, approximately 80 to approximately 195 amino acid substitutions, approximately 80 to approximately 190 amino acid substitutions amino acid substitution, about 80 amino acid substitutions to about 185 amino acid substitutions, about 80 amino acid substitutions to about 180 amino acids substitution, about 80 amino acid substitutions to about 175 amino acid substitutions, about 80 amino acid substitutions to about 170 amino acid substitutions, Approximately 80 to 165 amino acid substitutions, approximately 80 to 160 amino acid substitutions, approximately 80 Amino acid substitutions - approximately 155 amino acid substitutions, approximately 80 amino acid substitutions - approximately 150 amino acid substitutions, approximately 80 amino acid substitutions Substitutions ~ Approximately 145 amino acid substitutions, ~ Approximately 80 amino acid substitutions ~ Approximately 140 amino acid substitutions, ~ Approximately 80 amino acid substitutions Approximately 135 amino acid substitutions, approximately 80 amino acid substitutions to approximately 130 amino acid substitutions, approximately 80 amino acid substitutions to approximately 125 Amino acid substitution, about 80 amino acid substitutions to about 120 amino acid substitutions, about 80 amino acid substitutions to about 115 amino acid substitutions amino acid substitution, approximately 80 to approximately 110 amino acid substitution, approximately 80 to approximately 105 amino acid substitution , about 80 to about 100 amino acid substitutions, about 80 to about 95 amino acid substitutions, about 80 Amino acid substitutions up to about 90 amino acid substitutions, about 80 amino acid substitutions to about 85 amino acid substitutions, about 90 amino acids Substitutions: Approximately 240 amino acid substitutions, approximately 90 amino acid substitutions, approximately 235 amino acid substitutions, approximately 90 amino acid substitutions Approximately 230 amino acid substitutions, approximately 90 to approximately 225 amino acid substitutions, approximately 90 to approximately 220 Amino acid substitution, about 90 amino acid substitutions to about 215 amino acid substitutions, about 90 amino acid substitutions to about 210 amino acid substitutions amino acid substitution, approximately 90 to approximately 205 amino acid substitution, approximately 90 to approximately 200 amino acid substitution , about 90 to about 195 amino acid substitutions, about 90 to about 190 amino acid substitutions, about 90 Amino acid substitutions - approximately 185 amino acid substitutions, approximately 90 amino acid substitutions - approximately 180 amino acid substitutions, approximately 90 amino acid substitutions Approximately 175 amino acid substitutions, approximately 90 amino acid substitutions, approximately 170 amino acid substitutions, approximately 90 amino acid substitutions ~ about 165 amino acid substitutions, ~ about 90 amino acid substitutions ~ about 160 amino acid substitutions, ~ about 90 amino acid substitutions ~ about 15 5 amino acid substitutions, approximately 90 to approximately 150 amino acid substitutions, approximately 90 to approximately 145 amino acid substitutions amino acid substitution, about 90 amino acid substitution to about 140 amino acid substitution, about 90 amino acid substitution to about 135 amino acid substitution substitution, about 90 to about 130 amino acid substitutions, about 90 to about 125 amino acid substitutions, 90 amino acid substitutions to approximately 120 amino acid substitutions, approximately 90 amino acid substitutions to approximately 115 amino acid substitutions, approximately 90 amino acid substitutions Approximately 110 amino acid substitutions, approximately 90 amino acid substitutions, approximately 105 amino acid substitutions, approximately 90 amino acid substitutions substitutions to about 100 amino acid substitutions, about 90 amino acid substitutions to about 95 amino acid substitutions, about 100 amino acid substitutions to about 240 amino acid substitutions, approximately 100 to approximately 235 amino acid substitutions, approximately 100 to approximately 230 Amino acid substitution, about 100 amino acid substitutions to about 225 amino acid substitutions, about 100 amino acid substitutions to about 220 amino acid substitutions amino acid substitution, about 100 amino acid substitutions to about 215 amino acid substitutions, about 100 amino acid substitutions to about 210 amino acids Substitution, about 100 to about 205 amino acid substitutions, about 100 to about 200 amino acid substitutions , about 100 to about 195 amino acid substitutions, about 100 to about 190 amino acid substitutions, about 100 to about 185 amino acid substitutions, about 100 to about 180 amino acid substitutions, about 100 Amino acid substitutions - approximately 175 amino acid substitutions, approximately 100 amino acid substitutions - approximately 170 amino acid substitutions, approximately 100 amino acid substitutions Approximately 165 amino acid substitutions, approximately 100 amino acid substitutions, approximately 160 amino acid substitutions, approximately 100 amino acid substitutions Substitutions up to approximately 155 amino acid substitutions, approximately 100 amino acid substitutions to approximately 150 amino acid substitutions, approximately 100 amino acid substitutions ~ about 145 amino acid substitutions, ~ about 100 amino acid substitutions ~ about 140 amino acid substitutions, ~ about 100 amino acid substitutions ~ about 135 amino acid substitutions, approximately 100 to approximately 130 amino acid substitutions, approximately 100 to approximately 125 Amino acid substitution, about 100 amino acid substitutions to about 120 amino acid substitutions, about 100 amino acid substitutions to about 115 amino acid substitutions amino acid substitution, about 100 to about 110 amino acid substitution, about 100 to about 105 amino acid substitution Substitution, about 110 to about 240 amino acid substitutions, about 110 to about 235 amino acid substitutions , about 110 to about 230 amino acid substitutions, about 110 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 - approximately 210 amino acid substitutions, approximately 110 amino acid substitutions - approximately 205 amino acid substitutions, approximately 110 amino acid substitutions Approximately 200 amino acid substitutions, approximately 110 amino acid substitutions, approximately 195 amino acid substitutions, approximately 110 amino acid substitutions Substitutions up to approximately 190 amino acid substitutions, approximately 110 amino acid substitutions to approximately 185 amino acid substitutions, approximately 110 amino acid substitutions ~ about 180 amino acid substitutions, ~ about 110 amino acid substitutions ~ about 175 amino acid substitutions, ~ about 110 amino acid substitutions ~ about 170 amino acid substitutions, approximately 110 to approximately 165 amino acid substitutions, approximately 110 to approximately 160 Amino acid substitution, about 110 amino acid substitutions to about 155 amino acid substitutions, about 110 amino acid substitutions to about 150 amino acid substitutions amino acid substitution, about 110 amino acid substitution to about 145 amino acid substitution, about 110 amino acid substitution to about 140 amino acid substitution substitution, about 110 to about 135 amino acid substitutions, about 110 to about 130 amino acid substitutions , about 110 to about 125 amino acid substitutions, about 110 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 - approximately 235 amino acid substitutions, approximately 120 amino acid substitutions - approximately 230 amino acid substitutions, approximately 120 amino acid substitutions Approximately 225 amino acid substitutions, approximately 120 amino acid substitutions, approximately 220 amino acid substitutions, approximately 120 amino acid substitutions Substitutions up to approximately 215 amino acid substitutions, approximately 120 amino acid substitutions to approximately 210 amino acid substitutions, approximately 120 amino acid substitutions ~ about 205 amino acid substitutions, ~ about 120 amino acid substitutions ~ about 200 amino acid substitutions, ~ about 120 amino acid substitutions ~ about 195 amino acid substitutions, approximately 120 to approximately 190 amino acid substitutions, approximately 120 to approximately 185 Amino acid substitution, about 120 amino acid substitutions to about 180 amino acid substitutions, about 120 amino acid substitutions to about 175 amino acid substitutions amino acid substitution, about 120 amino acid substitutions to about 170 amino acid substitutions, about 100 amino acid substitutions to about 165 amino acids Substitution, about 120 to about 160 amino acid substitutions, about 120 to about 155 amino acid substitutions , about 120 to about 150 amino acid substitutions, about 120 to about 145 amino acid substitutions, about 120 to about 140 amino acid substitutions, about 120 to about 135 amino acid substitutions, about 120 Amino acid substitutions up to about 130 amino acid substitutions, about 120 amino acid substitutions to about 125 amino acid substitutions, about 130 amino acid substitutions Approximately 240 amino acid substitutions, approximately 130 amino acid substitutions, approximately 235 amino acid substitutions, approximately 130 amino acid substitutions Substitutions up to approximately 230 amino acid substitutions, approximately 130 amino acid substitutions to approximately 225 amino acid substitutions, approximately 130 amino acid substitutions ~ about 220 amino acid substitutions, ~ about 130 amino acid substitutions ~ about 215 amino acid substitutions, ~ about 130 amino acid substitutions ~ about 210 amino acid substitutions, approximately 130 to approximately 205 amino acid substitutions, approximately 130 to approximately 200 Amino acid substitution, about 130 amino acid substitutions to about 195 amino acid substitutions, about 130 amino acid substitutions to about 190 amino acid substitutions amino acid substitution, about 130 amino acid substitution to about 185 amino acid substitution, about 130 amino acid substitution to about 180 amino acid substitution Substitution, about 130 to about 175 amino acid substitutions, about 130 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 - approximately 145 amino acid substitutions, approximately 130 amino acid substitutions - approximately 140 amino acid substitutions, approximately 130 amino acid substitutions Approximately 135 amino acid substitutions, approximately 140 amino acid substitutions, approximately 240 amino acid substitutions, approximately 140 amino acid substitutions Substitutions up to approximately 235 amino acid substitutions, approximately 140 amino acid substitutions to approximately 230 amino acid substitutions, approximately 140 amino acid substitutions ~ about 225 amino acid substitutions, ~ about 140 amino acid substitutions ~ about 220 amino acid substitutions, ~ about 140 amino acid substitutions ~ about 215 amino acid substitutions, approximately 140 amino acid substitutions to approximately 210 amino acid substitutions, approximately 140 amino acid substitutions to approximately 205 Amino acid substitution, about 140 amino acid substitutions to about 200 amino acid substitutions, about 140 amino acid substitutions to about 195 amino acid substitutions amino acid substitution, about 140 amino acid substitutions to about 190 amino acid substitutions, about 140 amino acid substitutions to about 185 amino acids Substitution, about 140 to about 180 amino acid substitutions, about 140 to about 175 amino acid substitutions , about 140 to about 170 amino acid substitutions, about 140 to about 165 amino acid substitutions, about 140 to about 160 amino acid substitutions, about 140 to about 155 amino acid substitutions, about 140 Amino acid substitutions - approximately 150 amino acid substitutions, approximately 140 amino acid substitutions - approximately 145 amino acid substitutions, approximately 150 amino acid substitutions Approximately 240 amino acid substitutions, approximately 150 amino acid substitutions, approximately 235 amino acid substitutions, approximately 150 amino acid substitutions Substitutions up to approximately 230 amino acid substitutions, approximately 150 amino acid substitutions to approximately 225 amino acid substitutions, approximately 150 amino acid substitutions ~ about 220 amino acid substitutions, ~ about 150 amino acid substitutions ~ about 215 amino acid substitutions, ~ about 150 amino acid substitutions ~ about 210 amino acid substitutions, approximately 150 to approximately 205 amino acid substitutions, approximately 150 to approximately 200 Amino acid substitution, about 150 amino acid substitutions to about 195 amino acid substitutions, about 150 amino acid substitutions to about 190 amino acid substitutions amino acid substitution, about 150 amino acid substitution to about 185 amino acid substitution, about 150 amino acid substitution to about 180 amino acid substitution Substitution, 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 to about 160 amino acid substitutions, or or about 150 to about 155 amino acid substitutions, about 160 to about 240 amino acid substitutions, Approximately 160 to 235 amino acid substitutions, approximately 160 to 230 amino acid substitutions, approximately 16 0 amino acid substitutions to approximately 225 amino acid substitutions, approximately 160 amino acid substitutions to approximately 220 amino acid substitutions, approximately 160 amino acid substitutions 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 Approximately 205 amino acid substitutions, approximately 160 amino acid substitutions, approximately 200 amino acid substitutions, approximately 160 amino acid substitutions ~ Approximately 195 amino acid substitutions, ~ Approximately 160 amino acid substitutions ~ Approximately 190 amino acid substitutions, ~ Approximately 160 amino acid substitutions Approximately 185 amino acid substitutions, approximately 160 amino acid substitutions to approximately 180 amino acid substitutions, approximately 160 amino acid substitutions to approximately 17 5 amino acid substitutions, approximately 160 to approximately 170 amino acid substitutions, approximately 160 to approximately 165 amino acid substitutions amino acid substitution, about 170 amino acid substitutions to about 240 amino acid substitutions, about 170 amino acid substitutions to about 235 amino acid substitutions amino acid substitution, about 170 to about 230 amino acid substitution, about 170 to about 225 amino acid substitution substitution, about 170 amino acid substitutions to about 220 amino acid substitutions, about 170 amino acid substitutions to about 215 amino acid substitutions, Approximately 170 to 210 amino acid substitutions, approximately 170 to 205 amino acid substitutions, approximately 17 0 amino acid substitutions to approximately 200 amino acid substitutions, approximately 170 amino acid substitutions to approximately 195 amino acid substitutions, approximately 170 amino acid substitutions 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 185 amino acid substitutions Approximately 180 amino acid substitutions, approximately 170 amino acid substitutions, approximately 175 amino acid substitutions, approximately 180 amino acid substitutions Approximately 240 amino acid substitutions, approximately 180 amino acid substitutions, approximately 235 amino acid substitutions, approximately 180 amino acid substitutions Approximately 230 amino acid substitutions, approximately 180 to approximately 225 amino acid substitutions, approximately 180 to approximately 22 0 amino acid substitutions, about 180 to about 215 amino acid substitutions, about 180 to about 210 amino acid substitutions amino acid substitution, about 180 amino acid substitutions to about 205 amino acid substitutions, about 180 amino acid substitutions to about 200 amino acid substitutions amino acid substitution, about 180 to about 195 amino acid substitution, about 180 to about 190 amino acid substitution substitution, about 180 to about 185 amino acid substitutions, about 190 to about 240 amino acid substitutions, Approximately 190 to 235 amino acid substitutions, approximately 190 to 230 amino acid substitutions, approximately 19 0 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 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 Approximately 205 amino acid substitutions, approximately 190 amino acid substitutions, approximately 200 amino acid substitutions, approximately 190 amino acid substitutions Approximately 195 amino acid substitutions, approximately 200 amino acid substitutions, approximately 240 amino acid substitutions, approximately 200 amino acid substitutions Approximately 235 amino acid substitutions, approximately 200 amino acid substitutions to approximately 230 amino acid substitutions, approximately 200 amino acid substitutions to approximately 22 5 amino acid substitutions, approximately 200 to approximately 220 amino acid substitutions, approximately 200 to approximately 215 amino acid substitutions amino acid substitution, about 200 amino acid substitutions to about 210 amino acid substitutions, about 200 amino acid substitutions to about 205 amino acid substitutions amino acid substitution, about 205 to about 240 amino acid substitution, about 205 to about 235 amino acid substitution substitution, about 205 amino acid substitutions to about 230 amino acid substitutions, about 205 amino acid substitutions to about 225 amino acid substitutions, Approximately 205 to 220 amino acid substitutions, approximately 205 to 215 amino acid substitutions, approximately 20 5 amino acid substitutions to approximately 210 amino acid substitutions, approximately 210 amino acid substitutions to approximately 240 amino acid substitutions, approximately 210 amino acid substitutions 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 Acid substitutions ~ approximately 225 amino acid substitutions, Approximately 210 to 220 amino acid substitutions, approximately 210 to 215 amino acid substitutions, approximately 21 5 amino acid substitutions to approximately 240 amino acid substitutions, approximately 215 amino acid substitutions to approximately 235 amino acid substitutions, approximately 215 amino acid substitutions amino acid substitutions to about 230 amino acid substitutions, about 215 amino acid substitutions to about 225 amino acid substitutions, about 215 amino acid substitutions Approximately 220 amino acid substitutions, approximately 220 amino acid substitutions, approximately 240 amino acid substitutions, approximately 220 amino acid substitutions Approximately 235 amino acid substitutions, approximately 220 amino acid substitutions, approximately 230 amino acid substitutions, approximately 220 amino acid substitutions Approximately 225 amino acid substitutions, approximately 225 to approximately 240 amino acid substitutions, approximately 225 to approximately 23 5 amino acid substitutions, approximately 225 to approximately 230 amino acid substitutions, approximately 230 to approximately 240 amino acid substitutions A wild-type full-length otoferlinta containing approximately 230 to 235 amino acid substitutions. The sequence of the protein (e.g., wild-type human full-length otoferlin protein) can be included. The amino acids that are not conserved among wild-type otoferlin proteins from different species are , which can be mutated without losing activity, but wild-type otoferri derived from different species These amino acids, which are conserved among proteins, may be involved in the activity. amino acids that are not conserved between different species) and should not be mutated. Those skilled in the art will recognize that.

[0138] The active otoferlin protein may be, for example, from 1 amino acid to about 200 amino acids, from 1 amino acid to about 1 95 amino acids, 1 amino acid to approximately 190 amino acids, 1 amino acid to approximately 185 amino acids, 1 amino acid to approximately 180 Amino acids, 1 amino acid to about 175 amino acids, 1 amino acid to about 170 amino acids, 1 amino acid to about 165 Amino acids, 1 amino acid to about 160 amino acids, 1 amino acid to about 155 amino acids, 1 amino acid to about 150 amino acids amino acids, 1 amino acid to about 145 amino acids, 1 amino acid to about 140 amino acids, 1 amino acid to about 135 amino acids amino acids, 1 amino acid to about 130 amino acids, 1 amino acid to about 125 amino acids, 1 amino acid to about 120 amino acids Acid, 1 amino acid to about 115 amino acids, 1 amino acid to about 110 amino acids, 1 amino acid to about 105 amino acids , 1 amino acid to about 100 amino acids, 1 amino acid to about 95 amino acids, 1 amino acid to about 90 amino acids, 1 Amino acids up to about 85 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 75 amino acids, 1 amino acid 1 amino acid to about 70 amino acids, 1 amino acid to about 65 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid up to about 55 amino acids, 1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 4 0 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid 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 amino acid to about 9 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 7 amino acids, 1 amino acid 1 amino acid to about 6 amino acids, 1 amino acid to about 5 amino acids, 1 amino acid to about 4 amino acids, 1 amino acid to about 3 amino acids, about 2 amino acids to about 200 amino acids, about 2 amino acids to about 195 amino acids, about 2 amino acids Approximately 190 amino acids, approximately 2 to 185 amino acids, approximately 2 to 180 amino acids, approximately 2 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 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 to about 145 amino acids, about 2 to about 140 amino acids, about 2 to about 135 amino acids amino acids, about 2 to about 130 amino acids, about 2 to about 125 amino acids, about 2 to about 120 amino acids 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 1 05 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acids to about 95 amino acids, about 2 amino acids Approximately 90 amino acids, approximately 2 to approximately 85 amino acids, approximately 2 to approximately 80 amino acids, approximately 2 amino acids up to about 75 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to about 65 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 55 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 60 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 amino acids to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 20 amino acids, about 2 Amino acids up 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 up 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 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 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 amino acids, about 3 amino acids to about 165 amino acids, about 3 amino acids to about 160 amino acids, about 3 amino acids to about 155 amino acids 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 1 40 amino acids, about 3 amino acids to about 135 amino acids, about 3 amino acids to about 130 amino acids, about 3 amino acids ~ about 125 amino acids, ~ about 3 amino acids ~ about 120 amino acids, ~ about 3 amino acids ~ about 115 amino acids, ~ about 3 amino acids 3 to about 110 amino acids, about 3 to about 105 amino acids, about 3 to about 100 amino acids, about 3 Amino acids up 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 to about 65 amino acids, about 3 to about 60 amino acids, about 3 to about 55 amino acids , about 3 to about 50 amino acids, about 3 to about 45 amino acids, about 3 to about 40 amino acids amino acids, about 3 to about 35 amino acids, about 3 to about 30 amino acids, about 3 to about 25 amino acids amino acids, about 3 to about 20 amino acids, about 3 to about 15 amino acids, about 3 to about 10 amino acids amino acids, about 3 to about 9 amino acids, about 3 to about 8 amino acids, about 3 to about 7 amino acids amino acids, about 3 amino acids to about 6 amino acids, about 3 amino acids to about 5 amino acids, about 4 amino acids to about 200 amino acids about 4 amino acids to about 195 amino acids, about 4 amino acids to about 190 amino acids, about 4 amino acids to about 185 Amino acids, about 4 amino acids to about 180 amino acids, about 4 amino acids to about 175 amino acids, about 4 amino acids or more Approximately 170 amino acids, approximately 4 amino acids to approximately 165 amino acids, approximately 4 amino acids to approximately 160 amino acids, approximately 4 amino acids about 4 amino acids to about 155 amino acids, about 4 amino acids to about 150 amino acids, about 4 amino acids to about 145 amino acids, about 4 amino acids 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 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 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 7 0 amino acids, about 4 amino acids to about 65 amino acids, 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 About 40 amino acids, about 4 to about 35 amino acids, about 4 to about 30 amino acids, about 4 amino acids up 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 to about 10 amino acids, about 4 to about 9 amino acids, about 4 to about 8 amino acids, about 4 amino acids about 4 to about 6 amino acids, about 5 to about 200 amino acids, about 5 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 190 amino acids 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 amino acids, about 5 to about 150 amino acids, about 5 to about 145 amino acids, about 5 to about 140 amino acids 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 1 25 amino acids, about 5 to about 120 amino acids, about 5 to about 115 amino acids, about 5 amino acids ~ about 110 amino acids, ~ about 5 amino acids to about 105 amino acids, ~ about 5 amino acids to about 100 amino acids, ~ about 5 amino acids about 5 amino acids to about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 95 amino acids amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 Amino acids up to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids 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 Acid, about 5 to about 9 amino acids, about 5 to about 8 amino acids, about 5 to about 7 amino acids , about 6 amino acids to about 200 amino acids, about 6 amino acids to about 195 amino acids, about 6 amino acids to about 190 amino acids amino acids, about 6 amino acids to about 185 amino acids, about 6 amino acids to about 180 amino acids, about 6 amino acids to about 175 Amino acids, about 6 amino acids to about 170 amino acids, about 6 amino acids to about 165 amino acids, about 6 amino acids or more Approximately 160 amino acids, approximately 6 to 155 amino acids, approximately 6 to 150 amino acids, approximately 6 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 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 amino acids, about 6 to about 100 amino acids, about 6 to about 95 amino acids, about 6 to about 90 amino acids 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 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 4 5 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids or more About 15 amino acids, about 6 to about 10 amino acids, about 6 to about 9 amino acids, about 6 amino acids or more About 8 amino acids, about 7 to about 200 amino acids, about 7 to about 195 amino acids, about 7 amino acids ~ about 190 amino acids, ~ about 7 amino acids ~ about 185 amino acids, ~ about 7 amino acids ~ about 180 amino acids, ~ about 7 amino acids 7 to about 175 amino acids, about 7 to about 170 amino acids, about 7 to about 165 amino acids, about 7 Amino acids up 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 amino acids, about 7 amino acids to about 130 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 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 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 90 amino acids 7 to about 75 amino acids, about 7 to about 70 amino acids, about 7 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 Amino acids up to about 45 amino acids, about 7 amino acids to about 40 amino acids, about 7 amino acids 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 amino acids to about 200 amino acids, about 8 amino acids to about 195 amino acids, about 8 amino acids to about 190 amino acids about 8 amino acids to about 185 amino acids, about 8 amino acids to about 180 amino acids, about 8 amino acids to about 175 amino acids amino acids, about 8 amino acids to about 170 amino acids, about 8 amino acids to about 165 amino acids, about 8 amino acids to about 1 60 amino acids, about 8 amino acids to about 155 amino acids, about 8 amino acids to about 150 amino acids, about 8 amino acids ~ about 145 amino acids, ~ about 8 amino acids ~ about 140 amino acids, ~ about 8 amino acids ~ about 135 amino acids, ~ about 8 amino acids 8 to about 130 amino acids, about 8 to about 125 amino acids, about 8 to about 120 amino acids, about 8 Amino acids up to about 115 amino acids, about 8 amino acids to about 110 amino acids, about 8 amino acids to about 105 amino acids , about 8 amino acids to about 100 amino acids, about 8 amino acids to about 95 amino acids, 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 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 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 3 0 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 to about 10 amino acids, about 10 to about 200 amino acids, about 10 amino acids ~ about 195 amino acids, ~ about 10 amino acids, ~ about 190 amino acids, ~ about 10 amino acids, ~ about 185 amino acids, ~ about 10 Amino acids up to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids Acid, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 15 5 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 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 1 0 to about 125 amino acids, about 10 to about 120 amino acids, about 10 to about 115 amino acids amino acids, about 10 to about 110 amino acids, about 10 to about 105 amino acids, about 10 to about 10 0 amino acids, about 10 to about 95 amino acids, about 10 to about 90 amino acids, about 10 amino acids ~ about 85 amino acids, ~ about 10 amino acids ~ about 80 amino acids, ~ about 10 amino acids ~ about 75 amino acids, ~ about 10 amino acids 10 to about 70 amino acids, about 10 to about 65 amino acids, about 10 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 to about 30 amino acids, about 10 to about 25 amino acids, about 10 to about 20 amino acids Acid, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 200 amino acids, about 15 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 ~ about 180 amino acids, ~ about 15 amino acids ~ about 175 amino acids, ~ about 15 amino acids ~ about 170 amino acids, ~ about 15 Amino acids up to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids amino acids, about 15 to about 150 amino acids, about 15 to about 145 amino acids, about 15 to about 14 0 amino acids, about 15 amino acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15 amino acids about 15 to about 125 amino acids, about 15 to about 120 amino acids, about 15 to about 115 amino acids, about 1 5 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 to about 95 amino acids, about 15 to about 90 amino acids, about 15 to about 85 amino acids 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 to about 65 amino acids, about 15 to about 60 amino acids, about 15 amino acids about 55 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 45 amino acids, about 15 amino acids to about 50 amino acids about 15 to about 40 amino acids, about 15 to about 35 amino acids, about 15 to about 30 amino acids, about 15 Amino acids up 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 ~ about 170 amino acids, ~ about 20 amino acids ~ about 165 amino acids, ~ about 20 amino acids ~ about 160 amino acids, ~ about 20 Amino acids up to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids amino acids, about 20 to about 140 amino acids, about 20 to about 135 amino acids, about 20 to about 13 0 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 2 0 to about 100 amino acids, about 20 to about 95 amino acids, about 20 to about 90 amino acids , about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 Amino acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids or more About 45 amino acids, about 20 to about 40 amino acids, about 20 to about 35 amino acids, about 20 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 Amino acids up to about 195 amino acids, about 25 amino acids to about 190 amino acids, about 25 amino acids to about 185 amino acids amino acids, about 25 to about 180 amino acids, about 25 to about 175 amino acids, about 25 to about 17 0 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids about 25 to about 155 amino acids, about 25 to about 150 amino acids, about 25 to about 145 amino acids, about 2 5 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 to about 125 amino acids, about 25 to about 120 amino acids, about 25 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 about 25 to about 100 amino acids, about 25 to about 95 amino acids, about 25 to about 90 amino acids, about 25 Amino acids up 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 to about 70 amino acids, about 25 to about 65 amino acids, about 25 to about 60 amino acids about 25 to about 55 amino acids, about 25 to about 50 amino acids, about 25 to about 45 amino acids amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 to about 200 amino acids, about 30 to about 195 amino acids, about 30 amino acids about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 3 0 to about 175 amino acids, about 30 to about 170 amino acids, about 30 to about 165 amino acids amino acids, about 30 to about 160 amino acids, about 30 to about 155 amino acids, about 30 to about 15 0 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 3 0 to about 120 amino acids, about 30 to about 115 amino acids, about 30 to about 110 amino acids Acid, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 Amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids or more Approximately 80 amino acids, approximately 30 to approximately 75 amino acids, approximately 30 to approximately 70 amino acids, approximately 30 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 65 amino acids amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 200 amino acids, about 35 amino acids to about 195 amino acids amino acids, about 35 amino acids to about 190 amino acids, about 35 amino acids to about 185 amino acids, about 35 amino acids to about 18 0 amino acids, about 35 amino acids to about 175 amino acids, about 35 amino acids to about 170 amino acids, about 35 amino acids about 35 amino acids to about 160 amino acids, about 35 amino acids to about 155 amino acids, about 3 5 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 Acid, about 35 amino acids to about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino acids to about 12 5 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids about 35 to about 105 amino acids, about 35 to about 100 amino acids, about 3 5 to about 95 amino acids, about 35 to about 90 amino acids, about 35 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 amino acids, about 35 to about 65 amino acids, about 35 to about 60 amino acids, about 35 to about 55 Amino acids, about 35 to about 50 amino acids, about 35 to about 45 amino acids, about 35 amino acids or more About 40 amino acids, about 40 to about 200 amino acids, about 40 to about 195 amino acids, about 40 amino acids 40 to about 190 amino acids, about 40 to about 185 amino acids, about 40 to about 180 amino acids, About 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids 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 Approximately 150 amino acids, approximately 40 to 145 amino acids, approximately 40 to 140 amino acids, approximately 40 amino acids 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 to about 120 amino acids, about 40 to about 115 amino acids, about 40 to about 110 Amino acids, about 40 to about 105 amino acids, about 40 to about 100 amino acids, about 40 amino acids ~ about 95 amino acids, ~ about 40 amino acids, ~ about 90 amino acids, ~ about 40 amino acids, ~ about 85 amino acids, ~ about 40 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 Amino acids up to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, About 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 200 amino acids amino acids, about 45 amino acids to about 195 amino acids, about 45 amino acids 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 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 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 140 amino acids Approximately 130 amino acids, approximately 45 to approximately 125 amino acids, approximately 45 to approximately 120 amino acids, approximately 45 amino acids 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 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 to about 70 amino acids, about 45 to about 65 amino acids, about 45 amino acids ~ about 60 amino acids, ~ about 45 amino acids, ~ about 55 amino acids, ~ about 45 amino acids, ~ about 50 amino acids, about 50 to about 200 amino acids, about 50 to about 195 amino acids, about 50 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 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids Approximately 160 amino acids, approximately 50 to 155 amino acids, approximately 50 to 150 amino acids, approximately 50 amino acids 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 to about 130 amino acids, about 50 to about 125 amino acids, about 50 to about 120 Amino acids, about 50 to about 115 amino acids, about 50 to about 110 amino acids, about 50 amino acids ~ about 105 amino acids, ~ about 50 amino acids, ~ about 100 amino acids, ~ about 50 amino acids, ~ about 95 amino acids, ~ about 50 amino acids 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 to about 75 amino acids, about 50 to about 70 amino acids, about 50 to about 65 amino acids , about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 200 amino acids amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 190 amino acids Approximately 185 amino acids, approximately 55 to approximately 180 amino acids, approximately 55 to approximately 175 amino acids, approximately 55 amino acids amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids , about 55 amino acids to about 155 amino acids, about 55 amino acids 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 ~ about 130 amino acids, ~ about 55 amino acids ~ about 125 amino acids, ~ about 55 amino acids ~ about 120 amino acids, ~ about 55 Amino acids up to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids Acid, 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 or more About 75 amino acids, about 55 to about 70 amino acids, about 55 to about 65 amino acids, about 55 amino acids 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 up to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids Acid, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 16 5 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 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 6 0 to about 135 amino acids, about 60 to about 130 amino acids, about 60 to about 125 amino acids 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 11 0 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 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 95 amino acids 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 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 18 0 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 6 5 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 Acid, about 65 amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 12 5 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids about 65 to about 105 amino acids, about 65 to about 100 amino acids, about 6 5 to about 95 amino acids, about 65 to about 90 amino acids, about 65 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 amino acids, about 70 amino acids ~ about 200 amino acids, about 70 amino acids ~ Approximately 195 amino acids, approximately 70 to approximately 190 amino acids, approximately 70 to approximately 185 amino acids, approximately 70 amino acids 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 to about 150 amino acids, about 70 to about 145 amino acids, about 70 amino acids ~ about 140 amino acids, ~ about 70 amino acids ~ about 135 amino acids, ~ about 70 amino acids ~ about 130 amino acids, ~ about 70 Amino acids up to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids 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 10 0 amino acids, about 70 to about 95 amino acids, about 70 to about 90 amino acids, about 70 amino acids ~ about 85 amino acids, ~ about 70 amino acids, ~ about 80 amino acids, ~ about 70 amino acids, ~ about 75 amino acids, ~ about 75 amino acids about 75 to about 200 amino acids, about 75 to about 195 amino acids, about 75 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 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 165 amino acids Approximately 160 amino acids, approximately 75 to approximately 155 amino acids, approximately 75 to approximately 150 amino acids, approximately 75 amino acids 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 to about 115 amino acids, about 75 to about 110 amino acids, about 75 amino acids ~ about 105 amino acids, ~ about 75 amino acids, ~ about 100 amino acids, ~ about 75 amino acids, ~ about 95 amino acids, 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 approximately 200 amino acids, approximately 80 amino acids to approximately 195 amino acids, approximately 80 amino acids to approximately 190 amino acids 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 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 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 130 amino acids Approximately 120 amino acids, approximately 80 to 115 amino acids, approximately 80 to 110 amino acids, approximately 80 amino acids 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 to about 200 amino acids 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 85 to about 165 amino acids, 85 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 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 140 amino acids Approximately 130 amino acids, approximately 85 to approximately 125 amino acids, approximately 85 to approximately 120 amino acids, approximately 85 amino acids 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 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids Approximately 190 amino acids, approximately 90 to 185 amino acids, approximately 90 to 180 amino acids, approximately 90 amino acids amino acids to about 175 amino acids, about 90 amino acids to about 170 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 ~ about 135 amino acids, ~ about 90 amino acids, ~ about 130 amino acids, ~ about 90 amino acids, ~ about 125 amino acids, ~ about 90 Amino acids up to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids Acid, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 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 ~ about 190 amino acids, ~ about 95 amino acids, ~ about 185 amino acids, ~ about 95 amino acids, ~ about 180 amino acids, ~ about 95 Amino acids up to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids amino acids, about 95 to about 160 amino acids, about 95 to about 155 amino acids, about 95 to about 15 0 amino acids, about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino acids, about 95 amino acids about 95 amino acids to about 130 amino acids, about 95 amino acids to about 125 amino acids, about 9 5 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 to about 105 amino acids, about 95 to about 100 amino acids, about 100 to about 2 00 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids 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 Acid, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids Approximately 160 amino acids, approximately 100 to approximately 155 amino acids, approximately 100 to approximately 150 amino acids, approximately 100 Amino acids up to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids ~ about 120 amino acids, ~ about 100 amino acids ~ about 115 amino acids, ~ about 100 amino acids ~ about 110 amino acids, ~ about 1 00 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 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105 amino acids about 105 to about 170 amino acids, about 105 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 105 to 130 amino acids, 105 to 135 amino acids, 105 to 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 1 15 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids 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 Acid, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to Approximately 170 amino acids, approximately 110 to approximately 165 amino acids, approximately 110 to approximately 160 amino acids, approximately 110 Amino acids up to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 1 10 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 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids about 115 to about 170 amino acids, about 115 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 115 to 130 amino acids, 115 to 135 amino acids, 115 to 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 2 00 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids 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 Acid, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids Approximately 160 amino acids, approximately 120 to approximately 155 amino acids, approximately 120 to approximately 150 amino acids, approximately 120 Amino acids up to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids amino acids, about 120 to about 130 amino acids, about 120 to about 125 amino acids, about 125 amino acids ~ about 200 amino acids, ~ about 125 amino acids, ~ about 195 amino acids, ~ about 125 amino acids, ~ about 190 amino acids, 25 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 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids 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 130 to 190 amino acids, 130 to 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 1 70 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids 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 Acid, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids About 200 amino acids, about 135 to about 195 amino acids, about 135 to about 190 amino acids, about 135 Amino acids up to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids ~ about 160 amino acids, ~ about 135 amino acids, ~ about 155 amino acids, ~ about 135 amino acids, ~ about 150 amino acids, 35 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 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 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 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 145 to 160 amino acids, 145 to 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 1 95 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids 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 Acid, 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 to about 200 amino acids, about 155 to about 195 amino acids, about 155 Amino acids up to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids ~ about 165 amino acids, ~ about 155 amino acids ~ about 160 amino acids, ~ about 160 amino acids ~ about 200 amino acids, ~ about 1 60 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 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids 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 ~ about 170 amino acids, ~ about 170 amino acids ~ about 200 amino acids, ~ about 170 amino acids ~ 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 1 80 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids Acid, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids Approximately 195 amino acids, approximately 180 to approximately 190 amino acids, approximately 180 to approximately 185 amino acids, approximately 185 Amino acids up to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids acid, approx. 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 200 amino acids wild-type full-length otoferlin protein (e.g., wild-type human full-length otoferlin protein) lacking the amino acid The sequence of the wild-type full-length nucleotide sequence of the ... In some embodiments, two or more deletions are made from the sequence of the toferrin protein. The missing amino acids may be consecutive in the sequence of the wild-type full-length protein. In other examples where 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. Amino acids that are not conserved among wild-type full-length otoferlin proteins from different species can be deleted without loss of activity, but wild-type full-length otoconia from different species These amino acids, which are conserved among feline proteins, may be involved in their activity. amino acids that are not conserved among different species and therefore should not be deleted. Those skilled in the art will recognize that this is not the case.

[0139] In some instances, an active otoferlin protein may comprise, for example, from 1 amino acid to about 100 amino acids. Acid, 1 amino acid to about 95 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 85 amino acids, 1 Amino acids up to about 80 amino acids, 1 amino acid to about 75 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid 1 amino acid to about 65 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about 55 amino acids, 1 amino acid ~ about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 3 5 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 20 amino acids 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 amino acid 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 amino acids to about 95 amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids or more Approximately 85 amino acids, approximately 2 to approximately 80 amino acids, approximately 2 to approximately 75 amino acids, approximately 2 amino acids up to about 70 amino acids, about 2 amino acids to about 65 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 55 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 45 amino acids, about 2 amino acids to about 55 amino acids 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 15 amino acids, about 2 Amino acids up 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 up 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 amino acids to 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 up 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 to about 75 amino acids, about 3 to about 70 amino acids, about 3 to about 65 amino acids, About 3 to about 60 amino acids, about 3 to about 55 amino acids, about 3 to about 50 amino acids , about 3 to about 45 amino acids, about 3 to about 40 amino acids, about 3 to about 35 amino acids amino acids, about 3 to about 30 amino acids, about 3 to about 25 amino acids, about 3 to about 20 amino acids amino acids, about 3 to about 15 amino acids, about 3 to about 10 amino acids, about 3 to about 9 amino acids 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 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 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, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 55 amino acids, about 4 amino acids to about 5 0 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 About 20 amino acids, about 4 to about 15 amino acids, about 4 to about 10 amino acids, about 4 amino acids ~ 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 About 6 amino acids, about 5 to about 100 amino acids, about 5 to about 95 amino acids, about 5 amino acids up to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 75 amino acids 5 to about 60 amino acids, about 5 to about 55 amino acids, about 5 to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 Amino acids up to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids 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 100 amino acids, about 6 to about 95 amino acids, about 6 to about 90 amino acids, about 6 to about 85 amino acids, About 6 to about 80 amino acids, about 6 to about 75 amino acids, about 6 to about 70 amino acids , about 6 to about 65 amino acids, about 6 to about 60 amino acids, about 6 to about 55 amino acids about 6 to about 50 amino acids, about 6 to about 45 amino acids, about 6 to about 40 amino acids amino acids, about 6 to about 35 amino acids, about 6 to about 30 amino acids, about 6 to about 25 amino acids 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 amino acids to about 95 amino acids, about 7 amino acids to about 90 amino acids, about 7 amino acids to about 8 amino acids 5 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 About 55 amino acids, about 7 to about 50 amino acids, about 7 to about 45 amino acids, about 7 amino acids up to about 40 amino acids, about 7 amino acids to about 35 amino acids, about 7 amino acids to about 30 amino acids, about 7 amino acids to about 25 amino acids, about 7 amino acids to about 20 amino acids, about 7 amino acids to about 15 amino acids, about 7 amino acids to about 16 amino acids about 7 amino acids to about 9 amino acids, about 8 amino acids to about 100 amino acids, about 8 amino acids to about 10 amino acids to about 95 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids to about 85 amino acids, about 8 Amino acids up to about 80 amino acids, about 8 amino acids to about 75 amino acids, about 8 amino acids 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 , 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 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 9 0 amino acids, about 10 to about 85 amino acids, about 10 to about 80 amino acids, about 10 amino acids ~ about 75 amino acids, ~ about 10 amino acids ~ about 70 amino acids, ~ about 10 amino acids ~ about 65 amino acids, ~ about 10 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 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 to about 30 amino acids, about 10 to about 25 amino acids, about 10 to about 20 amino acids 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 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids or more About 80 amino acids, about 20 to about 75 amino acids, about 20 to about 70 amino acids, about 20 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 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids 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 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 to about 70 amino acids, about 30 to about 65 amino acids, about 30 amino acids ~ about 60 amino acids, ~ about 30 amino acids to about 55 amino acids, ~ about 30 amino acids to about 50 amino acids, ~ about 30 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 Amino acids up to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids , about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids 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 amino acids or more About 45 amino acids, about 50 to about 100 amino acids, about 50 to about 95 amino acids, about 50 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 Amino acids up to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, About 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 60 amino acids to about 100 amino acids 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 amino acids or more Approximately 70 amino acids, approximately 60 to approximately 65 amino acids, approximately 70 to approximately 100 amino acids, approximately 70 amino acids about 70 to about 95 amino acids, about 70 to about 90 amino acids, about 70 to about 85 amino acids, about 70 Amino acids up to about 80 amino acids, about 70 amino acids to about 75 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 amino acids, about 90 to about 100 amino acids, about 90 to about 95 amino acids, or about 95 amino acids a wild-type whole genome sequence having approximately 100 amino acids removed from its N-terminus and / or its C-terminus; It may comprise the sequence of the long otoferlin protein.

[0140] In some embodiments, an active otoferlin protein may be, for example, from 1 amino acid to 50 amino acids. amino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 35 amino acids, 1 amino acid 1 amino acid ~ 30 amino acids, 1 amino acid ~ 25 amino acids, 1 amino acid ~ 20 amino acids, 1 amino acid ~ 15 amino acids Amino acids, 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 Amino acid ~ 7 amino acids, 1 amino acid ~ 6 amino acids, 1 amino acid ~ 5 amino acids, 1 amino acid ~ 4 amino acids amino acids, 1 to 3 amino acids, approximately 2 to 50 amino acids, approximately 2 to 45 amino acids, approximately 2 to 40 amino acids, approximately 2 to 35 amino acids, approximately 2 to 30 amino acids, approximately 2 amino acids 2 to 25 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 amino acids ~10 amino acids, ~2 amino acids ~9 amino acids, ~2 amino acids ~8 amino acids, ~2 amino acids ~7 amino acids 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, approximately 3 to 45 amino acids, approximately 3 to 40 amino acids, approximately 3 amino acids Approximately 3 to 35 amino acids, approximately 3 to 30 amino acids, approximately 3 to 25 amino acids, approximately 3 amino acids ~20 amino acids, about 3 amino acids to 15 amino acids, about 3 amino acids to 10 amino acids, about 3 amino acids to 9 amino acids amino acids, about 3 to 8 amino acids, about 3 to 7 amino acids, about 3 to 6 amino acids, Approximately 3 to 5 amino acids, approximately 4 to 50 amino acids, approximately 4 to 45 amino acids, approximately 4 amino acids amino acids to 40 amino acids, about 4 amino acids to 35 amino acids, about 4 amino acids to 30 amino acids, about 4 amino acids About 4 to 25 amino acids, about 4 to 20 amino acids, about 4 to 15 amino acids, about 4 to 1 0 amino acids, about 4 to 9 amino acids, about 4 to 8 amino acids, about 4 to 7 amino acids Acid, about 4 amino acids to 6 amino acids, about 5 amino acids to 50 amino acids, about 5 amino acids to 45 amino acids, about 5 to 40 amino acids, approximately 5 to 35 amino acids, approximately 5 to 30 amino acids, approximately 5 amino acids Approximately 5 to 25 amino acids, approximately 5 to 20 amino acids, approximately 5 to 15 amino acids, approximately 5 amino acids ~10 amino acids, ~5 amino acids ~9 amino acids, ~5 amino acids ~8 amino acids, ~5 amino acids ~7 amino acids amino acids, approximately 6 to 50 amino acids, approximately 6 to 45 amino acids, approximately 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 Amino acids up to 20 amino acids, about 6 amino acids to 15 amino acids, about 6 amino acids to 10 amino acids, about 6 amino acids Approximately 6 to 8 amino acids, approximately 7 to 50 amino acids, approximately 7 to 4 5 amino acids, about 7 to 40 amino acids, about 7 to 35 amino acids, about 7 to 30 amino acids amino acids, approximately 7 to 25 amino acids, approximately 7 to 20 amino acids, approximately 7 to 15 amino acids , about 7 to 10 amino acids, about 7 to 9 amino acids, about 8 to 50 amino acids, about 8 Amino acids up to 45 amino acids, about 8 amino acids to 40 amino acids, about 8 amino acids to 35 amino acids, about 8 amino acids Approximately 8 to 30 amino acids, approximately 8 to 25 amino acids, approximately 8 to 20 amino acids, approximately 8 amino acids ~15 amino acids, about 8 amino acids to 10 amino acids, about 10 amino acids to 50 amino acids, about 10 amino acids to 4 amino acids 5 amino acids, approximately 10 to 40 amino acids, approximately 10 to 35 amino acids, approximately 10 to 30 Amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids amino acids, about 15 to 50 amino acids, about 15 to 45 amino acids, about 15 to 40 amino acids amino acids, approximately 15 to 35 amino acids, approximately 15 to 30 amino acids, approximately 15 to 25 amino acids Acid, 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, Approximately 20 to 25 amino acids, approximately 25 to 50 amino acids, approximately 25 to 45 amino acids, approximately 25-40 amino acids, approximately 25-35 amino acids, approximately 25-30 amino acids, approximately 30 Amino acids up to 50 amino acids, approximately 30 amino acids to 45 amino acids, approximately 30 amino acids to 40 amino acids, approximately 30 amino acids amino acids to 35 amino acids, about 35 amino acids to 50 amino acids, about 35 amino acids to 4 ... about 40 to 40 amino acids, about 40 to 50 amino acids, about 40 to 45 amino acids, or about 45 The sequence of the wild-type full-length otoferlin protein, which contains an insertion of approximately 50 amino acids, is In some instances, the inserted amino acids may comprise the amino acid sequence of the wild-type full-length protein. In some instances, amino acids may be inserted as a contiguous sequence into the sequence. As will be recognized in the art, , amino acids in a portion of the wild-type full-length protein sequence that is not well conserved among species. It can be inserted.

[0141] Ranges: Throughout this disclosure, various aspects of this invention may be presented in a range format. This description is merely for convenience and brevity and is not intended to be an inflexible limitation on the scope of the invention. It should be understood that the ranges should not be interpreted as a and all possible subranges and individual numerical values ​​within those ranges are considered to be specifically disclosed. For example, a range such as 1 to 6 should be expressed as 1-3, 1-4, 1-5, 2-4, etc. , subranges such as 2 to 6, 3 to 6, etc., and individual numbers within that range, e.g., 1, 2, 2.7 , 3, 4, 5, 5.3, and 6 should be considered to specifically disclose Therefore, a range such as 95-99% identity means having 95%, 96%, 97%, 98%, or 99% identity. and includes anything between 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99%. Includes subranges such as % identity. This applies regardless of the breadth of the range.

[0142] Unless otherwise defined, all technical and scientific terms used herein has the same meaning as commonly understood by a person skilled in the art in the technical field to which the present invention pertains. Methods and materials are described herein for use in the present invention; Other suitable methods and materials known in the art may also be used. Materials The methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and the like mentioned in this document are hereby incorporated by reference. and other references are incorporated by reference in their entireties. In case of conflict, this specification The book will rule, including the definition. DETAILED DESCRIPTION OF THE INVENTION

[0143] Detailed Description Mutations in OTOF result in DFNB9, a recessively inherited nonsyndromic prelingual hearing impairment Deficiency of otoferlin, a protein encoded by OTOF, leads to the development of auditory inner hair cells. Eliminates rapid exocytosis from the IHC. Due to the insufficient supply, no acoustic signals are transmitted to the brain, which explains the severe hearing loss. are.

[0144] that the method of the present invention results in the expression of full-length otoferlin in auditory inner hair cells; In otoferlin knockout mice, the ABR threshold for click stimulation was 30 Successful restoration of hearing with a range of 50 dB to 90 dB for pure tones and ABR thresholds of 50 dB to 90 dB for pure tones. In light of this discovery, it has been discovered that a defective otoferlin gene is and a composition for treating non-syndromic sensorineural hearing loss in an identified subject (e.g., a human). Products and methods are provided.

[0145] As used herein, a composition comprising at least two different nucleic acid vectors, At least two different vectors each encoding a different portion of the otoferlin protein. Each encoded portion contains a coding sequence that is at least 30 amino acids long. residues, and optionally, each amino acid sequence of the encoded portion is The amino acid sequence of at least two different Any single one of the vectors may contain an active otoferlin protein (e.g., the entire at least one of the coding sequences does not encode a , comprising a nucleotide sequence spanning two adjacent exons of otoferlin genomic DNA; and a mammalian chromosomal DNA lacking an intron sequence between the two adjacent exons; When introduced into a living cell, at least two different vectors interact with each other and with the cell's chromosome DNA. The active otoferlin protein undergoes homologous recombination with NA and thereby inserts into chromosomal DNA. A recombinant nucleic acid encoding a protein (e.g., a full-length otoferlin protein) is formed. The compositions are provided. Kits containing any of the compositions described herein are also provided. As used herein, a therapeutically effective amount of any of the compositions described herein is administered to the cochlea of ​​a mammal. Also provided is a method comprising the step of introducing into a cow.

[0146] As used herein, any of the compositions described herein can be introduced into mammalian cells. The method comprises the step of: producing an active otoferlin protein (e.g., full-length otoferlin) in mammalian cells; Also provided are methods for increasing expression of a ferrin protein. A method for treating a mammal comprising the step of introducing an effective amount of any of the compositions described herein into the cochlea of ​​the mammal. Active otoferlin protein (e.g., full-length otoferlin) is expressed in the inner hair cells of the mammalian cochlea. Also provided are methods for increasing the expression of a therapeutically effective phosphoprotein. A method for treating a defective cochlea comprising administering into the cochlea of ​​a subject an amount of any of the compositions described herein. Also provided are methods for treating non-syndromic sensorineural hearing loss in subjects identified as having the felin gene. It is served.

[0147] Further non-limiting aspects of the compositions, kits, and methods of the present invention are described herein. and can be used in any combination without limitation.

[0148] Otoferlin To date, several hundred mutations in the human OTOF gene that cause severe prelingual hearing loss (DFNB9) have been identified. Mutations have been identified that are autosomal recessive in various populations. It is the cause of hearing loss in 2-8% of people born with inherited nonsyndromic hearing loss (Rodriguez, z-Ballesteroset al. (2008) Hum. Mutat. 29 823-831; Choi et al. (2009) Clinical Genetics 75237-243; Duman et al. (2011) Genet Test Mol Biomarkers 15 29-33; Var ga et al. (2006) J Med Genet 43 576-581; Iwasa et al. (2013) BMC Med. Genet. 14 95) For example, at positions 490 and 515 in the conserved C2C domain of otoferlin, Two substitutions in exon 15 of DFNB9 cause DFNB9 (Mirqhomizadeh et al. (2002) Ne Urobiol. Dis.10(2): 157-164). Migliosi et al. We found a novel mutation, Q829X, in OTOF in a Chinese patient (Migliosi et al. (2002) ) J. Med. Genet. 39(7): 502-506).

[0149] Further exemplary mutations in the otoferlin gene detected in subjects with hearing loss and methods for sequencing nucleic acids encoding otoferlin are described, for example, in Rodriguez-Ball esteros 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: 950-9 53; Choi et al. (2009) Clin. Genet. 75(3): 237-243; and Marlin et al. (2010) Bi ochem BiophysRes Commun 394: 737-742.

[0150] Otoacoustic emissions from DFNB9 patients are normal for at least the first decade of life, and the morphology of the inner ear Indicating the integrity and proper function of outer hair cells. Except for the loss of the inner ear, the morphology and physiology of the inner ear remain unchanged in humans for at least the first decade of life. , which remains conserved in DFNB9.

[0151] Studies in mouse models have shown that synapses are structurally normal, and IHC reveals that synapses are normal within the first week of life. was found to retain normal synapse numbers in mice between P6 and P15. Approximately half of the synapses are lost (Roux et al. (2006) Cell 127 277-289). Animal model by recording changes in cell membrane capacitance and activity in the auditory nerve after vesicle fusion. This allowed us to study the effects of otoferlin mutations on synaptic transmission. In α-lin knockout (Otof- / -) mice, depolarization-induced C a2+ influx induced little exocytosis in IHCs (Rouxet (2006) Cell127 277-289). A patient with severe hearing loss who has a random point mutation in the C2F domain In pachanga (OtofPga / Pga) mice, brief (<10 ms) depolarization of IHCs Although sustained stimulation induced vesicle fusion of similar magnitude to that of wild-type mice, immediate release revealed a significant deficiency in vesicle recruitment to the available pool (Pangrsic et al. (201 0) Nat.Neurosci. 13 869-876). The elevation of the hearing threshold is mild, but the severity of speech comprehension is severely impaired. p.Ile in human patients with temperature-dependent deafness The 515Thr mutation was found (Varga et al. (2006) J Med Genet 43 576-581), which is revealed intermediate phenotypes when tested in a human model (Strenzke et al. (2016) E MBO J.35:2519-2535). These OtofI515T / I515T mice exhibited ABR with reduced I-wave amplitude. showed a moderate elevation of hearing thresholds when assessed by behavioral tests and single auditory nerve units. The recordings showed normal hearing thresholds. However, sustained exocytosis is reduced, although not as severely as OtofPga / Pga. In wild-type mice at warm temperature, 750 vesicles per second can fuse at each active site during sustained stimulation. However, this rate was reduced to 350 vesicles / sec / active site in OtofI515T / I515T mice, whereas Otof Pga / Pga IHC reduces to 200 vesicles / sec / active site (Pangrsicet et al. (2010) Nat. Ne urosci. 13869-876; Strenzke et al. (2016) EMBO J. 35 2519-2535). This correlates with a decrease in otoferlin protein levels in the vacuolar membrane, corresponds to exocytosis and hearing (Strenzke et al. (2016) EMBO J. 35 2519-2535).

[0152] 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, and the like. These include Southern blotting, Northern blotting, and Western blotting.

[0153] The OTOF gene is involved in the exocytosis of synaptic vesicles in cochlear hair cells. It encodes the protein otoferlin (see, e.g., Johnson and Chapman (2010) J. Ce ll Biol.191(1):187-198; and Heidrych et al. (2008) Hum. Mol. Genet. 17:3814-38 21).

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

[0155] Exemplary human wild-type otoferlin proteins include any one of SEQ ID NOs: 1-5. The human otoferlin protein isoform e (SEQ ID NO:5) is a mRNA sequence containing exon 48 but not exon 47 of the otoferlin gene. It is encoded by NA (Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000). In some embodiments, the active otoferlin protein has the sequence of SEQ ID NO:5 However, the RXR motif identified in Strenzke et al., EMBO J. 35(23):2499-2615, 2016 The nucleic acid encoding the wild-type otoferlin protein lacks 20 amino acids, including the A non-limiting example is any one of SEQ ID NOs: 7-11, or any of SEQ ID NOs: 7-11. As understood in the art, the sequences in SEQ ID NOs: 7-11 include any one of the sequences. At least some or all of the promoter sequences are designed to allow optimal expression in non-human mammals or humans. Orthologues of the human otoferlin protein are available in the art. It is well known in the art.

[0156] Human otoferlin protein cDNA sequence: Human canonical (long) isoform sequence (otoferlin protein) (S EQ ID NO:1) (also known as otoferlin isoform a) (NCBI accession no. AAD26117.1) Human isoform 2 (short type 1) (otoferlin protein) (SEQ ID NO: 2) Also known as telin isoform d (NCBI accession number NP_919304.1) Human isoform 3 (short type 2) (otoferlin protein) (SEQ ID NO: 3) Also known as telin isoform c (NCBI accession number NP_919303.1) Human isoform 4 (short form 3) (otoferlin protein) (SEQ ID NO: 4) Also known as telin isoform b (NCBI accession number NP_004793.2) Human isoform 5 (short form 4) (otoferlin protein) (SEQ ID NO: 5) Also known as telin isoform e (NCBI accession number NP_001274418.1) Complete cds (otoferlin cDNA) (www.ncbi.nlm.nih.gov / nuccore / AF107403.1) (SEQ ID N O: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)(SEQ ID NO:1 (encoding proteins) Human otoferlin transcript variant 2 (www.ncbi.nlm.nih.gov / nuccore / NM_004802.3)(SEQ ID NO:8)(SEQ ID NO:4) (encoding proteins) Human otoferlin transcript variant 3 (www.ncbi.nlm.nih.gov / nuccore / NM_194322.2)(SEQ ID NO:9)(SEQ ID NO:3 (encoding proteins) Human otoferlin transcript variant 4 (www.ncbi.nlm.nih.gov / nuccore / NM_194323.2)(SEQ ID NO:10)(SEQ ID NO:2 (encoding proteins) Human otoferlin transcript variant 5 (www.ncbi.nlm.nih.gov / nuccore / NM_001287489.1)(SEQ ID NO:11)(SEQ ID NO:5 (encoding proteins)

[0157] A non-limiting example of a human wild-type otoferlin genomic DNA sequence is SEQ ID NO: 12. The exons in NO:12 are: nucleotide positions 5001 to 5206 (exon 1); Nucleotide positions 25925 to 25983 (exon 2), nucleotide positions 35779 to 35867 (exon 3) ), nucleotide positions 44590 to 44689 (exon 4), nucleotide positions 47100 to 47281 (exon exon 5), nucleotide positions 59854–59927 (exon 6), nucleotide positions 61273–613 99 (exon 7), nucleotide positions 61891–61945 (exon 8), nucleotide position 6862 6 to 68757 (exon 9), nucleotide positions 73959 to 74021 (exon 10), nucleotide positions Nucleotide positions 74404 to 74488 (exon 11), nucleotide positions 79066 to 79225 (exon 12), Nucleotide positions 80051 to 80237 (exon 13), nucleotide positions 81107 to 81293 (exon 14) , nucleotide positions 82690 to 82913 (exon 15), nucleotide positions 83388 to 83496 (exon exon 16), nucleotide positions 84046–84226 (exon 17), nucleotide positions 84315–844 35 (exon 18), nucleotide positions 85950–86050 (exon 19), nucleotide position 86 193 to 86283 (exon 20), nucleotide positions 86411 to 86527 (exon 21), nucleotide Nucleotide positions 86656 to 86808 (exon 22), nucleotide positions 87382 to 87571 (exon 23), Nucleotide positions 87661 to 87785 (exon 24), nucleotide positions 88206 to 88340 (exon 25), nucleotide positions 89025–89186 (exon 26), nucleotide positions 89589–89708 ( exon 27), nucleotide positions 90132–90293 (exon 28), nucleotide position 90405 ~90567 (exon 29), nucleotide positions 91050 to 91180 (exon 30), nucleotide Positions 92549–92578 (exon 31), nucleotide positions 92978–93106 (exon 32), nucleotides Nucleotide positions 95225 to 95291 (exon 33), nucleotide positions 96198 to 96334 (exon 34) ), nucleotide positions 96466 to 96600 (exon 35), nucleotide positions 96848 to 96985 (exon exon 36), nucleotide positions 97623–97750 (exon 37), nucleotide positions 97857–9 8027 (exon 38), nucleotide positions 98670 to 98830 (exon 39), nucleotide positions 99593–99735 (exon 40), nucleotide positions 100128–100216 (exon 41), nucleotide Nucleotide positions 101518 to 101616 (exon 42), nucleotide positions 101762 to 102003 (exon 43), nucleotide positions 102669–102847 (exon 44), nucleotide positions 102952–1030 52 (exon 45), nucleotide positions 103494 to 103691 (exon 46), nucleotide positions 105479–106496 (exon 47), and exon 48 ( JPEG0007814280000002.jpg3127 the sequence beginning with ; Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000 See also Chthon's entry.

[0158] An intron is located between each pair of consecutive exons in SEQ ID NO:12, i.e., a nucleus. Nucleotide positions 100 to 5001 (intron 1), nucleotides 5207 to 25924 (intron 2), Nucleotide positions 25984 to 35778 (intron 3), nucleotide positions 35868 to 44589 (intron intron 4), nucleotide positions 44690 to 47099 (intron 5), nucleotide positions 47282 to 598 53 (intron 6), nucleotide positions 59928 to 61272 (intron 7), nucleotide position 6 1400 to 61890 (intron 8), nucleotide positions 61946 to 68625 (intron 9), nucleotide Nucleotide positions 68758 to 73958 (intron 10), nucleotide positions 74022 to 74403 (intron 11), nucleotide positions 74489 to 79065 (intron 12), nucleotide positions 79226 to 80050 (intron 13), nucleotide positions 80238 to 81106 (intron 14), nucleotide positions 81294 to 82689 (intron 15), nucleotide positions 82914 to 83387 (intron 16), nucleotide nucleotide positions 83497 to 84045 (intron 17), nucleotide positions 84227 to 84314 (intron Intron 18), nucleotide positions 84436 to 85949 (intron 19), nucleotide positions 86051 to 861 92 (intron 20), nucleotide positions 86284 to 86410 (intron 21), nucleotide positions Nucleotide positions 86528 to 86655 (intron 22), 86809 to 87381 (intron 23), Nucleotide positions 87572 to 87660 (intron 24), nucleotide positions 87786 to 88205 (intron intron 25), nucleotide positions 88341–89024 (intron 26), nucleotide positions 89187–89188 9588 (intron 27), nucleotide positions 89709 to 90131 (intron 28), nucleotide positions 90294 to 90404 (intron 29), nucleotide positions 90568 to 91049 (intron 30); Nucleotide positions 91181 to 92548 (intron 31), nucleotide positions 92579 to 92977 (intron 32), intron 32), nucleotide positions 93107 to 95224 (intron 33), nucleotide position 95292 ~96197 (intron 34), nucleotide positions 96335 to 96465 (intron 35), nucleobase nucleotide positions 96601 to 96847 (intron 36), nucleotide positions 96986 to 97622 (intron 37) ), nucleotide positions 97751 to 97856 (intron 38), nucleotide positions 98028 to 98669 ( intron 39), nucleotide positions 98831 to 99592 (intron 40), nucleotide position 99 736 to 100127 (intron 41), nucleotide positions 100217 to 101517 (intron 42), Nucleotide positions 101617 to 101761 (intron 43), nucleotide positions 102004 to 102668 (intron 44), intron 44), nucleotide positions 102848 to 102951 (intron 45), nucleotide position 10 3053 to 103494 (intron 46), nucleotide positions 103692 to 105478 (intron 47), and and located at nucleotide positions 106497 to 108496 (intron 48).

[0159] 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_00130069 6.1) 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: NC_000071; Region: complementary sequence (30367066..30462730) GPC_000000778; NCB Reference sequence: NC_000071.6 Zebrafish otoferlin A gene sequence (www.ncbi.nlm.nih.gov / gene / 557476) (SEQ ID NO:16)Accession NC_007131; Region: 31173357..31310109 GPC_000001574; NC BI reference sequence: NC_007131.7 Rhesus monkey otoferlin gene sequence (www.ncbi.nlm.nih.gov / gene / 696717) (SEQ ID NO :17) Accession: NC_027905; Region: Complementary sequence (26723411..26826586) GPC_000002 105; NCBI reference sequence: NC_027905.1 Canine otoferlin gene sequence (www.ncbi.nlm.nih.gov / gene / 607961) (SEQ ID NO: 18) Accession: NC 006599; Region: Complementary sequence (20518502..20619461) GPC_000000676; N CBI reference sequence: NC_006599.3 Chimpanzee otoferlin gene sequence (www.ncbi.nlm.nih.gov / gene / 459083) (SEQ ID N O:19) Accession: NC 006469; Region: Complementary sequence (27006052..27107747) GPC_00000 2338; 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) Baboontoferrin protein (SEQ ID NO:23)

[0160] vector As used herein, nucleic acid therapeutics, such as messenger RNA for auditory system polypeptides, are used. Compositions and methods of use for the treatment of diseases such as non-syndromic hearing loss are provided. Preferably, the nucleic acid of the auditory system polypeptide is delivered via an adeno-associated virus vector, adenovirus. viral vectors, lentiviral vectors, and retroviral vectors It is present in the vector.

[0161] Packaging the recombinant vector into a desired AAV capsid to generate the rAAV of the invention The above methods for preparing the hydroxylase inhibitor are not meant to be limiting, and other suitable methods may be known to those skilled in the art. It should be obvious.

[0162] The expression control sequences include appropriate transcription initiation sequences, transcription termination sequences, promoter sequences and endogenous sequences. splicing signals and polyadenylation (poly A) signals Efficient RNA processing signal; sequence that stabilizes cytoplasmic mRNA and enhances translation efficiency sequences (i.e., Kozak consensus sequences), sequences that enhance protein stability; and The native construct may contain sequences that enhance secretion of the encoded product, if desired. Numerous expression control sequences, including constitutive, inducible, and / or tissue-specific promoters are known in the art and may be utilized.

[0163] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences refer to nucleic acids. The expression or transcription of a sequence is under the influence or control of regulatory sequences. When nucleic acid sequences are linked in a functional manner, they are said to be "operably" linked. If it is desired that the two DNA sequences be translated into a protein, the promoter in the 5' regulatory sequence If induction of the promoter results in transcription of the coding sequence, and the nature of the linkage between the two DNA sequences (1) does not result in the introduction of frameshift mutations; (2) does not result in a process that directs transcription of the coding sequence. (3) does not interfere with the ability of the motor domain to produce the corresponding RNA transcript that is translated into protein. A promoter is said to be operably linked if it does not interfere with the ability of the promoter to The region is selected so that the resulting transcript is translated into the desired protein or polypeptide. Thus, a promoter region is a promoter of a nucleic acid sequence if it is capable of effecting transcription of that DNA sequence. Similarly, two or more coding regions may be operably linked by a common promoter. Their transcription from the promoter results in the expression of two or more proteins translated in frame. In some embodiments, the linkage is operably linked when the linkage results in Thus, the operably linked coding sequences result in a fusion protein. Thus, the operably linked coding sequences can be used to produce functional RNAs (e.g., shRNAs, miRNAs, miRNA inhibitors, etc.). Bitter).

[0164] In the case of nucleic acids encoding proteins, polyadenylation sequences are generally located at the end of the transgene sequence. The rAAV constructs useful in the present invention also desirably include a nucleotide sequence inserted after the 3' AAVITR sequence. Located between the promoter / enhancer sequence and the transgene, even if it contains introns One putative intron sequence is derived from SV40 and is referred to as the SV40T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). IRES sequences are used to generate two or more polypeptides from a single gene transcript. IRES sequences are used to produce proteins containing two or more polypeptide chains. The selection of these and other common vector elements is conventional and many of them are Such sequences are available [see, e.g., Sambrook et al. "Molecular Cloning. A Laboratory Manual."] toryManual", 2d ed., Cold Spring Harbor Laboratory, New York (1989), and its References cited therein, e.g., pages 3.18, 3.26 and 16.17, 16.27, and Ausube l et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1 989]. In some embodiments, the foot and mouth disease virus 2A sequence is included in the polyprotein. It contains small peptides that have been shown to mediate polyprotein cleavage. It is a nucleotide sequence (approximately 18 amino acids long) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattio n, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene T herapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 45 The cleavage activity of the 2A sequence has been demonstrated in plasmids and gene therapy vectors (AAV and retroviral vectors). This has been previously demonstrated in artificial systems, including viruses (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 Pla nt Journal,1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-20 8; de Felipe, Pet al., Human Gene Therapy, 2000; 11: 1921-1931; and Klump, H et al., GeneTherapy, 2001; 8: 811-817).

[0165] The precise nature of the regulatory sequences required for gene expression in host cells varies with species, tissue, and Although the sequence of the nucleotides may differ between cell types, they generally contain a TATA box, capsid, or other nucleotide sequences, as needed. transcription and translation initiation, respectively, such as the ribosomal ribosomal nucleotide sequence, the CAAT sequence, and an enhancer element. In particular, the 5' untranscribed and 5' untranslated sequences involved in the Non-transcriptional regulatory sequences include promoter sequences for transcriptional control of operably linked genes. The regulatory sequence may also optionally include an enhancer sequence or an upstream The vectors of the invention may optionally contain a 5' leader sequence or signal The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. It is within the enclosure.

[0166] Examples of constitutive promoters include, but are not limited to, the retrovirus Rous sarcoma virus RSV LTR promoter (optionally with RSV enhancer), cytomegalovirus a CMV promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al. ( 1985) Cell, 41:521-530], SV40 promoter, dihydrofolate reductase promoter promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter and the EF1α promoter [Invitrogen].

[0167] Inducible promoters allow for the regulation of gene expression and can be mediated by exogenously supplied compounds, temperature, etc. or the presence of certain physiological conditions, e.g., acute phase, specific differentiation of cells. Inducible promoters can be regulated only by the presence of a certain condition or during cell replication. and inducible systems include, but are not limited to, those manufactured by Invitrogen, Clontech, and Ariad. Many other systems have been described and are available from a variety of commercial sources, including Induction regulated by an exogenously supplied promoter can be easily selected for. Examples of promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dextromethorphan (DT) promoter, and the ovine ... Dex-inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter motor system (WO98 / 10088); ecdysone insect promoter (No et al. (1996) Proc. Na tl. Acad. Sci. USA, 93:3346-3351), tetracycline-repressible system (Gossen et al. Pr oc. Natl. Acad.Sci. USA, 89:5547-5551 (1992)), tetracycline-inducible system (Gos sen et al.Science, 268:1766-1769 (1995), Harvey et al. Curr. Opin. Chem. Biol., 2:512-518 (1998)), RU486-inducible system (Wang et al. Nat. Biotech., 1 5:239-243 (1997) and Wang et al. Gene Ther., 4:432-441 (1997)), and rapamycin and a synthase-inducible system (Magariet et al. J. Clin. Invest., 100:2865-2872 (1997)). Yet other types of inducible promoters that may be useful in this context are specific physiological conditions, such as temperature, acute phase, specific differentiation state of cells, or during cell replication Only the

[0168] In another embodiment, the native promoter of the transgene is used. A suitable promoter is desirable so that the expression of the transgene should mimic the native expression. Native promoters may be preferred when transgene expression is delayed. They may be regulated intermittently or developmentally, in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, an enhancer element may be used when the gene must be segmented. other native expression control elements, such as polyadenylation sites or Kozak consensus sequences. Genes may be used to mimic native expression.

[0169] In some embodiments, the regulatory sequences confer tissue-specific gene expression capabilities. In the case of tissue-specific regulatory sequences, tissue-specific transcription factors 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: These include, but are not limited to, the following tissue-specific promoters: liver-specific thyroxine-binding globulin (LGB) TBG promoter, insulin promoter, glucagon promoter, somatostatin tatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter Motor, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter -, α-myosin heavy chain (α-MHC) promoter, or cardiac troponin T (cTnT) promoter Other exemplary promoters include the β-actin promoter, the hepatitis B virus core promoter, promoter, Sandiget et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP ) promoter, Arbuthnotet et al., Hum. Gene Ther., 7:1503-14 (1996), Osteo calcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialic acid promoter (B. lo protein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansalet et al., J. Immunol., 161:1063-8 (1998)); immunoglobulin Heavy chain promoter; T cell receptor α chain promoter; neuron-specific enolase (NSE) promoter Neuronal promoters such as the promoter of the ribosomal protein (Andersen et al., Cell. Mol. Neurobiol., 13:503-1 5 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Picc ioli et al., Neuron, 15:373-84 (1995)), among others, which will be apparent to those skilled in the art. There will be.

[0170] In some embodiments, one or more binding sites for one or more miRNAs , one of the subjects harboring the transgene incorporated into the rAAV vector, or Inhibits expression of a transgene in multiple tissues. Those skilled in the art will appreciate that the binding site is tissue-specifically induced. It will be appreciated that the expression of the transgene may be selected to control expression of the transgene. For example, The binding site for the specific miR-122 was used to inhibit the expression of the transgene in the liver. The target site in the mRNA may be located in the 5' UTR, 3' UTR, or It may be located in the coding region. Typically, the target site is located in the 3' UTR of the mRNA. Furthermore, the transgene may be expressed in mRNA by multiple miRNAs recognizing the same or multiple sites. The presence of multiple miRNA binding sites may be designed to regulate the cooperation of multiple RISCs. The target site sequence is synthesized. The target site sequence may contain a total of 5 to 100, 10 to 60, or more nucleotides. The target gene binding site sequence may comprise at least 5 nucleotides of the target gene binding site sequence.

[0171] Auditory system polypeptides and auditory system polypeptide transgene coding sequences Composition of the auditory system polypeptide transgene sequence in the rAAV vector to generate the resulting vector Thus, the present invention relates to a method for treating a disease state associated with hearing loss in a mammalian subject. one or more auditory system polypeptides, peptides, or compounds useful in the treatment or prevention of auditory conditions Exemplary therapeutic proteins include delivery of rAAV vectors encoding the therapeutic proteins. Contains otoferlin; Ca v 1.3; bassoon, piccolo, ribeye ( a scaffolding protein selected from ribeye, and harmonin; Vglut3; synapse Ptotagmin; vesicle tethering / docking proteins; vesicle priming proteins; one or more selected from the group consisting of: vesicle fusion protein; GluA2 / 3; or GluA4 Polypeptides are included.

[0172] The AAV composition optionally includes a polypeptide reporter protein. Reporter sequences that can be provided in the present invention include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescence Protein (GFP), chloramphenicol acetyltransferase (CAT), Lucif Included are DNA sequences encoding enzymes, and others known in the art. When associated with regulatory elements that drive their expression, reporter sequences are capable of producing enzymatic, X radiographic, colorimetric, fluorescent or other spectroscopic assays, fluorescence-activated cell sorting assays, etc. as well as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry Provide a signal that is detectable by conventional means, including immunological assays, including For example, if the marker sequence is a LacZ gene, the presence of a vector carrying the signal The transgene is detected by assaying for β-galactosidase activity. If the signal is a color fluorescent protein or luciferase, the vector carrying the signal is Such reporters can be measured visually by color or light production in a luminometer. For example, to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of rAAV. This may be useful for

[0173] In another example, the transgene is intended for use in generating an animal model of a disease. Suitable transgene coding sequences may be selected by those skilled in the art. It is clear in

[0174] The rAAV vectors can be used to target auditory system polypeptide genes or functional pathways for auditory system polypeptides. to treat a disease associated with reduced expression, absence of expression, or dysfunction of another gene in a gene or genes encoding an auditory system polypeptide to be transferred to a subject for the purpose of It may include some.

[0175] In a first aspect, the therapeutic composition is administered to a human subject to induce the production of auditory system receptors in target cells. Several adeno-associated viruses (AAV) have the ability to construct messenger RNAs of polypeptides. A therapeutic composition is provided comprising a plurality of AAV vectors. The therapeutic composition is administered to target cells of a human subject, and the target cells express the full-length auditory system polypeptide messenger. The multiple AAV vectors include a first AAV vector and a second AAV vector. The first and second AAV vectors are independently packaged into packages of less than about 6 kb. Messenger RNA for auditory system polypeptides may be, for example, those described herein. The group consisting of otoferlin and its orthologs or homologs, as provided in The AAV vector encodes an auditory system polypeptide selected from the group consisting of CBA, CMV, and C B7 promoter, or one or more cochlear-specific promoters. It contains at least one promoter sequence.

[0176] The compositions provided herein comprise at least two (e.g., two, three, four, five) or six) nucleic acid vectors, each of the at least two different vectors being , comprising coding sequences encoding different portions of the otoferlin protein, Each of the amino acids is at least 30 amino acids long (e.g., from about 30 amino acids to about 1950 amino acids). , about 30 amino acids to about 1900 amino acids, about 30 amino acids to about 1850 amino acids, about 30 amino acids to about 18 00 amino acids, about 30 amino acids to about 1750 amino acids, about 30 amino acids to about 1700 amino acids, about 30 amino acids 1650 amino acids, 30 amino acids to 1600 amino acids, 30 amino acids to 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 14 00 amino acids, about 30 amino acids to about 1350 amino acids, about 30 amino acids to about 1300 amino acids, about 30 amino acids 1250 amino acids, 30 amino acids to 1200 amino acids, 30 amino acids to 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 10 00 amino acids, about 30 amino acids to about 950 amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 3 0 to approximately 700 amino acids, approximately 30 to approximately 650 amino acids, approximately 30 to approximately 600 amino acids Acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 45 0 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids Approximately 30 to 250 amino acids, approximately 30 to 200 amino acids, approximately 3 0 to about 150 amino acids, about 30 to about 100 amino acids, about 30 to about 50 amino acids 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, approximately 50 amino acids to approximately 1800 amino acids, approximately 50 amino acids to approximately 1750 amino acids, approximately 50 amino acids amino acids to approximately 1700 amino acids, approximately 50 amino acids to approximately 1650 amino acids, approximately 50 amino acids to approximately 1600 amino acids 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 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 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 amino acids to approximately 900 amino acids, approximately 50 amino acids to approximately 850 amino acids, approximately 50 amino acids to approximately 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 ~ about 500 amino acids, ~ about 50 amino acids ~ about 450 amino acids, ~ about 50 amino acids ~ about 400 amino acids, ~ about 50 Amino acids up to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids amino acids, about 50 to about 200 amino acids, about 50 to about 150 amino acids, about 50 to about 10 0 amino acids, about 100 amino acids to about 1950 amino acids, about 100 amino acids to about 1900 amino acids, about 100 amino acids amino acids to about 1850 amino acids, about 100 amino acids to about 1800 amino acids, about 100 amino acids to about 1750 amino acids amino acids, about 100 amino acids to about 1700 amino acids, about 100 amino acids to about 1650 amino acids, about 100 amino acids Approximately 1600 amino acids, approximately 100 amino acids to approximately 1550 amino acids, approximately 100 amino acids to approximately 1500 amino acids , about 100 amino acids to about 1450 amino acids, about 100 amino acids to about 1400 amino acids, about 100 amino acids to Approximately 1350 amino acids, approximately 100 to approximately 1300 amino acids, approximately 100 to approximately 1250 amino acids, approximately 100 amino acids to approximately 1200 amino acids, approximately 100 amino acids to approximately 1150 amino acids, approximately 100 amino acids to approximately 110 0 amino acids, about 100 amino acids to about 1050 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids amino acids to approximately 950 amino acids, approximately 100 amino acids to approximately 900 amino acids, approximately 100 amino acids to approximately 850 amino acids Acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids Approximately 700 amino acids, approximately 100 to approximately 650 amino acids, approximately 100 to approximately 600 amino acids, approximately 100 Amino acids up to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids ~ about 300 amino acids, ~ about 100 amino acids ~ about 250 amino acids, ~ about 100 amino acids ~ about 200 amino acids, ~ about 1 00 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 1850 amino acids, about 150 amino acids to about 1800 amino acids, about 150 amino acids 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 amino acids, about 150 amino acids to about 1600 amino acids, about 150 amino acids to about 1550 amino acids, about 150 amino acids Approximately 1500 amino acids, approximately 150 amino acids to approximately 1450 amino acids, approximately 150 amino acids to approximately 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 Approximately 1250 amino acids, approximately 150 to approximately 1200 amino acids, approximately 150 to approximately 1150 amino acids, approximately 150 amino acids to about 1100 amino acids, about 150 amino acids to about 1050 amino acids, about 150 amino acids to about 100 0 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids Approximately 150 to approximately 850 amino acids, approximately 150 to approximately 800 amino acids, approximately 150 to approximately 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 6 00 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids 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 Acid, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids Approximately 200 amino acids, approximately 200 to approximately 1950 amino acids, approximately 200 to approximately 1900 amino acids, approximately 2 00 amino acids to approximately 1850 amino acids, approximately 200 amino acids to approximately 1800 amino acids, approximately 200 amino acids to approximately 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 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 amino acids, about 200 amino acids to about 1450 amino acids, about 200 amino acids to about 1400 amino acids, about 200 amino acids Approximately 1350 amino acids, approximately 200 amino acids to approximately 1300 amino acids, approximately 200 amino acids to approximately 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 Approximately 1100 amino acids, approximately 200 to approximately 1050 amino acids, approximately 200 to approximately 1000 amino acids, approximately 200 amino acids to approximately 950 amino acids, approximately 200 amino acids to approximately 900 amino acids, approximately 200 amino acids to approximately 850 amino acids amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids 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, Approximately 200 to 550 amino acids, approximately 200 to 500 amino acids, approximately 200 to 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 Approximately 300 amino acids, approximately 200 amino acids to approximately 250 amino acids, approximately 250 amino acids to approximately 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 Approximately 1800 amino acids, approximately 250 to approximately 1750 amino acids, approximately 250 to approximately 1700 amino acids, approximately 250 amino acids to approximately 1650 amino acids, approximately 250 amino acids to approximately 1600 amino acids, approximately 250 amino acids to approximately 155 0 amino acids, about 250 amino acids to about 1500 amino acids, about 250 amino acids to about 1450 amino acids, about 250 amino acids 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 amino acids, about 250 amino acids to about 1250 amino acids, about 250 amino acids to about 1200 amino acids, about 250 amino acids Approximately 1150 amino acids, approximately 250 amino acids to approximately 1100 amino acids, approximately 250 amino acids to approximately 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 amino acids to approximately 750 amino acids, approximately 250 amino acids to approximately 700 amino acids, approximately 250 amino acids to approximately 650 amino acids Acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 550 amino acids, about 250 amino acids Approximately 500 amino acids, approximately 250 to approximately 450 amino acids, approximately 250 to approximately 400 amino acids, approximately 250 Amino acids up to about 350 amino acids, about 250 amino acids to about 300 amino acids, about 300 amino acids to about 1950 amino acids amino acids, about 300 amino acids to about 1900 amino acids, about 300 amino acids to about 1850 amino acids, about 300 amino acids Approximately 1800 amino acids, approximately 300 amino acids to approximately 1750 amino acids, approximately 300 amino acids to approximately 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 Approximately 1550 amino acids, approximately 300 to approximately 1500 amino acids, approximately 300 to approximately 1450 amino acids, approximately 300 amino acids to approximately 1400 amino acids, approximately 300 amino acids to approximately 1350 amino acids, approximately 300 amino acids to approximately 130 0 amino acids, about 300 amino acids to about 1250 amino acids, about 300 amino acids to about 1200 amino acids, about 300 amino acids amino acids to about 1150 amino acids, about 300 amino acids to about 1100 amino acids, about 300 amino acids to about 1050 amino acids amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids ~ about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 3 00 amino acids to approximately 750 amino acids, approximately 300 amino acids to approximately 700 amino acids, approximately 300 amino acids to approximately 650 amino acids amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, Approximately 300 amino acids to approximately 350 amino acids, approximately 350 amino acids to approximately 1950 amino acids, approximately 350 amino acids to approximately 19 00 amino acids, about 350 amino acids to about 1850 amino acids, about 350 amino acids to about 1800 amino acids, about 350 Amino acids up to approximately 1750 amino acids, approximately 350 amino acids to approximately 1700 amino acids, approximately 350 amino acids to approximately 1650 amino acids amino acids, about 350 amino acids to about 1600 amino acids, about 350 amino acids to about 1550 amino acids, about 350 amino acids Approximately 1500 amino acids, approximately 350 amino acids to approximately 1450 amino acids, approximately 350 amino acids to approximately 1400 amino acids Acid, about 350 amino acids to about 1350 amino acids, about 350 amino acids to about 1300 amino acids, about 350 amino acids up to about 1250 amino acids, about 350 amino acids to about 1200 amino acids, about 350 amino acids to about 1150 amino acids, Approximately 350 amino acids to approximately 1100 amino acids, approximately 350 amino acids to approximately 1050 amino acids, approximately 350 amino acids to approximately 1 000 amino acids, about 350 amino acids to about 950 amino acids, about 350 amino acids to about 900 amino acids, about 350 amino acids amino acids to approximately 850 amino acids, approximately 350 amino acids to approximately 800 amino acids, approximately 350 amino acids to approximately 750 amino acids Acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids Approximately 600 amino acids, approximately 350 to approximately 550 amino acids, approximately 350 to approximately 500 amino acids, approximately 350 Amino acids up to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 1950 amino acids amino acids, about 400 amino acids to about 1900 amino acids, about 400 amino acids to about 1850 amino acids, about 400 amino acids Approximately 1800 amino acids, approximately 400 amino acids to approximately 1750 amino acids, approximately 400 amino acids to approximately 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 Approximately 1550 amino acids, approximately 400 to approximately 1500 amino acids, approximately 400 to approximately 1450 amino acids, approximately 400 amino acids to approximately 1400 amino acids, approximately 400 amino acids to approximately 1350 amino acids, approximately 400 amino acids to approximately 130 0 amino acids, about 400 amino acids to about 1250 amino acids, about 400 amino acids to about 1200 amino acids, about 400 amino acids amino acids to about 1150 amino acids, about 400 amino acids to about 1100 amino acids, about 400 amino acids to about 1050 amino acids amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids ~ about 900 amino acids, ~ about 400 amino acids ~ about 850 amino acids, ~ about 400 amino acids ~ about 800 amino acids, ~ about 4 00 amino acids to approximately 750 amino acids, approximately 400 amino acids to approximately 700 amino acids, approximately 400 amino acids to approximately 650 amino acids amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 1950 amino acids, Approximately 450 amino acids to approximately 1900 amino acids, approximately 450 amino acids to approximately 1850 amino acids, approximately 450 amino acids to approximately 1 800 amino acids, about 450 amino acids to about 1750 amino acids, about 450 amino acids to about 1700 amino acids, about 450 Amino acids up to approximately 1650 amino acids, approximately 450 amino acids to approximately 1600 amino acids, approximately 450 amino acids to approximately 1550 amino acids amino acids, about 450 amino acids to about 1500 amino acids, about 450 amino acids to about 1450 amino acids, about 450 amino acids 1400 amino acids, 450 amino acids to 1350 amino acids Acid, about 450 amino acids to about 1300 amino acids, about 450 amino acids to about 1250 amino acids, about 450 amino acids up to about 1200 amino acids, about 450 amino acids to about 1150 amino acids, about 450 amino acids to about 1100 amino acids, Approximately 450 amino acids to approximately 1050 amino acids, approximately 450 amino acids to approximately 1000 amino acids, approximately 450 amino acids to approximately 9 50 amino acids, about 450 amino acids to about 900 amino acids, about 450 amino acids to about 850 amino acids, about 450 amino acids amino acids to approximately 800 amino acids, approximately 450 amino acids to approximately 750 amino acids, approximately 450 amino acids to approximately 700 amino acids Acid, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids Approximately 550 amino acids, approximately 450 to approximately 500 amino acids, approximately 500 to approximately 1950 amino acids, approximately 50 0 amino acids to approximately 1900 amino acids, approximately 500 amino acids to approximately 1850 amino acids, approximately 500 amino acids to approximately 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 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 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1450 amino acids, about 500 amino acids Approximately 1400 amino acids, approximately 500 amino acids to approximately 1350 amino acids, approximately 500 amino acids to approximately 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 Approximately 1150 amino acids, approximately 500 to approximately 1100 amino acids, approximately 500 to approximately 1050 amino acids, approximately 500 amino acids to approximately 1000 amino acids, approximately 500 amino acids to approximately 950 amino acids, approximately 500 amino acids to approximately 900 Amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids Approximately 750 amino acids, approximately 500 amino acids to approximately 700 amino acids, approximately 500 amino acids to approximately 650 amino acids , about 500 to about 600 amino acids, about 500 to about 550 amino acids, about 550 to about 1 950 amino acids, approximately 550 to approximately 1900 amino acids, approximately 550 to approximately 1850 amino acids, approximately 550 Amino acids up to approximately 1800 amino acids, approximately 550 amino acids to approximately 1750 amino acids, approximately 550 amino acids to approximately 1700 amino acids amino acids, about 550 amino acids to about 1650 amino acids, about 550 amino acids to about 1600 amino acids, about 550 amino acids Approximately 1550 amino acids, approximately 550 amino acids to approximately 1500 amino acids, approximately 550 amino acids to approximately 1450 amino acids Acid, about 550 amino acids to about 1400 amino acids, about 550 amino acids to about 1350 amino acids, about 550 amino acids up to about 1300 amino acids, about 550 amino acids to about 1250 amino acids, about 550 amino acids to about 1200 amino acids, Approximately 550 amino acids to approximately 1150 amino acids, approximately 550 amino acids to approximately 1100 amino acids, approximately 550 amino acids to approximately 1 0.50 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 Amino acids up to approximately 900 amino acids, approximately 550 amino acids to approximately 850 amino acids, approximately 550 amino acids to approximately 800 amino acids amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids ~ about 650 amino acids, ~ about 550 amino acids ~ about 600 amino acids, ~ about 600 amino acids ~ about 1950 amino acids, 600 amino acids to approximately 1900 amino acids, approximately 600 amino acids to approximately 1850 amino acids, approximately 600 amino acids to approximately 180 0 amino acids, about 600 amino acids to about 1750 amino acids, about 600 amino acids to about 1700 amino acids, about 600 amino acids amino acids to about 1650 amino acids, about 600 amino acids to about 1600 amino acids, about 600 amino acids to about 1550 amino acids amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1450 amino acids, about 600 amino acids Approximately 1400 amino acids, approximately 600 amino acids to approximately 1350 amino acids, approximately 600 amino acids to approximately 1300 amino acids , about 600 amino acids to about 1250 amino acids, about 600 amino acids to about 1200 amino acids, about 600 amino acids to Approximately 1150 amino acids, approximately 600 to approximately 1100 amino acids, approximately 600 to approximately 1050 amino acids, approximately 600 to approximately 1000 amino acids, approximately 600 to approximately 950 amino acids, approximately 600 to approximately 900 Amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids Approximately 750 amino acids, approximately 600 amino acids to approximately 700 amino acids, approximately 600 amino acids to approximately 650 amino acids , about 650 amino acids to about 1950 amino acids, about 650 amino acids to about 1900 amino acids, about 650 amino acids to Approximately 1850 amino acids, approximately 650 to approximately 1800 amino acids, approximately 650 to approximately 1750 amino acids, approximately 650 amino acids to approximately 1700 amino acids, approximately 650 amino acids to approximately 1650 amino acids, approximately 650 amino acids to approximately 160 0 amino acids, about 650 amino acids to about 1550 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids amino acids to about 1450 amino acids, about 650 amino acids to about 1400 amino acids, about 650 amino acids to about 1350 amino acids amino acids, about 650 amino acids to about 1300 amino acids, about 650 amino acids to about 1250 amino acids, about 650 amino acids Approximately 1200 amino acids, approximately 650 amino acids to approximately 1150 amino acids, approximately 650 amino acids to approximately 1100 amino acids , about 650 amino acids to about 1050 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to Approximately 950 amino acids, approximately 650 to approximately 900 amino acids, approximately 650 to approximately 850 amino acids, approximately 650 Amino acids up to approximately 800 amino acids, approximately 650 amino acids to approximately 750 amino acids, approximately 650 amino acids to approximately 700 amino acids amino acids, about 700 amino acids to about 1950 amino acids, about 700 amino acids to about 1900 amino acids, about 700 amino acids Approximately 1850 amino acids, approximately 700 amino acids to approximately 1800 amino acids, approximately 700 amino acids to approximately 1750 amino acids , about 700 amino acids to about 1700 amino a...

Claims

1. below: a) a first AAV vector comprising a 5' inverted repeat translocation (ITR), a promoter, a gene encoding the N-terminal portion of an otoferlin polypeptide located at the 3' end of the promoter, a splicing donor signal sequence located at the 3' end of the gene encoding the N-terminal portion of the otoferlin polypeptide, an F1 phage recombination region located at the 3' end of the splicing donor signal sequence, and a 3' ITR; and b) A second AAV vector comprising a 5' ITR, an F1 phage recombination region, a splice acceptor signal sequence located at the 3' end of the F1 phage recombination region, a gene encoding the C-terminal portion of an otoferlin polypeptide located at the 3' end of the splice acceptor signal sequence, a polyadenylation signal located at the 3' end of the gene encoding the C-terminal portion of the otoferlin polypeptide, and a 3' ITR. wherein the amino acid sequences of each of the encoded portions do not overlap with each other, and wherein no single vector encodes the full-length otoferlin protein and is capable of constituting the full-length otoferlin messenger RNA.

2. The multiple AAV vectors of claim 1 , wherein the otoferlin polypeptide is an otoferlin isoform 5 polypeptide.

3. The multiple AAV vectors of claim 2 , wherein the otoferlin polypeptide is a human otoferlin isoform 5 polypeptide.

4. 4. The multiple AAV vectors of any one of claims 1 to 3, wherein the first and second vectors each comprise a total number of nucleotides of up to about 5 kb.

5. The plurality of AVV vectors according to any one of claims 1 to 4, wherein the F1 phage recombination region is a 77 base pair recombination region from the F1 phage genome.

6. The multiple AAV vectors according to any one of claims 1 to 5, wherein the promoter is a CAG promoter or a CAG / CBA promoter.

7. 2. The AAV vectors of claim 1, wherein the polyadenylation signal is a bovine growth hormone polyadenylation sequence.

8. 8. The multiple AAV vectors of any one of claims 1 to 7, wherein the 5' and 3' ITRs are AAV2 serotype ITRs.

9. 9. The multiple AAV vectors of any one of claims 1 to 8, wherein the first and second AAV vectors are encapsulated by AAV capsids, and the AAV capsids are AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rhlO, rh39, rh43, or Anc80.

10. 10. The plurality of AAV vectors of claim 9, wherein the first and second AAV vectors are encapsulated by Anc80 capsids, and the Anc80 capsids are Anc80L65 capsids.

11. 11. The multiple AAV vectors of any one of claims 1 to 10, wherein the otoferlin coding sequence is split at the exon 21-exon 22 junction.

12. 12. The multiple AAV vectors of any one of claims 1 to 11, wherein each portion of the gene encoding an otoferlin polypeptide encodes up to 70% of SEQ ID NO:

5.

13. A plurality of AAV vectors described in any one of claims 1 to 12, wherein the first and second AAVs are capable of undergoing homologous recombination and splicing between a splicing donor signal sequence and a splicing acceptor signal sequence to reconstitute a full-length otoferlin messenger RNA.

14. The full-length otoferlin messenger RNA is any of the following: (i) recombination and splicing via the ITRs between the splicing donor signal sequence and the splicing acceptor signal sequence; or (ii) recombination and splicing between the splicing donor signal sequence and the splicing acceptor signal sequence induced by the F1 phage recombination region.

2. The plurality of AAV vectors of claim 1, produced according to

15. 15. A composition comprising a plurality of AAV vectors according to any one of claims 1 to 14, wherein the composition is formulated for intracochlear administration.

16. 15. The multiple AAV vectors of any one of claims 1 to 14, wherein the F1 phage recombination region comprises the nucleotide sequence of positions 2 to 78 of SEQ ID NO:39.

Images