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

Abstract

This article provides compositions comprising at least two different nucleic acid vectors, each of which contains a coding sequence encoding a different portion of an otopathic protein, and the use of these compositions to treat hearing loss in subjects.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 979,792, filed February 21, 2020, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list that has been electronically submitted in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on February 21, 2020, named 2013615-0002_SL.txt, and is 1,298,432 bytes in size. Technical Field

[0005] This disclosure generally relates to the use of nucleic acid to treat hearing loss in human subjects. Background of the Invention

[0007] The ear is a complex organ, classically described as comprising the outer ear, middle ear, inner ear, auditory (auditory) nerve, and auditory system (which processes sound as it travels from the ear to the brain). Besides detecting sound, the ear also helps maintain balance. Therefore, conditions of the inner ear can lead to hearing loss, tinnitus, vertigo, and imbalance. Vertigo is a hallucination of motion and is a major symptom of vestibular system disorders. Vertigo can be caused by problems in the inner ear or central nervous system. Common inner ear causes of vertigo include: vestibular neuritis (sudden unilateral vestibular loss), Meniere's disease (episodic vertigo), benign paroxysmal positional vertigo (BPPV), and bilateral vestibular loss. Common central nervous system causes of vertigo include: post-concussion syndrome, cervical vertigo, vestibular migraine, cerebrovascular disease, and acoustic neuroma.

[0008] Hearing loss is one of the most common sensory impairments and can occur for a variety of reasons. Some people may be born with hearing loss, while others may gradually lose their hearing over time. Presbycusis (also spelled presbyacusis) is age-related hearing loss. Approximately 36 million U.S. adults report some degree of hearing loss, and one in three people over the age of 60 and half of those over the age of 85 have experienced hearing loss.

[0009] Hearing loss can result from environmental factors or a combination of genetic and environmental factors. About half of people with tinnitus—who experience phantom noises (ringing, buzzing, chirping, humming, or beating) in their auditory system—also have an excessive sensitivity / reduced tolerance to certain sound frequencies and volume ranges, a condition known as hyperacusis (also spelled hyperacousis). Williams syndrome (also known as Williams-Beuren syndrome) is a multisystem disorder caused by a hemizygous deletion in a 1.6 Mb region at 7q11.23, which contains about 26 genes, including one encoding LIM kinase 1 (LIMK1). Individuals with Williams syndrome exhibit hyperacusis and progressive hearing loss, and the early onset of hyperacusis suggests it may be associated with one of the deleted genes.

[0010] Environmental causes of hearing loss include certain medications, specific infections before or after birth, and prolonged exposure to loud noise. Hearing loss can also be caused by noise affecting specific parts of the ear, ototoxic drugs, age-related hearing loss, disease, infection, or cancer. Ischemic damage can cause hearing loss through pathophysiological mechanisms. As another example, autoimmune inner ear disease (AIED) is characterized by rapidly progressive bilateral sensorineural hearing loss, which occurs when the body's immune system attacks cells in the inner ear that are mistaken for viruses or bacteria.

[0011] Approximately 1.5 out of every 1,000 children are born with severe hearing loss, and approximately 2 to 3 out of every 1,000 children are born with partial hearing loss (Smith et al., 2005, Lancet 365:879-890). More than half of these cases are attributed to genetic predisposition (Di Domenico, et al., 2011, J.Cell.Physiol.226:2494-2499).

[0012] Nonsyndromic hearing loss is hearing loss that is unrelated to other signs and symptoms. In contrast, syndromic hearing loss involves hearing loss that occurs in connection with abnormalities in other parts of the body. The majority of cases of hereditary hearing loss (70% to 80%) are nonsyndromic; the remaining cases are caused by specific genetic syndromes.

[0013] Hearing loss can be conductive (caused by damage to the ear canal or middle ear), sensorineural (caused by damage to the inner ear or auditory nerve), or a combination of both. Most forms of nonsyndromic hearing loss are associated with permanent hearing loss due to damage to the structures of the inner ear (sensory hearing loss). The vast majority of sensorineural hearing loss is caused by abnormalities in the hair cells of the organ Corti in the cochlea. There are also very unusual sensorineural hearing losses involving the eighth cranial nerve (vestiocochlear nerve) or the auditory part of the brain. In the rarest case of this type of hearing loss, only the auditory center of the brain is affected. In this case, cortical hearing loss may occur, where sounds can be heard at normal thresholds, but the perceived sound quality is so poor that speech cannot be understood. However, most sensorineural hearing loss is due to poor hair cell function. Hair cells may be abnormal at birth or damaged throughout an individual's life. Damage can have both external causes, such as noise trauma and infection, and internal abnormalities, such as congenital mutations in genes that play an important role in the anatomy or physiology of the cochlea.

[0014] Hearing loss caused by changes in the middle ear is called conductive hearing loss. Some forms of nonsyndromic hearing loss involve changes in both the inner and middle ear, and are called mixed hearing loss. Hearing loss that occurs before a child learns to speak is classified as prelingual or congenital. Hearing loss that occurs after speech development is classified as postlingual. Most autosomal recessive loci result in prelingual severe to profound hearing loss.

[0015] Nonsyndromic hearing loss can have different inheritance patterns and can occur at any age. Types of nonsyndromic hearing loss are named according to their inheritance pattern. The autosomal dominant form is called DFNA, the autosomal recessive form is called DFNB, and the X-linked form is called DFN. Each type is also numbered according to the order in which it was described. For example, DFNA1 is the first described autosomal dominant type of nonsyndromic hearing loss.

[0016] Between 75% and 80% of cases are inherited in an autosomal recessive pattern, meaning that both copies of the gene in each cell are mutated. Typically, each parent of an individual with autosomal recessive deafness is a carrier of one copy of the mutated gene but is not affected by this form of hearing loss.

[0017] Another 20% to 25% of non-syndromic hearing loss cases are autosomal dominant, meaning that one copy of the altered gene in each cell is sufficient to cause hearing loss. Individuals with autosomal dominant hearing loss most often inherit the altered copy of the gene from parents who also have hearing loss.

[0018] Between 1% and 2% of cases show an X-linked inheritance pattern, meaning the mutated gene causing the condition is located on the X chromosome (one of the two sex chromosomes). Men with X-linked nonsyndromic hearing loss tend to experience more severe hearing loss earlier in life compared to women who inherit a copy of the same gene mutation. A characteristic of X-linked inheritance is that the father cannot pass the X-linked trait to his son.

[0019] Mitochondrial nonsyndromic deafness, caused by changes in mitochondrial DNA, occurs in less than 1% of cases in the United States. The altered mitochondrial DNA is passed down from the mother to all her sons and daughters. This type of deafness is not inherited from the father.

[0020] Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment characterized by normal outer hair cell function and abnormal or absent auditory brainstem responses. It is one of the most common causes of hearing and speech communication impairments in infants and young children. Approximately 10% of children with permanent hearing loss may have ANSD. The OTOF gene was the first gene identified as an autosomal recessive nonsyndromic ANSD, and mutations in OTOF have been found to account for approximately 5% of all autosomal recessive nonsyndromic hearing loss cases in some populations (Rodriguez-Ballesteros et al. 2008 Human Mut 29(6):823-831).

[0021] The causes of nonsyndromic hearing loss are complex. Researchers have identified more than 30 genes that, when altered, are associated with nonsyndromic hearing loss. However, some of these genes are not yet fully characterized. Different mutations in the same gene can be associated with different types of hearing loss, and some genes are associated with both syndromic and nonsyndromic hearing loss.

[0022] For example, genes associated with nonsyndromic hearing loss include, but are not limited to, ATP2B2, ACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB3, KCNQ4, LHFPL5, MYO1A, MYO15A, MYO6, MYO7A, OTOF, PCDH15, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIOBP, USH1C, and WFS1.

[0023] The most common cause of hearing loss is nonsyndromic hearing loss and deafness (DFNB1) (also known as GJB2-associated DFNB1 nonsyndromic hearing loss and deafness; autosomal recessive deafness 1; neurosensory nonsyndromic recessive deafness 1). Nonsyndromic hearing loss and deafness (DFNB1) is characterized by congenital, non-progressive, mild to profound sensorineural hearing impairment. It is caused by mutations in GJB2 (which encodes the protein connexin 26) and GJB6 (which encodes the protein connexin 30). Diagnosis of DFNB1 depends on molecular genetic testing to identify deafening mutations in GJB2 and altered upstream cis-regulatory elements of the gap junction β-2 protein (connexin 26). Molecular genetic testing for GJB2 detects deafening mutations in over 99% of these genes. Unlike some other forms of hearing loss, DFNB1 nonsyndromic hearing loss and deafness does not affect balance or movement. The degree of hearing loss is difficult to predict based on which gene mutation an individual has. Even if members of the same family are affected by DFNB1 nonsyndromic hearing loss and deafness, the degree of their hearing loss may vary.

[0024] Mutations in the genes encoding connexin 26 (Cx26) and / or Cx30 are associated with approximately half of all cases of autosomal nonsyndromic prelingual hearing loss. Cx26 and Cx30 are two major Cx isoforms found in the cochlea, which assemble together to form hybrid (heterogeneous and heterogeneous) gap junctions (GJ) (Ahmad, et al., Proc. Natl. Acad. Sci., 2007, 104(4):1337-1341). Nonsyndromic hearing loss and deafness DFNA3 is caused by dominant-negative pathogenic variants in the GJB2 or GJB6 genes, which alter the proteins connexin 26 (Cx26) or connexin 30 (Cx30), respectively, and is characterized by prelingual or postlingual, mild to profound, progressive high-frequency sensorineural hearing loss.

[0025] OTOF-related hearing loss (DFNB9 nonsyndromic hearing loss) is characterized by two phenotypes: prelingual nonsyndromic hearing loss and the less common temperature-sensitive nonsyndromic auditory neuropathy (TS-NSAN). Another form of progressive hearing loss is associated with mutations in the otoferlin gene (e.g., I1573T or P1987R mutations, and / or E1700Q mutations) or temperature insensitivity.

[0026] Pendred syndrome / DFNB4 (deafness with goiter) is an autosomal recessive disorder and accounts for 7.5% of all cases of congenital deafness. Pendred syndrome is associated with mutations in the PDS gene (also known as DFNB4, EVA, PDS, TDH2B, and solute carrier family 26, member 4, SLC26A4) on the long arm (7q31) of chromosome 7, which encodes the pendrin protein. Mutations in this gene also lead to enlarged vestibular aqueduct syndrome (EVA or EVAS), another congenital cause of deafness. Specific mutations are more likely to cause EVAS, while others are associated with Pendred syndrome. (Azaiez, et al. (December 2007), Hum. Genet. 122(5):451-7).

[0027] The transmembrane protease serine 3 is an enzyme encoded by the TMPRSS3 gene (also known as DFNB10, DFNB8, ECHOS1, and TADG12). This gene was identified through its association with congenital and childhood-onset autosomal recessive hearing loss. Mutations in TMPRSS3 are associated with postlingual and rapidly progressive hearing loss. The protein encoded by the TMPRSS3 gene contains a serine protease domain, a transmembrane domain, an LDL receptor-like domain, and a cysteine-rich scavenger receptor domain. Serine proteases are known to be involved in a variety of biological processes, and their dysfunction often leads to diseases and conditions in humans. This gene is expressed in the fetal cochlea and many other tissues and is believed to be involved in the development and maintenance of the inner ear or perilymph and endolymph contents. This gene has also been identified as a tumor-associated gene overexpressed in ovarian tumors. Four alternative splicing variants have been described, two of which encode the same product.

[0028] DFN3 deafness is caused by a mutation in the POU3F4 gene located on the X chromosome. In people with this condition, one of the small bones in the middle ear (the stapes) cannot move normally, which interferes with hearing. This characteristic symptom of DFN3 is called stapes fixation. At least four other regions of the X chromosome are associated with hearing loss, but the responsible gene has not yet been identified. DFNB59 (deafness, autosomal recessive 59), also known as Pejvakin or PJVK, is a 352-amino acid protein belonging to the gasdermin family in vertebrates. DFNB59 is encoded by a gene located on human chromosome 2q31.2, which is essential for the normal function of neurons in the auditory pathway and the function of outer hair cells. Mutations in DFNB59 are thought to cause nonsyndromic sensorineural deafness autosomal recessive 59, a type of sensorineural hearing impairment characterized by the absence or severe abnormality of auditory brainstem responses, but with normal otoacoustic emissions (auditory neuropathy or auditory asynchrony). DFNB59 and DFNA5 are significantly similar, indicating that these genes share a common origin.

[0029] Alport syndrome is caused by mutations in the COL4A3, COL4A4, and COL4A5 genes involved in collagen biosynthesis. Mutations in any of these genes prevent the proper production or assembly of the type IV collagen network, an important structural component of the basement membrane in the kidneys, inner ear, and eye. One of the criteria for diagnosing Alport syndrome is bilateral sensorineural hearing loss in the 2000 to 8000 Hz range. The hearing loss develops gradually, is not present in early infancy, and typically appears before age 30.

[0030] Ion channel defects are associated with hearing loss: DFNA2 nonsyndromic hearing loss is inherited as an autosomal dominant mutation in the KCNQ4 gene, which encodes member 4 of the potassium voltage-gated channel subfamily KQT, also known as voltage-gated potassium channel subunit Kv7.4. DFNA2 nonsyndromic hearing loss is characterized by symmetry, primarily high-frequency sensorineural hearing loss (SNHL), and is progressive across all frequencies. In younger individuals, hearing loss tends to be mild in the low frequencies and moderate in the high frequencies; in older individuals, hearing loss is moderate in the low frequencies and severe to profound in the high frequencies. Although hearing impairment is frequently found in routine hearing assessments of school-aged children, hearing loss may have been present at birth, especially at high frequencies. Most affected individuals initially require hearing aids to help amplify sound between the ages of 10 and 40. By age 70, all individuals with DFNA2 hearing loss have severe to profound hearing impairment.

[0031] Mutations in the KCNE1 and KCNQ1 genes cause Jervell and Lange-Nielsen syndrome (JLNS), a type of long QT syndrome associated with severe bilateral hearing loss. This condition is an autosomal recessive disorder affecting an estimated 1.6 to 6 children per million, and accounts for less than 10% of all long QT syndrome cases. It has a significantly higher incidence in Norway and Sweden, reaching as high as 1 in 200,000. Proteins produced by the KCNE1 and KCNQ1 genes work together to form potassium channels, transporting positively charged potassium ions out of cells. The movement of potassium ions through these channels is essential for maintaining normal function of the inner ear and heart muscle.

[0032] EAST / SeSAME syndrome is characterized by intellectual disability, ataxia, seizures, hearing loss, and renal salt waste, and is believed to be caused by mutations in the KCNJ10 inward rectifier potassium channel.

[0033] Subjects with Bartter syndrome with sensorineural hearing loss type 4 (also known as Bartter syndrome IV or BSND) have a defect in the Cl-channel helper subunit.

[0034] Mutations in the ATP6V1B1 gene, expressed in the kidneys and cochlea, are associated with distal renal tubular acidosis (DRTA). A significant proportion of children with autosomal recessive DRTA have also been found to experience progressive bilateral sensorineural hearing loss.

[0035] Usher syndrome (also known as Hallgren syndrome, Usher-Hallgren syndrome, retinitis pigmentosa-hearing loss syndrome, and dystrophic retinal hearing loss syndrome) is a rare condition caused by mutations in any of at least ten genes, resulting in a combination of hearing loss and progressive vision impairment, and is a leading cause of deafness and blindness. Hearing loss is caused by a defective inner ear, while vision loss is caused by retinitis pigmentosa (RP), a degeneration of retinal cells. Usher syndrome has three clinical subtypes, designated I, II, and III. Individuals with Usher I are born severely deaf and begin to lose vision in the first decade of their lives. Due to problems with the vestibular system, they learn to walk slowly like children and exhibit balance difficulties. Individuals with Usher II are not born deaf but do have hearing loss, but do not appear to have significant balance problems; they also begin to lose vision later in life (in the second decade), and may retain some vision even into middle age. Individuals with Usher syndrome III are not born deaf but experience progressive hearing and vision loss; they may or may not have balance difficulties.

[0036] A mouse model of congenital deafness has been generated by making null mutations in the gene encoding vesicle glutamate transporter-3 (VGLUT3). Recently, hearing has been restored in VGLUT3 knockout mice using virus-mediated gene therapy (Akil, et al., 2012, Neuron 75:283-293).

[0037] Math1 (the mouse homolog of ATH1); also known as HATH1 or Atonal, the Drosophila homolog of (ATOH1), is essential for the development of inner ear hair cells; therefore, it is proposed that Math1 acts as an "anterior hair cell gene" in the developing sensory epithelium (Bermingham et al., 1999, Science 284:1837-1841). Several studies have now demonstrated that viral-mediated Math1 delivery leads to the regeneration of hair cells in the damaged cochlea of ​​mice and improvements in hearing and balance (Baker et al., 2009, Adv. Otorhinolaryngol. 66:52-63; Husseman and Raphael, 2009, Adv. Otorhinolaryngol. 66:37-51; Izumikawa et al., 2008, Hear. Res. 240:52-56; Kawamoto et al., 2003, J. Neurosci. 23:4395-4400; Praetorius et al., 2010, Acta Otolaryngol. 130:215-222; Staecker et al., 2007, Otol. Neurotol. 28:223-231).

[0038] Mutations in the WFS1 gene cause more than 90% of cases of Wolfram syndrome type 1. Wolfram syndrome is a condition affecting many systems of the body, most commonly characterized by high blood sugar levels due to a lack of the hormone insulin (diabetes) and progressive vision loss due to degeneration of the nerves that transmit information from the eyes to the brain (optic atrophy). However, people with Wolfram syndrome often also suffer from pituitary dysfunction, leading to excessive urine output (diabetes insipidus), hearing loss due to inner ear changes (sensory deafness), urinary tract problems, decreased levels of the male sex hormone testosterone (hypogonadism), or neurological or psychiatric disorders. Approximately 65% ​​of people with Wolfram syndrome have sensorineural hearing loss, ranging in severity from deafness at birth to mild hearing loss beginning in adolescence, which worsens over time. In addition, about 60% of people with Wolfram syndrome develop neurological or psychiatric disorders, most commonly balance and coordination problems (ataxia), which usually begin in early adulthood.

[0039] The WFS1 gene encodes a protein called wolframin, which is thought to regulate calcium levels in cells. When Wolfram syndrome is caused by a mutation in the WFS1 gene, it is inherited in an autosomal recessive pattern, and the wolframin protein has reduced or absent function. As a result, intracellular calcium levels are not regulated, and the endoplasmic reticulum (ER) cannot function properly. When the ER does not have enough functional wolframin, the cell triggers its own cell death (apoptosis). The death of cells in the pancreas, especially insulin-producing cells (β cells), leads to diabetes in people with Wolfram syndrome. The gradual loss of cells along the optic nerve eventually leads to blindness in affected individuals. Cell death in other body systems can cause a variety of signs and symptoms of type 1 Wolfram syndrome.

[0040] Mutations in the mitochondrial genes MT-TS1 and MT-RNR1 have been found to increase the risk of developing nonsyndromic hearing loss. Nonsyndromic mitochondrial hearing loss and deafness is characterized by moderate to profound hearing loss. Pathogenic variants of MT-TS1 are commonly associated with the onset of sensorineural hearing loss in childhood. Pathogenic variants of MT-RNR1 are associated with susceptibility to hearing loss if exposed to certain antibiotics known as aminoglycosides (ototoxicity) and / or delayed sensorineural hearing loss; however, some individuals with MT-RNR1 gene mutations develop hearing loss even without exposure to these antibiotics. Hearing loss associated with aminoglycoside ototoxicity is bilateral and severe to profound, occurring within days to weeks following administration of any amount (even a single dose) of aminoglycoside antibiotics such as gentamicin, tobramycin, amikacin, kanamycin, or streptomycin.

[0041] Treatment for hearing loss currently includes hearing amplification for mild to severe loss and cochlear implantation for severe to profound loss (Kral and O'Donoghue, 2010, N. Engl. J. Med. 363: 1438-1450). To date, most research in this field has focused on cochlear hair cell regeneration, applicable to the most common forms of hearing loss, including age-related hearing loss, noise-induced hearing loss, infection, and ototoxicity.

[0042] In animal models of cochlear ischemia, ischemic injury can be prevented by compounds such as insulin-like growth factor (IGF-1), AM-111 (an inhibitor of apoptosis), edarabone (a free radical scavenger), ginsenoside RB1 (Kappo), glial cell-derived neurotrophic factor (GDNF), BDNF, CNTF, SOD1, SOD2, Necrostatin-1, DFNA5, and MSRB3. However, it appears that combinations of multiple substances may be more effective than single compounds (e.g., complementary therapies that modulate oxidative stress, exotoxicity, blood flow, calcium and stimulus overload, apoptosis pathways, neurotrophic or hormonal control mechanisms).

[0043] Inhibition of JNK-1-induced apoptosis (mitochondrial-induced) can be prevented by compounds such as dominant-negative JNK-1 and d-stereoisomer JNK-1 (Mol. Pharmacol. 2007 March; 71(3):654-66; the contents of which are incorporated herein by reference in their entirety).

[0044] For a long time, people have needed medications and methods to prevent or reverse deafness.

[0045] Brief description of the attached figures

[0046] Figure 1 This is an exemplary schematic diagram of the genetic maps of 5' and 3' vectors used for dual AAV transduction in inner hair cells (IHC) using a trans-splicing method. In the 5' vector, the CMV enhancer (CMVe) and the human β-actin promoter (hbA) drive the transcription of mRNA encoding eGFP and the P2A peptide, which is cleaved during translation. The 5' vector also contains cDNA encoding the N-terminal portion of ototoxicin and the splice donor site (SD). The SD DNA sequence was provided by Trapani et al. (2014) EMBO MolMed 6 194–211. In the 3' vector, the splice acceptor site (SA) is subcloned after the first inverted terminal repeat (ITR) and before the 3' portion of the ototoxicin coding sequence. This is followed by the mRNA stable sequence, the marmot hepatitis virus posttranscriptional regulatory element (WPRE), and the polyadenylation signal (pA).

[0047] Figure 2 This is an exemplary schematic diagram of the genetic map of the 5' and 3' vectors undergoing dual AAV transduction in IHC using a heterozygous approach. Besides... Figure 1In addition to the elements described, highly recombinogenic sequences from the F1 phage (AK) sequence are also included at the 3' end of the '5' vector and the 5' end of the 3' vector to force correct vector assembly (Trapani et al. (2014) EMBO Mol Med 6 194–211).

[0048] Figure 3 It displays nontransductive Otof - / - Mice (n=25; fine black line) and Otof after double AAV-mediated otomorphin expression - / - A graph of auditory brainstem response (ABR) in mice (n=14; gray lines represent individual animals, and thick black lines represent the average response of all animals).

[0049] Figure 4 It displays nontransductive Otof - / - Mice (n=25; long dashed line), Otof after double AAV-mediated otomorphin expression - / - Mice (n=14; dashed line) and wild-type Otof mice after dual AAV transduction with enhanced green fluorescent protein (eGFP) wt / wt A graph showing the change in ABR amplitude over time for mice (n=5; thin black lines represent individual animals, thick black lines represent the average amplitude of all animals).

[0050] Figure 5 This is an exemplary graph showing the variation of the ABR threshold (in dB SPL) in response to pure tone or click stimuli as a function of frequency (in kHz). For individuals with dual AAV-mediated otopathic protein expression, Otof... - / - Animals (white circles represent individual animals, and the diagonally filled circles represent the average threshold of all animals), for untransduced Otof - / - Animals (black circles), and Otof after double AAV transduction with eGFP. wt / wt Animals (circles filled with squares), responding to pure tone or short sound stimuli, demonstrate the minimum sound pressure level (SPL) at which the wave can be detected.

[0051] Figure 6 It is a graph showing the transduction rate of the IHC along the cochlea (e.g., the entire Corti, the apical turn, and the midbasal / basal turn) by immunohistochemistry using two different antibodies, one binding to the N-terminal auricularin fragment and the other binding to the C-terminal portion of the auricularin.

[0052] Figure 7 It is Otof derived from the expression of otopathic protein mediated by dual AAV. - / -A set of immunohistochemical images of an organ from the mouse Corti. Calcium-binding protein was used as a marker for inner and outer hair cells. Cells expressing eGFP indicate viral transduction of at least 5' virus. The left and middle insets show anti-otomarin antibody staining (Abcam) in the N-terminal half of the otocin. The right inset shows C-terminal anti-otomarin antibody staining (Synaptic Systems). In summary, all three insets demonstrate full-length otocin expression in the IHC. Importantly, although AAV transduces several cell types in the Corti organ (indicated by eGFP fluorescence), otocin expression is limited to inner hair cells. Scale bar: 100 μm.

[0053] Figure 8 It displays Otof - / - IHC (white; n=10), Otof after dual AAV-mediated otopathic protein expression - / - IHC (diagonal; n=8) and wild-type IHC (black; n=6) from background-matched control animals with Ca2+ depolarizing stimulation 2+ A graph of electric current.

[0054] Figure 9 It is displayed in Otof - / - IHCs from mice with dual AAV transduction (black lines, individual IHCs; rhombuses with diagonal padding: mean ± sem; n = 8), and Otof injections. - / - A graph showing plasma membrane capacitance (ΔCm) during depolarization in two individual mice with dual AAV transduction (dashed lines, showing only minimal exocytosis) and a background-matched wild-type control (black rhombuses; n=6). Wild-type data from Strenzke et al. (2016) EMBO J.35 2519–2535 are shown with black circles for comparison.

[0055] Figure 10 It displays Qreal in Otof - / - IHC (white diamond shape), Otof - / - A representative graph of the duration (ms) of depolarization in IHC (black circles) of mice with dual AAV transduction and background-matched wild-type controls (black diamonds).

[0056] Figure 11 This is a representative plasmid map of pAAV-AK-SA-3'mOTOF-EWB.

[0057] Figure 12 This is a representative plasmid map of pAAV-SA-3'mOTOF-EWB.

[0058] Figure 13This is a representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD.

[0059] Figure 14 This is a representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD-AK.

[0060] Figure 15 It is for display purposes. Figure 2 The two carrier transduction Otof shown - / - IHC in mice (medium shaded; thick line), IHC in wild-type mice (dark shaded; medium thick line), and Otof - / - A graph of plasma membrane capacitance (ΔCm) of IHC in mice during the duration of depolarization.

[0061] Figure 16 It displays Qreal in Otof - / - IHC (white diamond shape), used Figure 2 The two carrier transduction Otof shown - / - A representative graph of depolarization duration (ms) in the IHC of mice (gray rhombuses) and the IHC of wild-type controls (black rhombuses) matched to the background.

[0062] Figure 17 This is a representative plasmid map of pAAV-AK-SA-3'mOTOF-EWB.

[0063] Figure 18 This is a representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD.

[0064] Figure 19 This is a representative plasmid map of pAAV-HBA-eGFP-P2A-5'mOTOF-SD-AK.

[0065] Figure 20 This is a representative plasmid map of pAAV-SA-3'mOTOF-EWB.

[0066] Figure 21 This is a representative schematic diagram of a part of pAKOS102 (SEQ ID NO:43).

[0067] Figure 22 This is a representative schematic diagram of a part of pAKOS103 (SEQ ID NO:44).

[0068] Figure 23 This is a representative schematic diagram of a part of pAKOS103.

[0069] Figure 24This is a representative plasmid map of pAKOS104 (SEQ ID NO:45).

[0070] Figure 25 This is a representative schematic diagram of a part of pAKOS104-DHFR (SEQ ID NO:46).

[0071] Figure 26 This is a representative schematic diagram of a part of pAKOS104-DHFR.

[0072] Figure 27 This is a representative schematic diagram of a part of pAKOS105 (SEQ ID NO:47).

[0073] Figure 28 This is a representative schematic diagram of a part of pAKOS105.

[0074] Figure 29 This is a representative schematic diagram of a portion of pAKOS105_GFP (SEQ ID NO:48).

[0075] Figure 30 This is a representative schematic diagram of a part of pAKOS106 (SEQ ID NO:49).

[0076] Figure 31 This is a representative schematic diagram of a part of pAKOS106.

[0077] Figure 32 This is a representative schematic diagram of a part of pAKOS107 (SEQ ID NO:50).

[0078] Figure 33 This is a representative schematic diagram of a part of pAKOS107.

[0079] Figure 34 This is a representative schematic diagram of a part of pAKOS108 (SEQ ID NO:51).

[0080] Figure 35 This is a representative schematic diagram of a part of pAKOS108.

[0081] Figure 36 This is a representative schematic diagram of a part of pAKOS109 (SEQ ID NO:52).

[0082] Figure 37 This is a representative schematic diagram of factor VIII filler (SEQ ID NO.54-57).

[0083] Figure 38This is a representative schematic diagram of p109 (SEQ ID NO:84).

[0084] Figure 39 This is a representative schematic diagram of p105 (SEQ ID NO:85).

[0085] Figure 40 This is a representative schematic diagram of 105.WPRE.

[0086] Figure 41 This is a representative schematic diagram on page 108.

[0087] Figure 42 This is a representative schematic diagram of 1OTOF18.CL1.

[0088] Figure 43 This is a representative schematic diagram of 19OTOF48.

[0089] Figure 44 This is a representative schematic diagram of 1OTOF20.CL1.

[0090] Figure 45 This is a representative schematic diagram of 21OTOF48.WPRE.

[0091] Figure 46 This is a representative schematic diagram of 1OTOF21.CL1.

[0092] Figure 47 This is a representative schematic diagram of 22OTOF48.WPRE.

[0093] Figure 48 This is a representative schematic diagram of 105.pA.NTF3.CMVd.

[0094] Figure 49 This is an immunoblot showing the expression of full-length human otomorphin in transfected HEK293FT cells using different indicated plasmids.

[0095] Figure 50 It shows that it comes from Figure 49 The table shows the quantitative expression of full-length human otomorphin in three replicates of the experiment described in the paper.

[0096] Figure 51 It is wild-type Otof that has not been treated with a vector, or Otof that has not been treated with a vector, or Otof that has been treated with dual AAV Anc80.h Otof vectors (p105 and p109 vectors). - / - A graph of short-tone ABR thresholds in mice. Otof values ​​were measured at 26–28 days and 91 days post-treatment after administration of dual AAV Anc80.hOtof vectors (p105 and p109 vectors). - / - The hearing of mice.

[0097] Figure 52 It is wild-type Otof that has not been treated with a vector, or Otof that has not been treated with a vector, or Otof that has been treated with dual AAV Anc80.h Otof vectors (p105 and p109 vectors). - / - A graph of tone burst ABR thresholds in mice. Otof values ​​were measured at 26–28 days and 91 days post-treatment after administration of dual AAV Anc80.hOtof vectors (p105 and p109 vectors). - / - The hearing of mice.

[0098] Figure 53 This is a representative schematic diagram of a portion of pAAV-HBA-eGFP-P2A-5'mOTOF.SD (SEQ ID NO:87).

[0099] Figure 54 This is a representative schematic diagram of a part of pAAV-SA-3'mOTOF.WPRE (SEQ ID NO:88).

[0100] Figure 55 This is a representative schematic diagram of a portion of pAAV-HBA-eGFP-P2A-5'mOTOF.SD-AK.

[0101] Figure 56 This is a representative schematic diagram of a part of pAAV-AK-SA-3'mOTOF.WPRE.

[0102] Figure 57 This is a representative schematic diagram of a part of pAAV-CMV-5'hOTOF-SD-AK.

[0103] Figure 58 This is a representative schematic diagram of a part of pAAV-HBA-5'hOTOF-SD-AP.

[0104] Figure 59 This is a representative schematic diagram of a part of pAAV-HBA-5'hOTOF-SD-AK.

[0105] Figure 60 This is a representative schematic diagram of a part of pAAV-HBA-5'hOTOF codop-SD-AK.

[0106] Figure 61 This is a representative schematic diagram of a part of pAAV-HBA-5'hOTOF codop-SD.

[0107] Figure 62 This is a representative schematic diagram of a part of pAAV-CMV-5'hOTOF codop-SD.

[0108] Figure 63 This is a representative schematic diagram of a part of pAAV-CMV-5'hOTOF codop-SD-AK.

[0109] Figure 64 This is a representative schematic diagram of a part of pAAV-CBA-5'hOTOF codop-SD-AK.

[0110] Figure 65 This is a representative schematic diagram of a part of pAAV-CBA-5'hOTOF-SD.

[0111] Figure 66 This is a representative schematic diagram of a part of pAAV-SA-3'OTOF.

[0112] Figure 67 This is a representative schematic diagram of a part of pAAV-AP-SA-3'OTOF.

[0113] Figure 68 This is a representative schematic diagram of a part of pAAV-AK-SA-3'OTOFcodop.

[0114] Figure 69 This is a representative immunoblot showing the use of DNA transfection reagents. Expression of full-length human ototoxicity protein in HEK293FT cells transfected with (polyplus), indicating different plasmid pairs at 600 ng each. Lane 1 contains a pre-stained protein ladder. Lane 2 contains proteins expressed using the vector pAKOS104 (e.g., ...). Figure 24 and 59 (as shown) and the carrier pAKOS105 (as shown) Figure 27 , 28 Protein samples from HEK293FT cells transfected with the vector pAKOS108 (as shown in Figure 39). Lane 3 contains protein samples from HEK293FT cells transfected with the vector pAKOS108 (as shown in Figure 39). Figure 34 , 35 (as shown in 41 and 57) and the carrier pAKOS105 (as shown in 41 and 57) Figure 27 , 28 Protein samples from HEK293FT cells transfected with the vector pAKOS109 (as shown in Figure 39). Lane 4 contains protein samples from HEK293FT cells transfected with the vector pAKOS109 (as shown in Figure 39). Figure 36 and 38 (as shown) and the carrier pAKOS105 (as shown) Figure 27 , 28 Protein samples from HEK293FT cells transfected with pAAV-HBA-5'hOTOFcodop-SD-AK (as shown in Figure 39). Lane 5 contained protein samples from HEK293FT cells transfected with pAAV-HBA-5'hOTOFcodop-SD-AK (as shown in Figure 39). Figure 60(as shown) and the carrier pAAV-AK-SA-3'OTOFcodop (as shown) Figure 68 Protein samples from HEK293FT cells transfected (as shown). Lane 6 contains proteins from cells transfected with the vector pAAV-CMV-5'hOTOFcodop-SD-AK (e.g., ...). Figure 63 (as shown) and the carrier pAAV-AK-SA-3'OTOFcodop (as shown) Figure 68 (As shown) Protein samples from HEK293FT cells transfected with the vector pAAV_CBA-5'hOTOFcodop-SD-AK (e.g., ...). Lane 7 contains protein samples from HEK293FT cells transfected with the vector pAAV_CBA-5'hOTOFcodop-SD-AK (e.g., ...). Figure 64 (as shown) and the carrier pAAV-AK-SA-3'OTOFcodop (as shown) Figure 68 Protein samples from HEK293FT cells transfected (as shown). Lane 8 contains proteins from cells transfected with the vector pAKOS102 (e.g., ...). Figure 21 (as shown) and carrier pAKOS103 (as shown) Figure 22 , 23 Protein samples from HEK293FT cells transfected with CBA.TS vector and pAKOS103 vector (as shown in Figure 66). Lane 9 contains protein samples from HEK293FT cells transfected with CBA.TS vector and pAKOS103 vector (as shown in Figure 66). Figure 22 , 23 Protein samples from HEK293FT cells transfected with pAKOS106 (as shown in Figure 66). Lane 10 contains protein samples from HEK293FT cells transfected with pAKOS106 (as shown in Figure 66). Figure 30 , 31 (as shown in Figure 58) and pAKOS107 (as shown in Figure 58) Figure 32 , 33 Protein samples from HEK293FT cells transfected (shown in Figure 67) were analyzed. Cells were harvested 96 hours post-transfection, lysed with RIPA buffer, and analyzed in 4–12% Bolt protein gels (which were subsequently transferred to nitrocellulose membranes). Human ototoxicity protein was detected using an anti-OTOF polyclonal antibody (Thermo PA5-52935). Human β-actin was used as a primary antibody for loading controls between lanes. The experiment was performed in triplicate. Relative quantitative measurements for each experiment are provided under immunoblotting, along with mean measurements and standard deviation (STDEV).

[0115] Figure 70 This is an immunoblot showing the expression of full-length human otomorphin in transfected HEK293FT cells at different multiplicity of infection (MOI) using different indicated plasmids. HEK293FT cells were cultured in 96-well plates at 4 x 10⁻⁶ wells. 4Cells / well were seeded overnight. Six hours after seeding, a dual vector was added to each well. 96 hours after transfection, cells were harvested, lysed with RIPA buffer, and analyzed in 4–12% Bolt protein gels (which were subsequently transferred to nitrocellulose membranes). Human otomorphin was detected using an anti-OTOF polyclonal antibody (Thermo PA5-52935). Human β-actin was used as a primary antibody for loading controls between lanes. Lane 1: CBA.OTOF(AK), MOI 503,000; Lane 2: CBA.OTOF(AK), MOI 1,510,000; Lane 3: CBA.OTOF(AK), MOI 100,000; Lane 4: CBA.OTOF(AK), MOI 303,000; Lane 5: CMV.OTOF(AK), MOI 638,000; Lane 6: CMV.OTOF(AK), MOI 1,910,000; Lane 7: CMV.OTOF(AK), MOI 127,000; Lane 8: CMV.OTOF(AK), MOI 382,000; Lane 9: Negative control.

[0116] Figure 71 A set of immunohistochemical images of an organ from an Otof- / - mouse, age P17, following administration of dual AAV vectors (p105 and 109 vectors) expressing CBA.hOTOF(AK) into the unilateral cochlea. Protein expression was analyzed in three distinct frequency regions (base-apex) using immunohistochemistry after dissection of the ipsilateral cochlea.

[0117] Figure 72 This demonstrates the effects of dual AAV-TS (n=10 mice) and dual AAV-Hyb (n=9 mice) injections of CD1B6F1-Otof - / - A graph showing the percentage of N- and C-terminal ototoxicin-labeled inner hair cells (IHC) in mice (P18–P30 years of age). Individual animals are depicted with hollow symbols. Data are presented as mean ± standard error of mean (sem), ns. P>0.05; *P≤0.05; **P≤0.01; ***P≤0.001, Wilcoxon paired signed-rank test, and unpaired t-test with Welch correction.

[0118] Figure 73 It is an Otof that shows dual AAV transduction from mice (P23-30 years old). - / -A graph showing the mean N-terminal and C-terminal otocin immunofluorescence levels in wild-type inner hair cells (IHC). Otocin levels were normalized to the immunofluorescence levels in non-transduced B6 wild-type IHC for each antibody. The number of quantified IHCs is shown in the bars. Data are presented as mean ± standard error of mean (sem), ns. P>0.05; *P≤0.05; **P≤0.01; ***P≤0.001, Kruskal-Wallis test followed by Dunn's multiple comparison test.

[0119] Figure 74 This shows wild-type (B6: n=48 IHC, CD1B6F1: n=108 IHC) transduced Otof from injected (-AAV injected ear, n=65 IHC) and contralateral non-injected (-AAV non-injected ear, n=46 IHC) ears (from P25-P29 age mice). - / - (Dual AAV-TS: n = 59 IHCs, Dual AAV-Hyb: n = 37 IHCs) and non-transductive Otof - / - A graph showing the number of synaptic bands quantified from the inner hair cells in the apical cochlear apex. Individual animals are depicted with hollow symbols. Data are presented as mean ± standard error of mean (sem), ns. P>0.05, **P≤0.001 (Kruskal-Wallis test, followed by Dunn's multiple comparison test).

[0120] Figure 75 It shows B6 wild-type (P6: n = 53 IHCs; P14: n = 73 IHCs) and B6 Otof at two different developmental stages (P6 and P14). - / - (P6: n = 62 IHCs; P14: n = 65 IHCs) A graph of the number of synapses quantified from inner hair cells at the apical corner of mice. Individual animals are depicted with hollow symbols. Data are presented as mean ± standard error of mean (sem), ns. P > 0.05, **P ≤ 0.001 (Kruskal-Wallis test, followed by Dunn's multiple comparison test).

[0121] Figure 76 This indicates double AAV-injection of otodermal protein in individuals aged 23-30 years, without Otof injection. - / - A graph showing the sum of auditory brainstem response (ABR) wave amplitudes at different short sound intensities in wild-type and control mice. Number of mice analyzed: CD1B6F1 wild-type animals (+AAV.eGFP: n = 12 mice, dual AAV-TS: n = 6 mice) and CD1B6F1 Otof - / -Animals (double AAV-TS: n = 16 mice, double AAV-Hyb: n = 8 mice, =AAV: n = 38 mice). Data are expressed as mean ± standard error of mean (sem). Individual animals are depicted with hollow symbols.

[0122] Figure 77 It is CD1B6F1 Otof that displays individual dual AAV-TS processing. - / - Animals (n = 8 animals; from) Figure 76 The sum of auditory brainstem response (ABR) wave IV amplitudes relative to their full-length inner hair cell (IHC) transduction rates (from...) Figure 5 A graph plotted using C-terminal auricularin. r: correlation coefficient. = 8 mice, = AAV: n = 38 mice). Individual animals are depicted with hollow symbols. r ≥ 0.5 is a positive correlation (70 dB SPL and 90 dB SPL: Pearson correlation test; 50 dB SPL: Spearman correlation test).

[0123] Figure 78 This is a schematic diagram of an exemplary dual AAV carrier system of the present disclosure, which includes "upstream" and "downstream" carriers (AKhOTOF5 and AKhOTOF3, respectively).

[0124] Figure 79 This is a schematic diagram of the "upstream" carrier AKhOTOF5.

[0125] Figure 80 This is a schematic diagram of the "downstream" carrier AKhOTOF3.

[0126] Figure 81 A perspective view of a device for delivering fluid to the inner ear according to aspects of this disclosure is illustrated.

[0127] Figure 82 A side view of a bent needle assembly according to an aspect of this disclosure is shown.

[0128] Figure 83 A perspective view of a device for delivering fluid to the inner ear according to aspects of this disclosure is illustrated.

[0129] Figure 84 A perspective view illustrating a bent needle assembly connected to a remote end of a device according to an aspect of this disclosure is shown. Invention Overview

[0131] This disclosure is based on the discovery that a composition comprising at least two different nucleic acid vectors, each of the at least two different vectors comprising a coding sequence encoding a different portion of an otopathic protein, can be used to generate a sequence encoding an active otopathic protein (e.g., full-length otopathic protein) in mammalian cells, thereby treating non-syndromic sensorineural hearing loss in subjects who require this treatment.

[0132] This document provides compositions comprising at least two different nucleic acid vectors, wherein: each of the at least two different vectors includes a coding sequence encoding a different portion of an ototoxic protein, each of the encoded portions being at least 30 amino acid residues in length, wherein the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; none of the at least two different vectors encodes the full-length ototoxic protein; at least one of the coding sequences includes a nucleotide sequence spanning two adjacent exons of the ototoxic protein genomic DNA and lacks an intron sequence between the two adjacent exons; and when introduced into mammalian cells, the at least two different vectors undergo polymerization or homologous recombination with each other to form a recombinant nucleic acid encoding the full-length ototoxic protein. In some embodiments of any of the compositions described herein, each of the at least two different vectors is a plasmid, transposon, granulosome, artificial chromosome, or viral vector. In some embodiments of any of the compositions described herein, each of the at least two different vectors is a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC). In some embodiments of any of the compositions described herein, each of at least two different vectors is a viral vector selected from adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, or retroviral vectors. In some embodiments of any of the compositions described herein, each of at least two different vectors is an AAV vector.

[0133] In some embodiments of any of the compositions described herein, the amino acid sequence of one of the encoded portions overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, the length of the overlapping amino acid sequence is between about 30 amino acid residues and about 1000 amino acid residues.

[0134] In some embodiments of any of the compositions described herein, the vector comprises two distinct vectors, each comprising a distinct segment of an intron, wherein the intron comprises the nucleotide sequence of an intron present in the otomorphin genomic DNA, and wherein the two distinct segments overlap in sequence by at least 100 nucleotides. In some embodiments of any of the compositions described herein, the two distinct segments overlap in sequence by about 100 nucleotides to about 800 nucleotides. In some embodiments of any of the compositions described herein, the length of the nucleotide sequence of each of the at least two distinct vectors is between about 500 nucleotides and about 10,000 nucleotides. In some embodiments of any of the compositions described herein, the length of the nucleotide sequence of each of the at least two distinct vectors is between 500 nucleotides and 5,000 nucleotides.

[0135] In some embodiments of any of the compositions described herein, the number of different vectors in the composition is two. In some embodiments of any of the compositions described herein, the first of the two different vectors includes a coding sequence encoding the N-terminal portion of an ototoxic protein. In some embodiments of any of the compositions described herein, the length of the N-terminal portion of the ototoxic protein is between 30 and 1600 amino acids. In some embodiments of any of the compositions described herein, the length of the N-terminal portion of the ototoxic protein is between 200 and 1500 amino acids. In some embodiments of any of the compositions described herein, the first vector further includes one or both of a promoter and a Kozak sequence. In some embodiments of any of the compositions described herein, the first vector includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.

[0136] In some embodiments of any of the compositions described herein, one of the two vectors comprises SEQ ID NO:39 (or comprises at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:39), and the second of the two vectors comprises SEQ ID NO:40 (or comprises at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:40). In some embodiments of any of the compositions described herein, one of the two vectors comprises SEQ ID NO:41 (or comprises at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:41), and the second of the two vectors comprises SEQ ID NO:42 (or comprises at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:42). In some embodiments of any of the compositions described herein, one of the two vectors comprises SEQ ID NO:84 (or comprises at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:84), and the second of the two vectors comprises SEQ ID NO:85 (or comprises at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:85).

[0137] In some embodiments of any of the compositions described herein, one of at least two different vectors contains a sequence encoding the NTF3 protein.

[0138] In some embodiments of any of the compositions described herein, the sequence encoding the NTF3 protein is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO:78.

[0139] In some embodiments of any of the compositions described herein, the first vector further includes a coding sequence encoding a destabilizing region, wherein the destabilizing region is located at the 3' of a coding sequence encoding the N-terminal portion of ototoxicity protein.

[0140] In some embodiments of any of the compositions described herein, the coding sequence for the N-terminal portion of ototoxicin comprises exons 1-21 of isotype 5 of the human ototoxicin gene.

[0141] In some embodiments of any of the compositions described herein, the second of the two different vectors comprises a coding sequence encoding the C-terminal portion of an ototoxicin. In some embodiments of any of the compositions described herein, the C-terminal portion of the ototoxicin is between 30 and 1600 amino acids in length. In some embodiments of any of the compositions described herein, the C-terminal portion of the ototoxicin is between 200 and 1500 amino acids in length. In some embodiments of any of the compositions described herein, the second vector further comprises a poly(dA) signal sequence. In some embodiments of any of the compositions described herein, the coding sequence encoding the C-terminal portion of the ototoxicin comprises exons 22-48 of isotype 5 of the human ototoxicin gene. In some embodiments of any of the compositions described herein, the second vector further comprises a sequence for mRNA stabilization. In some embodiments of any of the compositions described herein, a pharmaceutically acceptable excipient is further included.

[0142] This document also provides kits that include any of the compositions described herein. Some embodiments of any of the kits described herein further include a pre-filled syringe containing the composition.

[0143] This document also provides methods comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​a mammal. In some embodiments of any of the compositions described herein, the mammal is a human. In some embodiments of any of the compositions described herein, the mammal has been previously identified as having a defective ototoxicity protein gene.

[0144] This document also provides methods for increasing the expression of active otopathic proteins (e.g., full-length otopathic proteins) in mammalian cells, comprising introducing any of the compositions described herein into mammalian cells. In some embodiments of any of the methods described herein, the mammalian cells are cochlear hair cells. In some embodiments of any of the methods described herein, the mammalian cells are human cells. In some embodiments of any of the methods described herein, the mammalian cells have been previously identified as having defective otopathic protein genes.

[0145] This document also provides methods for increasing the expression of active cochlear proteins (e.g., full-length cochlear proteins) in the inner hair cells of the mammalian cochlea, comprising: introducing a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​a mammal. In some embodiments of any of the methods described herein, the mammal has been previously identified as having a defective ototoxic protein gene. In some embodiments of any of the methods described herein, the mammal is a human.

[0146] This document also provides a method for treating nonsyndromic sensorineural hearing loss in a subject identified as having a defective ototoxicin gene, the method comprising administering a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​the subject. In some embodiments of any of the methods described herein, the subject is a human being. Some embodiments of any of the methods described herein further include, prior to the administration step, determining that the subject has a defective ototoxicin gene.

[0147] This article also provides a composition comprising two different nucleic acid vectors, wherein: the first nucleic acid vector comprises a promoter, a first coding sequence encoding the N-terminal portion of ototoxicity protein located at the 3' end of the promoter, and a splice donor signal sequence located at the 3' end of the first coding sequence; the second nucleic acid vector comprises a splice acceptor signal sequence, a second coding sequence encoding the C-terminal portion of ototoxicity protein located at the 3' end of the splice acceptor signal sequence, and a polyadenylated sequence located at the 3' end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the amino acid sequences of the encoded portions do not overlap, wherein no single vector in the two different vectors encodes the full-length ototoxicity protein, and when the coding sequence is transcribed in mammalian cells to produce an RNA transcript, splicing occurs between the splice donor signal sequence on one transcript and the splice acceptor signal sequence on the other transcript, thereby forming a recombinant RNA molecule encoding the full-length ototoxicity protein. In some embodiments of any of the compositions described herein, at least one of the vectors comprises a nucleotide sequence spanning two adjacent exons of the ototoxicity protein genomic DNA and lacks an intron sequence between the two adjacent exons.

[0148] This document also provides compositions comprising: a first nucleic acid vector comprising a promoter, a first coding sequence encoding an N-terminal portion of an otoxin located at the 3' end of the promoter, a splice donor signal sequence located at the 3' end of the first coding sequence, and a first detectable biomarker gene located at the 3' end of the splice donor signal sequence; and a second nucleic acid vector, distinct from the first nucleic acid vector, comprising a second detectable biomarker gene, a splice acceptor signal sequence located at the 3' end of the second detectable biomarker gene, a second coding sequence encoding an C-terminal portion of an otoxin located at the 3' end of the splice acceptor signal sequence, and a polyadenylated sequence located at the 3' end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the corresponding amino acid sequences of the encoded portions do not overlap with each other, wherein no single vector in the two different vectors encodes the full-length otoxin, and wherein, when the coding sequence is transcribed in mammalian cells to produce an RNA transcript, splicing occurs between the splice donor signal on one transcript and the splice acceptor signal on the other transcript, thereby forming a recombinant RNA molecule encoding the full-length otoxin. In some embodiments of any of the compositions described herein, at least one of the coding sequences in the vector comprises a nucleotide sequence spanning two adjacent exons of the ototoxicin genomic DNA and lacks an intron sequence between the two adjacent exons. In some embodiments of any of the compositions described herein, a first or second detectable biomarker gene encodes alkaline phosphatase. In some embodiments of any of the compositions described herein, the first and second detectable biomarker genes are identical.

[0149] This document also provides compositions comprising a first nucleic acid vector comprising a promoter, a first coding sequence encoding the N-terminal portion of an ototoxic protein located at the promoter 3', a splice donor signal sequence located at the end of the first coding sequence 3', and a highly recombination-inducing sequence located at the splice donor signal sequence 3' (e.g., an F1 phage recombination region, e.g., SEQ ID NO:66); and a second nucleic acid vector, distinct from the first nucleic acid vector, comprising a second highly recombination-inducing sequence (e.g., an F1 phage recombination region, e.g., SEQ ID NO:67, or an alkaline phosphatase recombination region, e.g., SEQ ID NO:67). IDNO:89), a 3' splice acceptor signal sequence located at the second high recombination origin (e.g., the F1 phage recombination origin or alkaline phosphatase recombination origin), a second coding sequence located at the 3' end of the splice acceptor signal sequence encoding the C-terminal portion of the ototoxic protein, and a polyadenylated sequence located at the 3' end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the corresponding amino acid sequences of the encoded portions do not overlap with each other, wherein no single vector in either of the two different vectors encodes the full-length ototoxic protein, and, when the coding sequence is transcribed in mammalian cells to produce an RNA transcript, splicing occurs between the splice donor signal of one transcript and the splice acceptor signal on the other transcript, thereby forming a recombinant RNA molecule encoding the full-length ototoxic protein.

[0150] In some embodiments of any of the compositions described herein, at least one of the vectors comprises a nucleotide sequence spanning two adjacent exons of the ototoxicity protein genomic DNA and lacks an intron sequence between the two adjacent exons.

[0151] This document also provides kits comprising any of the compositions described herein. Some embodiments of any of the kits described herein further include a pre-filled syringe containing the composition.

[0152] This document also provides methods comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​a mammal. In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal has been previously identified as having a defective ototoxicity protein gene.

[0153] This document also provides methods for increasing the expression of full-length ototoxicity protein in mammalian cells, including introducing any of the compositions described herein into mammalian cells. In some embodiments of any of the methods described herein, the mammalian cells are cochlear hair cells. In some embodiments of any of the methods described herein, the mammalian cells are human cells. In some embodiments of any of the methods described herein, the mammalian cells have been previously identified as having the defective ototoxicity protein gene.

[0154] This document also provides methods for increasing the expression of full-length ototoxicity protein in the inner hair cells of the mammalian cochlea, comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea. In some embodiments of any of the methods described herein, the mammal has been previously identified as having a defective ototoxicity protein gene. In some embodiments of any of the methods described herein, the mammal is a human.

[0155] This article also provides a method for treating asymptomatic sensorineural hearing loss in a subject identified as having a defective ototoxicin gene, the method comprising administering a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​the subject. In some embodiments of any of the methods described herein, the subject is a human being. Some embodiments of any of the methods described herein further include, prior to the administration step, determining that the subject has a defective ototoxicin gene.

[0156] This article provides a therapeutic composition comprising multiple adeno-associated virus (AAV) vectors, wherein the multiple AAV vectors are capable of forming auditory polypeptide messenger RNA in target cells of human subjects to which the therapeutic composition has been administered.

[0157] In some embodiments of any of the therapeutic compositions described herein, multiple AAV vectors are capable of forming full-length auditory polypeptide messenger RNA in the target cells of a human subject to which the therapeutic composition has been administered.

[0158] Some embodiments of any therapeutic composition described herein further include a first AAV carrier and a second AAV carrier, wherein the first and second AAV carriers independently contain a packaging capacity of less than about 6 kb.

[0159] In some embodiments of any of the therapeutic compositions described herein, the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from otopathic proteins and their orthologs or homologs.

[0160] Some embodiments of any therapeutic composition described herein may further include a nucleic acid (e.g., a vector) comprising a nucleic acid sequence encoding an auditory polypeptide messenger RNA, wherein the RNA encodes an auditory polypeptide selected from the group consisting of: Ca v 1.3 Scaffold proteins selected from bassoon, piccolo, ribeye, and harmonin, Vglut3, synaptic binding proteins, vesicle tethering / docking proteins, vesicle initiation proteins, vesicle fusion proteins, GluA2 / 3, and GluA4.

[0161] In some embodiments of any therapeutic composition described herein, the first AAV vector further comprises at least one promoter sequence selected from CBA, CMV, or CB7 promoters.

[0162] In some embodiments of any therapeutic composition described herein, the first AAV carrier further comprises at least one promoter sequence selected from cochlear-specific promoters.

[0163] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated for intracochlear administration. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to comprise lipid nanoparticles. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to comprise polymer nanoparticles. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to comprise small circular DNA. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to comprise CELID DNA. In some embodiments of any of the compositions described herein, the therapeutic composition is formulated to comprise a synthetic perilymph solution. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to comprise a synthetic perilymph solution comprising 20-200 mM NaCl; 1-5 mM potassium chloride; 0.1-10 mM CaCl2; 1-10 mM glucose; and 2-50 mM HEPES; and having a pH between about 6 and about 9.

[0164] This article also provides therapeutic compositions containing auditory peptide messenger RNA.

[0165] This article also provides therapeutic compositions comprising one or more adenovirus (AV) vectors, wherein the one or more AV vectors are capable of forming auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition has been administered.

[0166] In some embodiments of any of the therapeutic compositions described herein, one or more AV carriers are capable of forming full-length auditory polypeptide messenger RNA in the target cells of a human subject to which the therapeutic composition has been administered.

[0167] This article also provides therapeutic compositions comprising one or more lentiviral vectors, wherein the one or more lentiviral vectors are capable of forming auditory polypeptide messenger RNA in target cells of human subjects to whom the therapeutic composition has been administered.

[0168] In some embodiments of any of the therapeutic compositions described herein, one or more lentiviral vectors are capable of constituting active, for example, full-length auditory polypeptide messenger RNA, in the target cells of a human subject to which the therapeutic composition has been administered.

[0169] This article also provides a surgical method comprising the following steps: i) making a first incision at a first incision point into the cochlea of ​​a human subject; ii) administering an effective dose of a therapeutic composition (e.g., any therapeutic composition described herein) into the cochlea.

[0170] In some embodiments of any of the methods described herein, the therapeutic composition is applied to the subject at a first incision site. In some embodiments of any of the methods described herein, the therapeutic composition is applied to the subject through or into the first incision.

[0171] In some embodiments of any of the methods described herein, the therapeutic composition is administered to a subject via or through the round window membrane of the cochlea.

[0172] In some embodiments of any of the methods described herein, a medical device capable of creating multiple incisions in a circular window membrane is used to administer the therapeutic composition.

[0173] In some embodiments of any of the methods described herein, the medical device includes a plurality of microneedles. In some embodiments of any of the methods described herein, the medical device includes a plurality of microneedles comprising a generally circular first aspect, wherein each microneedle has a diameter of at least about 10 micrometers.

[0174] In some embodiments of any of the methods described herein, the medical device includes a base and / or reservoir capable of receiving a therapeutic composition. In some embodiments of any of the methods described herein, the medical device includes a plurality of hollow microneedles, each of which includes a lumen capable of transferring the therapeutic composition.

[0175] In some embodiments of any of the methods described herein, the medical device includes means for generating at least a partial vacuum.

[0176] This article also provides a therapeutic delivery system comprising i) a medical device capable of creating multiple incisions in the round window membrane of the inner ear of a subject in need, and ii) a therapeutic composition containing multiple adeno-associated virus (AAV) vectors, wherein the multiple AAV vectors are capable of constituting active (e.g., full-length) auditory polypeptide messenger RNA in target cells of the inner ear.

[0177] This document also provides means for performing a surgical method comprising the following steps: i) administering an effective dose of a therapeutic composition (e.g., any therapeutic composition described herein) into the cochlea of ​​a human subject in need of such treatment, wherein the therapeutic composition can be administered by means of a) means for creating multiple incisions in the round window membrane and b) an effective dose of the therapeutic composition.

[0178] In some embodiments of any device used to perform the surgical methods described herein, the medical device comprises a plurality of microneedles.

[0179] This article also provides a therapeutic composition comprising a single adeno-associated virus (AAV) vector, wherein the AAV vector is capable of forming auditory polypeptide messenger RNA in target cells of a human subject to which the therapeutic composition has been administered.

[0180] In some embodiments of any of the therapeutic compositions described herein, a single AAV vector is capable of forming a full-length auditory polypeptide messenger RNA in the target cells of a human subject to which the therapeutic composition has been administered. In some embodiments of any of the methods described herein, a single vector (e.g., any vector described herein) including a sequence encoding an active otopathic protein (e.g., any full-length or truncated active otopathic protein described herein) may be administered to the subject.

[0181] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition further comprises a second carrier different from the AAV carrier, wherein the individual AAV carrier and the second carrier independently contain a packaging capacity of less than about 6 kb.

[0182] In some embodiments of any of the therapeutic compositions described herein, the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group consisting of otopathic proteins and their orthologs or homologs.

[0183] In some embodiments of any of the therapeutic compositions described herein, the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group consisting of otopathic proteins and their truncated mutants.

[0184] In some embodiments of any of the therapeutic compositions described herein, the otomorphin truncated mutant includes at least a single C2 domain:

[0185]

[0186] In some embodiments of any of the therapeutic compositions described herein, the otopathic protein truncated mutant does not include the C-terminal region of the endogenous otopathic protein polypeptide.

[0187] In some embodiments of any of the therapeutic compositions described herein, the auditory peptide messenger RNA encodes an auditory peptide selected from the group consisting of: Ca v 1.3 Scaffold proteins selected from bassoon, piccolo, ribeye, and harmonin, Vglut3, synaptic binding proteins, vesicle tethering / docking proteins, vesicle initiation proteins, vesicle fusion proteins, GluA2 / 3, and GluA4.

[0188] In some embodiments of any of the therapeutic compositions described herein, a single AAV vector further comprises at least one promoter sequence selected from CBA, CMV, or CB7 promoters.

[0189] In some embodiments of any of the therapeutic compositions described herein, a single AAV carrier further comprises at least one promoter sequence selected from cochlear-specific promoters.

[0190] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated for intracochlear administration.

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

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

[0193] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include small circular DNA.

[0194] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include CELID DNA.

[0195] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include a synthetic perilymphatic solution.

[0196] In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include a synthetic perilymph solution comprising 20-200 mM NaCl; 1-5 mM potassium chloride; 0.1-10 mM CaCl2; 1-10 mM glucose; 2-50 mM HEPES; and having a pH between about 6 and about 9.

[0197] This article also provides therapeutic compositions comprising auditory polypeptide messenger RNA encoding a truncated mutant of ototoxic protein.

[0198] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0199] The terms “a” and “an” refer to one or more (i.e., at least one) grammatical object in an article. For example, “an element” includes one element and more than one element.

[0200] The term “about” when referring to measurable values ​​such as quantity or time interval is intended to cover variations of up to ±20%. These variations can be, for example, up to ±10% of the specified value, or in some cases ±5% of the specified value, or in some cases ±1% of the specified value, or in some cases ±0.1% of the specified value, because such variations are appropriate for performing the disclosed methods.

[0201] The term "signal transduction domain" refers to the functional portion of a protein that functions as an effector by transmitting information within the cell to regulate cellular activity through the generation of second messengers or in response to such messengers via defined signal transduction pathways. Otocin is considered a calcium sensor required for exocytosis in inner hair cells and neurotransmitter release in immature outer hair cells. Five of the six C2 domains of otocin (C2B-F) bind calcium ions in solution with moderate and low affinity constants (Kd = 25–95 μM and 400–700 μM, respectively) (Padmanarayana et al. 2014 Biochem 53:5023–5033). In the presence of phosphatidylserine (PS), a calcium concentration of 10 μM leads to significant C2-liposome interactions in the C2C-C2E domains of otocin. Thus, otocin possesses domains that appear to operate using an "electrostatic switch" mechanism, as well as calcium-independent binding domains. PI(4,5)P2 is a major presynaptic signaling molecule that has been shown to interact with the C2C and C2F domains of otodermal protein in a calcium-independent manner (Padmanarayana et al. 2014 Biochem 53: 5023-5033).

[0202] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that binds specifically to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to an antigen-binding domain sufficient to confer recognition and specific binding to a target such as an antigen, such as the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (VL or VH), camel VHH domains, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are continuously linked via a short, flexible polypeptide linker and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise stated, as used herein, scFv may have VL and VH variable regions in any order; for example, with respect to the N-terminus and C-terminus of a polypeptide, scFv may contain VL-connector-VH or may contain VH-connector-VL.

[0203] The term "recombinant polypeptide" refers to a polypeptide generated using recombinant DNA technology, such as a polypeptide expressed by a viral vector expression system. The term should also be interpreted as a polypeptide generated by synthesizing a DNA molecule encoding the polypeptide, wherein the DNA molecule expresses a protein or specifies the amino acid sequence of the polypeptide, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequencing technologies available in the art and well-known.

[0204] The term "mutation in the otoblastin gene" refers to a modification in the wild-type otoblastin gene that results in an otoblastin having one or more of the following characteristics compared to the wild-type otoblastin: deletion of one or more amino acids, substitution of one or more amino acids, and insertion of one or more amino acids, and / or resulting in a reduced expression level of the otoblastin encoded in mammalian cells compared to the expression level of the otoblastin encoded in mammalian cells without the mutation. In some embodiments, the mutation may result in an otoblastin having the deletion of one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids). In some embodiments, the mutation may result in a frameshift in the otoblastin gene. The term "frameshift" is known in the art to encompass any mutation in the coding sequence that results in a shift of the reading frame of the coding sequence. In some embodiments, the frameshift may result in a nonfunctional protein. In some embodiments, a point mutation may be a nonsense mutation (i.e., resulting in an early stop codon in a gene exon). Nonsense mutations can result in truncated proteins that may or may not be functional (compared to the corresponding wild-type protein). In some embodiments, mutations can result in loss (or reduced) expression of otoblastin mRNA or both otoblastin mRNA and protein. In some embodiments, mutations can result in otoblastin with alterations to its biological activity (function) that are lost or reduced compared to wild-type otoblastin.

[0205] In some embodiments, the mutation involves inserting one or more nucleotides into the ototoxicin gene. In some embodiments, the mutation is in the regulatory sequence of the ototoxicin gene, i.e., not part of the coding sequence. In some embodiments, the mutation in the regulatory sequence can be in the promoter or enhancer region and prevent or reduce the proper transcription of the ototoxicin gene.

[0206] Modifications can be introduced into nucleotide sequences using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0207] The term "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of this disclosure using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0208] The term "coding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, which acts as a template for synthesizing a defined amino acid sequence according to the genetic code. Therefore, if the transcription and translation of mRNA corresponding to a gene, cDNA, or RNA produces a protein, then that gene, cDNA, or RNA encodes a protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand used as a template for transcription can be referred to as encoding the protein product.

[0209] The term "homology" or "identity" refers to the subunit sequence identity between two polymer molecules, such as two nucleic acid molecules, two DNA molecules, two RNA molecules, or two polypeptide molecules. Homology or identity occurs when subunit positions in both molecules are occupied by the same monomeric subunit; for example, if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half the positions in two sequences (e.g., five positions out of ten subunits in the polymer) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., nine out of ten) are matching or homologous, then the two sequences are 90% homologous.

[0210] Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and therefore encode the same amino acid sequence. Nucleotide sequences encoding proteins may also include introns.

[0211] The term "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

[0212] The term "exogenous" refers to any material introduced from outside or produced outside of an organism, cell, tissue, or system.

[0213] The term "isolated" refers to something altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "isolated," but the same nucleic acids or peptides that are partially or completely separated from their native coexisting material are "isolated." Isolated nucleic acids or proteins can exist in a substantially purified form or can exist in non-natural environments, such as, for example, host cells.

[0214] The term "fundamentally purified cells" refers to cells that are substantially free of other cell types. Fundamentally purified cells also refer to cells that have been isolated from other cell types typically associated with them in their natural state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term refers only to cells that have been isolated from cells naturally associated with them in their natural state. In some respects, the cells are cultured in vitro. In other respects, the cells are not cultured in vitro.

[0215] The terms "transfected," "transformed," or "transduced" refer to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected," "transformed," or "transduced" cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary test cells and their progeny.

[0216] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0217] As used in this article, “transient” refers to a period of time during which non-integrated transgenes are expressed, ranging from hours to days to weeks. If the transgene is integrated into the genome or contained within a stable plasmid replicon in the host cell, the expression period is shorter than the gene expression period.

[0218] The term "subject" is intended to include any living organism (e.g., a mammal, a human) in which an immune response can be elicited. In some embodiments, the subject is a rodent (e.g., a rat or mouse), rabbit, sheep, dog, cat, horse, non-human primate, or human. In some embodiments, the subject has or is at risk of developing non-syndromic hearing loss. In some embodiments, the subject has previously been identified as having a mutation in the ototoxicin gene. In some embodiments, the subject has been identified as having a mutation in the ototoxicin gene and has been diagnosed with asymptomatic sensorineural hearing loss. In some embodiments, the subject has been identified as having asymptomatic sensorineural hearing loss.

[0219] As used in this article, the term "therapeutic" refers to treatment. Therapeutic effects are achieved by reducing, suppressing, alleviating, or eradicating a disease state.

[0220] As used in this article, the term "prevention" refers to preventive or protective treatment of a disease or disease state. In this context, "prevention" includes reducing the likelihood that a subject will experience the disease.

[0221] The terms “effective amount” or “therapeutic effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition that effectively achieves a particular biological outcome as described herein. In some embodiments, a therapeutically effective amount of the composition may result in an increase in the expression level of an active otopathic protein (e.g., wild-type, full-length otopathic protein, or an otopathic protein variant with the desired activity) (e.g., compared to the expression level prior to treatment with the composition). In some embodiments, a therapeutically effective amount of the composition may result in an increase in the expression level of an active otopathic protein (e.g., wild-type, full-length otopathic protein, or an active variant) in target cells (e.g., cochlear inner hair cells). In some embodiments, a therapeutically effective amount of the composition may result in different cellular localizations of an active otopathic protein (e.g., wild-type, full-length otopathic protein, or an active variant) in target cells (e.g., cochlear inner hair cells). In some embodiments, a therapeutically effective amount of the composition may result in an increased expression level of active otopathic protein (e.g., wild-type, full-length otopathic protein, or active variant), and / or an increased activity of one or more otopathic proteins in target cells (e.g., compared to reference levels, such as levels in pre-treatment subjects, levels in subjects with mutations in the otopathic protein gene, or levels in subjects or subject populations with asymptomatic sensorineural hearing loss).

[0222] The term “parenteral” administration of the composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0223] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and their polymers. Unless otherwise specified, the term covers nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise stated, a specific nucleic acid sequence also implicitly covers variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0224] In some embodiments of the nucleic acid described herein, the nucleic acid is DNA. In some embodiments of the nucleic acid described herein, the nucleic acid is RNA.

[0225] In the context of this disclosure, the following abbreviations are used to denote common nucleic acid bases: “A” for adenosine, “C” for cytosine, “G” for guanosine, “T” for thymidine, and “U” for uridine.

[0226] As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro, preferably mRNA. Typically, in vitro transcribed RNA is generated using an in vitro transcription vector. The in vitro transcription vector includes a template for generating in vitro transcribed RNA.

[0227] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein, comprising two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains and longer chains; short chains are also commonly referred to in the art, for example, as peptides, oligopeptides, and oligomers, while longer chains are commonly referred to in the art, for example, as proteins, which include various types. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0228] The term "signal transduction pathway" refers to the biochemical relationships between various signal transduction molecules that play a role in the transmission of signals from one part of the cell to another. The phrase "cell surface receptors" includes molecules and molecular complexes capable of receiving signals and transmitting them across the cell membrane.

[0229] The term "active otopathic protein" refers to a DNA-encoded protein that, if it replaces two wild-type alleles encoding a full-length otopathic protein in the auditory hair cells (e.g., inner auditory hair cells) of a wild-type mammal, and if expressed in the auditory hair cells of that mammal, results in a hearing level in that mammal that is close to the normal hearing level of a fully wild-type similar mammal. A non-limiting example of an active otopathic protein is a full-length otopathic protein (e.g., any full-length otopathic protein described herein).

[0230] For example, active otopathic proteins may include the sequence of wild-type full-length otopathic proteins (e.g., wild-type human full-length otopathic proteins), including 1 amino acid substitution to about 240 amino acid substitutions, 1 amino acid substitution to about 235 amino acid substitutions, 1 amino acid substitution to about 230 amino acid substitutions, 1 amino acid substitution to about 225 amino acid substitutions, 1 amino acid substitution to about 220 amino acid substitutions, 1 amino acid substitution to about 215 amino acid substitutions, 1 amino acid substitution to about 210 amino acid substitutions, 1 amino acid substitution to about 205 amino acid substitutions, 1 amino acid substitution to about 200 amino acid substitutions, 1 amino acid substitution to about 195 amino acid substitutions, 1 amino acid substitution to about 190 amino acids. Amino acid substitution, 1 amino acid substitution to about 185 amino acid substitutions, 1 amino acid substitution to about 180 amino acid substitutions, 1 amino acid substitution to about 175 amino acid substitutions, 1 amino acid substitution to about 170 amino acid substitutions, 1 amino acid substitution to about 165 amino acid substitutions, 1 amino acid substitution to about 160 amino acid substitutions, 1 amino acid substitution to about 155 amino acid substitutions, 1 amino acid substitution to about 150 amino acid substitutions, 1 amino acid substitution to about 145 amino acid substitutions, 1 amino acid substitution to about 140 amino acid substitutions, 1 amino acid substitution to about 135 amino acid substitutions, 1 amino acid substitution to about 130 amino acid substitutions, 1 amino acid substitution to about 125 amino acid substitutions Amino acid substitution, 1 amino acid substitution to about 120 amino acid substitutions, 1 amino acid substitution to about 115 amino acid substitutions, 1 amino acid substitution to about 110 amino acid substitutions, 1 amino acid substitution to about 105 amino acid substitutions, 1 amino acid substitution to about 100 amino acid substitutions, 1 amino acid substitution to about 95 amino acid substitutions, 1 amino acid substitution to about 90 amino acid substitutions, 1 amino acid substitution to about 85 amino acid substitutions, 1 amino acid substitution to about 80 amino acid substitutions, 1 amino acid substitution to about 75 amino acid substitutions, 1 amino acid substitution to about 70 amino acid substitutions, 1 amino acid substitution to about 65 amino acid substitutions, 1 amino acid substitution to about 60 amino acid substitutions, 1 One amino acid substitution to about 55 amino acid substitutions, one amino acid substitution to about 50 amino acid substitutions, one amino acid substitution to about 45 amino acid substitutions, one amino acid substitution to about 40 amino acid substitutions, one amino acid substitution to about 35 amino acid substitutions, one amino acid substitution to about 30 amino acid substitutions, one amino acid substitution to about 25 amino acid substitutions, one amino acid substitution to about 20 amino acid substitutions, one amino acid substitution to about 15 amino acid substitutions, one amino acid substitution to about 10 amino acid substitutions, one amino acid substitution to about 9 amino acid substitutions, one amino acid substitution to about 8 amino acid substitutions, one amino acid substitution to about 7 amino acid substitutions, one amino acid substitution to about 6 amino acid substitutions.One amino acid substitution to about five amino acid substitutions; one amino acid substitution to about four amino acid substitutions; one amino acid substitution to about three amino acid substitutions; about two amino acid substitutions to about 240 amino acid substitutions; about two amino acid substitutions to about 235 amino acid substitutions; about two amino acid substitutions to about 230 amino acid substitutions; about two amino acid substitutions to about 225 amino acid substitutions; about two amino acid substitutions to about 220 amino acid substitutions; about two amino acid substitutions to about 215 amino acid substitutions; about two amino acid substitutions to about 210 amino acid substitutions; about two amino acid substitutions to about 205 amino acid substitutions; about two amino acid substitutions to about 200 amino acid substitutions; about two amino acid substitutions to about 195 amino acids. Amino acid substitution, about 2 amino acid substitutions to about 190 amino acid substitutions, about 2 amino acid substitutions to about 185 amino acid substitutions, about 2 amino acid substitutions to about 180 amino acid substitutions, about 2 amino acid substitutions to about 175 amino acid substitutions, about 2 amino acid substitutions to about 170 amino acid substitutions, 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 to about 145 amino acid substitutions, about 2 amino acid substitutions to about 140 amino acid substitutions, about 2 amino acid substitutions to about 135 amino acid substitutions, about 2 Amino acid substitution to about 130 amino acid substitutions, about 2 amino acid substitutions to about 125 amino acid substitutions, about 2 amino acid substitutions to about 120 amino acid substitutions, about 2 amino acid substitutions to about 115 amino acid substitutions, about 2 amino acid substitutions to about 110 amino acid substitutions, about 2 amino acid substitutions to about 105 amino acid substitutions, about 2 amino acid substitutions to about 100 amino acid substitutions, about 2 amino acid substitutions to about 95 amino acid substitutions, about 2 amino acid substitutions to about 90 amino acid substitutions, about 2 amino acid substitutions to about 85 amino acid substitutions, about 2 amino acid substitutions to about 80 amino acid substitutions, about 2 amino acid substitutions to about 75 amino acid substitutions, about 2 amino acid substitutions to about 70 amino acid substitutions. Substitution, about 2 amino acid substitutions to about 65 amino acid substitutions, about 2 amino acid substitutions to about 60 amino acid substitutions, about 2 amino acid substitutions to about 55 amino acid substitutions, about 2 amino acid substitutions to about 50 amino acid substitutions, about 2 amino acid substitutions to about 45 amino acid substitutions, about 2 amino acid substitutions to about 40 amino acid substitutions, about 2 amino acid substitutions to about 35 amino acid substitutions, about 2 amino acid substitutions to about 30 amino acid substitutions, about 2 amino acid substitutions to about 25 amino acid substitutions, about 2 amino acid substitutions to about 20 amino acid substitutions, about 2 amino acid substitutions to about 15 amino acid substitutions, about 2 amino acid substitutions to about 10 amino acid substitutions, about 2 amino acid substitutions to about 9 amino acid substitutions.About 2 amino acid substitutions to about 8 amino acid substitutions, about 2 amino acid substitutions to about 7 amino acid substitutions, about 2 amino acid substitutions to about 6 amino acid substitutions, about 2 amino acid substitutions to about 5 amino acid substitutions, about 2 amino acid substitutions to about 4 amino acid substitutions, about 3 amino acid substitutions to about 240 amino acid substitutions, about 3 amino acid substitutions to about 235 amino acid substitutions, about 3 amino acid substitutions to about 230 amino acid substitutions, about 3 amino acid substitutions to about 225 amino acid substitutions, about 3 amino acid substitutions to about 220 amino acid substitutions, about 3 amino acid substitutions to about 215 amino acid substitutions, about 3 amino acid substitutions to about 210 amino acid substitutions, about 3 amino acid substitutions to about 205 amino acids. Acid substitution, about 3 amino acid substitutions to about 200 amino acid substitutions, about 3 amino acid substitutions to about 195 amino acid substitutions, about 3 amino acid substitutions to about 190 amino acid substitutions, about 3 amino acid substitutions to about 185 amino acid substitutions, about 3 amino acid substitutions to about 180 amino acid substitutions, about 3 amino acid substitutions to about 175 amino acid substitutions, about 3 amino acid substitutions to about 170 amino acid substitutions, about 3 amino acid substitutions to about 165 amino acid substitutions, about 3 amino acid substitutions to about 160 amino acid substitutions, about 3 amino acid substitutions to about 155 amino acid substitutions, about 3 amino acid substitutions to about 150 amino acid substitutions, about 3 amino acid substitutions to about 145 amino acid substitutions, about 3 amino... Acid substitution to about 140 amino acid substitutions, about 3 amino acid substitutions to about 135 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 120 amino acid substitutions, about 3 amino acid substitutions to about 115 amino acid substitutions, about 3 amino acid substitutions to about 110 amino acid substitutions, about 3 amino acid substitutions to about 105 amino acid substitutions, about 3 amino acid substitutions to about 100 amino acid substitutions, about 3 amino acid substitutions to about 95 amino acid substitutions, about 3 amino acid substitutions to about 90 amino acid substitutions, about 3 amino acid substitutions to about 85 amino acid substitutions, about 3 amino acid substitutions to about 80 amino acid substitutions. Substitution of approximately 3 amino acids to approximately 75 amino acid substitutions, approximately 3 amino acid substitutions to approximately 70 amino acid substitutions, approximately 3 amino acid substitutions to approximately 65 amino acid substitutions, approximately 3 amino acid substitutions to approximately 60 amino acid substitutions, approximately 3 amino acid substitutions to approximately 55 amino acid substitutions, approximately 3 amino acid substitutions to approximately 50 amino acid substitutions, approximately 3 amino acid substitutions to approximately 45 amino acid substitutions, approximately 3 amino acid substitutions to approximately 40 amino acid substitutions, approximately 3 amino acid substitutions to approximately 35 amino acid substitutions, approximately 3 amino acid substitutions to approximately 30 amino acid substitutions, approximately 3 amino acid substitutions to approximately 25 amino acid substitutions, approximately 3 amino acid substitutions to approximately 20 amino acid substitutions, approximately 3 amino acid substitutions to approximately 15 amino acid substitutions.About 3 amino acid substitutions to about 10 amino acid substitutions, about 3 amino acid substitutions to about 9 amino acid substitutions, about 3 amino acid substitutions to about 8 amino acid substitutions, about 3 amino acid substitutions to about 7 amino acid substitutions, about 3 amino acid substitutions to about 6 amino acid substitutions, about 3 amino acid substitutions to about 5 amino acid substitutions, about 4 amino acid substitutions to about 240 amino acid substitutions, about 4 amino acid substitutions to about 235 amino acid substitutions, about 4 amino acid substitutions to about 230 amino acid substitutions, about 4 amino acid substitutions to about 225 amino acid substitutions, about 4 amino acid substitutions 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. Substitution, about 4 amino acids substituted to about 205 amino acids substituted, about 4 amino acids substituted to about 200 amino acids substituted, about 4 amino acids substituted to about 195 amino acids substituted, about 4 amino acids substituted to about 190 amino acids substituted, about 4 amino acids substituted to about 185 amino acids substituted, about 4 amino acids substituted to about 180 amino acids substituted, about 4 amino acids substituted to about 175 amino acids substituted, about 4 amino acids substituted to about 170 amino acids substituted, about 4 amino acids substituted to about 165 amino acids substituted, about 4 amino acids substituted to about 160 amino acids substituted, about 4 amino acids substituted to about 155 amino acids substituted, about 4 amino acids substituted to about 150 amino acids substituted, about 4 amino acids Substitution to about 145 amino acid substitutions, about 4 amino acid substitutions to about 140 amino acid substitutions, about 4 amino acid substitutions to about 135 amino acid substitutions, about 4 amino acid substitutions to about 130 amino acid substitutions, about 4 amino acid substitutions to about 125 amino acid substitutions, about 4 amino acid substitutions to about 120 amino acid substitutions, about 4 amino acid substitutions to about 115 amino acid substitutions, about 4 amino acid substitutions to about 110 amino acid substitutions, about 4 amino acid substitutions to about 105 amino acid substitutions, about 4 amino acid substitutions to about 100 amino acid substitutions, about 4 amino acid substitutions to about 95 amino acid substitutions, about 4 amino acid substitutions to about 90 amino acid substitutions, about 4 amino acid substitutions to about 85 amino acid substitutions. Substitution, approximately 4 amino acid substitutions to approximately 80 amino acid substitutions, approximately 4 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 to approximately 65 amino acid substitutions, approximately 4 amino acid substitutions to approximately 60 amino acid substitutions, approximately 4 amino acid substitutions to approximately 55 amino acid substitutions, approximately 4 amino acid substitutions to approximately 50 amino acid substitutions, approximately 4 amino acid substitutions to approximately 45 amino acid substitutions, approximately 4 amino acid substitutions to approximately 40 amino acid substitutions, approximately 4 amino acid substitutions to approximately 35 amino acid substitutions, 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 amino acid substitutions to about 15 amino acid substitutions, about 4 amino acid substitutions to about 10 amino acid substitutions, 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 7 amino acid substitutions, about 4 amino acid substitutions to about 6 amino acid substitutions, about 5 amino acid substitutions to about 240 amino acid substitutions, about 5 amino acid substitutions to about 235 amino acid substitutions, about 5 amino acid substitutions to about 230 amino acid substitutions, about 5 amino acid substitutions to about 225 amino acid substitutions, about 5 amino acid substitutions to about 220 amino acid substitutions, about 5 amino acid substitutions to about 215 amino acid substitutions, about 5 amino acid substitutions to about 210 amino acid substitutions. Substitution, about 5 amino acids substituted to about 205 amino acids substituted, about 5 amino acids substituted to about 200 amino acids substituted, about 5 amino acids substituted to about 195 amino acids substituted, about 5 amino acids substituted to about 190 amino acids substituted, about 5 amino acids substituted to about 185 amino acids substituted, about 5 amino acids substituted to about 180 amino acids substituted, about 5 amino acids substituted to about 175 amino acids substituted, about 5 amino acids substituted to about 170 amino acids substituted, about 5 amino acids substituted to about 165 amino acids substituted, about 5 amino acids substituted to about 160 amino acids substituted, about 5 amino acids substituted to about 155 amino acids substituted, about 5 amino acids substituted to about 150 amino acids substituted, about 5 amino acids Substitution to about 145 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 130 amino acid substitutions, about 5 amino acid substitutions to about 125 amino acid substitutions, about 5 amino acid substitutions to about 120 amino acid substitutions, about 5 amino acid substitutions to about 115 amino acid substitutions, about 5 amino acid substitutions to about 110 amino acid substitutions, about 5 amino acid substitutions to about 105 amino acid substitutions, about 5 amino acid substitutions to about 100 amino acid substitutions, about 5 amino acid substitutions to about 95 amino acid substitutions, about 5 amino acid substitutions to about 90 amino acid substitutions, about 5 amino acid substitutions to about 85 amino acid substitutions. Substitution, approximately 5 amino acid substitutions to approximately 80 amino acid substitutions, approximately 5 amino acid substitutions to approximately 75 amino acid substitutions, approximately 5 amino acid substitutions to approximately 70 amino acid substitutions, approximately 5 amino acid substitutions to approximately 65 amino acid substitutions, approximately 5 amino acid substitutions to approximately 60 amino acid substitutions, approximately 5 amino acid substitutions to approximately 55 amino acid substitutions, approximately 5 amino acid substitutions to approximately 50 amino acid substitutions, approximately 5 amino acid substitutions to approximately 45 amino acid substitutions, approximately 5 amino acid substitutions to approximately 40 amino acid substitutions, approximately 5 amino acid substitutions to approximately 35 amino acid substitutions, approximately 5 amino acid substitutions to approximately 30 amino acid substitutions, approximately 5 amino acid substitutions to approximately 25 amino acid substitutions, approximately 5 amino acid substitutions to approximately 20 amino acid substitutions.About 5 amino acid substitutions to about 15 amino acid substitutions, about 5 amino acid substitutions to about 10 amino acid substitutions, about 5 amino acid substitutions 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, about 6 amino acid substitutions to about 240 amino acid substitutions, about 6 amino acid substitutions to about 235 amino acid substitutions, about 6 amino acid substitutions to about 230 amino acid substitutions, about 6 amino acid substitutions to about 225 amino acid substitutions, about 6 amino acid substitutions to about 220 amino acid substitutions, about 6 amino acid substitutions to about 215 amino acid substitutions, about 6 amino acid substitutions to about 210 amino acid substitutions, about 6 amino acid substitutions to about 205 amino acid substitutions. Amino acid substitution, about 6 amino acid substitutions to about 200 amino acid substitutions, about 6 amino acid substitutions to about 195 amino acid substitutions, about 6 amino acid substitutions to about 190 amino acid substitutions, about 6 amino acid substitutions to about 185 amino acid substitutions, about 6 amino acid substitutions to about 180 amino acid substitutions, about 6 amino acid substitutions to about 175 amino acid substitutions, about 6 amino acid substitutions to about 170 amino acid substitutions, about 6 amino acid substitutions to about 165 amino acid substitutions, about 6 amino acid substitutions to about 160 amino acid substitutions, about 6 amino acid substitutions to about 155 amino acid substitutions, about 6 amino acid substitutions to about 150 amino acid substitutions, about 6 amino acid substitutions to about 145 amino acid substitutions, about 6 amino acids Amino acid substitution to about 140 amino acid substitutions, about 6 amino acid substitutions to about 135 amino acid substitutions, about 6 amino acid substitutions to about 130 amino acid substitutions, about 6 amino acid substitutions to about 125 amino acid substitutions, about 6 amino acid substitutions to about 120 amino acid substitutions, about 6 amino acid substitutions to about 115 amino acid substitutions, about 6 amino acid substitutions to about 110 amino acid substitutions, about 6 amino acid substitutions to about 105 amino acid substitutions, about 6 amino acid substitutions to about 100 amino acid substitutions, about 6 amino acid substitutions to about 95 amino acid substitutions, about 6 amino acid substitutions to about 90 amino acid substitutions, about 6 amino acid substitutions to about 85 amino acid substitutions, about 6 amino acid substitutions to about 80 amino acid substitutions. Substitution, about 6 amino acids substituted to about 75 amino acids substituted, about 6 amino acids substituted to about 70 amino acids substituted, about 6 amino acids substituted to about 65 amino acids substituted, about 6 amino acids substituted to about 60 amino acids substituted, about 6 amino acids substituted to about 55 amino acids substituted, about 6 amino acids substituted to about 50 amino acids substituted, about 6 amino acids substituted to about 45 amino acids substituted, about 6 amino acids substituted to about 40 amino acids substituted, about 6 amino acids substituted to about 35 amino acids substituted, about 6 amino acids substituted to about 30 amino acids substituted, about 6 amino acids substituted to about 25 amino acids substituted, about 6 amino acids substituted to about 20 amino acids substituted, about 6 amino acids substituted to about 15 amino acids substituted.About 6 amino acid substitutions to about 10 amino acid substitutions, about 6 amino acid substitutions to about 9 amino acid substitutions, about 6 amino acid substitutions to about 8 amino acid substitutions, about 7 amino acid substitutions to about 240 amino acid substitutions, about 7 amino acid substitutions to about 235 amino acid substitutions, about 7 amino acid substitutions to about 230 amino acid substitutions, about 7 amino acid substitutions to about 225 amino acid substitutions, about 7 amino acid substitutions to about 220 amino acid substitutions, about 7 amino acid substitutions to about 215 amino acid substitutions, about 7 amino acid substitutions to about 210 amino acid substitutions, about 7 amino acid substitutions to about 205 amino acid substitutions, about 7 amino acid substitutions to about 200 amino acid substitutions, about 7 amino acid substitutions to about 19... 5 amino acid substitutions, about 7 amino acid substitutions to about 190 amino acid substitutions, about 7 amino acid substitutions to about 185 amino acid substitutions, about 7 amino acid substitutions to about 180 amino acid substitutions, about 7 amino acid substitutions to about 175 amino acid substitutions, about 7 amino acid substitutions to about 170 amino acid substitutions, about 7 amino acid substitutions to about 165 amino acid substitutions, about 7 amino acid substitutions to about 160 amino acid substitutions, about 7 amino acid substitutions to about 155 amino acid substitutions, about 7 amino acid substitutions to about 150 amino acid substitutions, about 7 amino acid substitutions to about 145 amino acid substitutions, about 7 amino acid substitutions to about 140 amino acid substitutions, about 7 amino acid substitutions to about 135 amino acid substitutions, about 7 amino acid substitutions to about 130 amino acid substitutions, about 7 amino acid substitutions to about 125 amino acid substitutions, about 7 amino acid substitutions to about 120 amino acid substitutions, about 7 amino acid substitutions to about 115 amino acid substitutions, about 7 amino acid substitutions to about 110 amino acid substitutions, about 7 amino acid substitutions to about 105 amino acid substitutions, about 7 amino acid substitutions to about 100 amino acid substitutions, about 7 amino acid substitutions to about 95 amino acid substitutions, about 7 amino acid substitutions to about 90 amino acid substitutions, about 7 amino acid substitutions to about 85 amino acid substitutions, about 7 amino acid substitutions to about 80 amino acid substitutions, about 7 amino acid substitutions to about 75 amino acid substitutions, about 7 amino acid substitutions to about 70 amino groups. 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 55 amino acid substitutions, about 7 amino acid substitutions to about 50 amino acid substitutions, about 7 amino acid substitutions to about 45 amino acid substitutions, about 7 amino acid substitutions to about 40 amino acid substitutions, about 7 amino acid substitutions to about 35 amino acid substitutions, about 7 amino acid substitutions to about 30 amino acid substitutions, about 7 amino acid substitutions to about 25 amino acid substitutions, about 7 amino acid substitutions to about 20 amino acid substitutions, about 7 amino acid substitutions to about 15 amino acid substitutions, about 7 amino acid substitutions 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 substitutions, about 8 amino acid substitutions to about 235 amino acid substitutions, about 8 amino acid substitutions to about 230 amino acid substitutions, 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 to about 205 amino acid substitutions, about 8 amino acid substitutions to about 200 amino acid substitutions, about 8 amino acid substitutions to about 195 amino acid substitutions, about 8 amino acid substitutions to about 190 amino acid substitutions, about 8 amino acid substitutions to about 185 amino acid substitutions, about 8 amino groups Acid substitution to about 180 amino acid substitutions, about 8 amino acid substitutions to about 175 amino acid substitutions, about 8 amino acid substitutions to about 170 amino acid substitutions, about 8 amino acid substitutions to about 165 amino acid substitutions, about 8 amino acid substitutions to about 160 amino acid substitutions, about 8 amino acid substitutions to about 155 amino acid substitutions, about 8 amino acid substitutions to about 150 amino acid substitutions, about 8 amino acid substitutions to about 145 amino acid substitutions, about 8 amino acid substitutions to about 140 amino acid substitutions, about 8 amino acid substitutions to about 135 amino acid substitutions, about 8 amino acid substitutions to about 130 amino acid substitutions, about 8 amino acid substitutions to about 125 amino acid substitutions, about 8 amino acid substitutions to about 12 0 amino acid substitutions, about 8 amino acid substitutions to about 115 amino acid substitutions, about 8 amino acid substitutions to about 110 amino acid substitutions, about 8 amino acid substitutions to about 105 amino acid substitutions, about 8 amino acid substitutions to about 100 amino acid substitutions, about 8 amino acid substitutions to about 95 amino acid substitutions, about 8 amino acid substitutions to about 90 amino acid substitutions, about 8 amino acid substitutions to about 85 amino acid substitutions, about 8 amino acid substitutions to about 80 amino acid substitutions, about 8 amino acid substitutions to about 75 amino acid substitutions, 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 60 amino acid substitutions, about 8 amino acid substitutions Substitution to approximately 55 amino acid substitutions, approximately 8 amino acid substitutions to approximately 50 amino acid substitutions, approximately 8 amino acid substitutions to approximately 45 amino acid substitutions, approximately 8 amino acid substitutions to approximately 40 amino acid substitutions, approximately 8 amino acid substitutions to approximately 35 amino acid substitutions, approximately 8 amino acid substitutions to approximately 30 amino acid substitutions, approximately 8 amino acid substitutions to approximately 25 amino acid substitutions, approximately 8 amino acid substitutions to approximately 20 amino acid substitutions, approximately 8 amino acid substitutions to approximately 15 amino acid substitutions, approximately 8 amino acid substitutions to approximately 10 amino acid substitutions, approximately 10 amino acid substitutions to approximately 240 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.About 10 amino acids substituted to about 225 amino acids substituted, about 10 amino acids substituted to about 220 amino acids substituted, about 10 amino acids substituted to about 215 amino acids substituted, about 10 amino acids substituted to about 210 amino acids substituted, about 10 amino acids substituted to about 205 amino acids substituted, about 10 amino acids substituted to about 200 amino acids substituted, about 10 amino acids substituted to about 195 amino acids substituted, about 10 amino acids substituted to about 190 amino acids substituted, about 10 amino acids substituted to about 185 amino acids substituted, about 10 amino acids substituted to about 180 amino acids substituted, about 10 amino acids substituted to about 175 amino acids substituted, about 10 amino acids substituted to about 170 amino groups. Acid substitution, about 10 amino acid substitutions to about 165 amino acid substitutions, about 10 amino acid substitutions to about 160 amino acid substitutions, about 10 amino acid substitutions to about 155 amino acid substitutions, about 10 amino acid substitutions to about 150 amino acid substitutions, about 10 amino acid substitutions to about 145 amino acid substitutions, about 10 amino acid substitutions to about 140 amino acid substitutions, about 10 amino acid substitutions to about 135 amino acid substitutions, about 10 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 to about 120 amino acid substitutions, about 10 amino acid substitutions to about 115 amino acid substitutions, about 10 amino acid substitutions to about 11 0 amino acid substitutions, about 10 amino acid substitutions to about 105 amino acid substitutions, about 10 amino acid substitutions to about 100 amino acid substitutions, about 10 amino acid substitutions to about 95 amino acid substitutions, about 10 amino acid substitutions to about 90 amino acid substitutions, 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 to about 65 amino acid substitutions, about 10 amino acid substitutions to about 60 amino acid substitutions, about 10 amino acid substitutions to about 55 amino acid substitutions, about 10 amino acid substitutions to about 50 amino acid substitutions. The substitutions are as follows: approximately 10 amino acid substitutions to approximately 45 amino acid substitutions; approximately 10 amino acid substitutions to approximately 40 amino acid substitutions; approximately 10 amino acid substitutions to approximately 35 amino acid substitutions; approximately 10 amino acid substitutions to approximately 30 amino acid substitutions; approximately 10 amino acid substitutions to approximately 25 amino acid substitutions; approximately 10 amino acid substitutions to approximately 20 amino acid substitutions; approximately 10 amino acid substitutions to approximately 15 amino acid substitutions; approximately 15 amino acid substitutions to approximately 240 amino acid substitutions; approximately 15 amino acid substitutions to approximately 235 amino acid substitutions; approximately 15 amino acid substitutions to approximately 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.About 15 amino acid substitutions to about 215 amino acid substitutions, about 15 amino acid substitutions to about 210 amino acid substitutions, about 15 amino acid substitutions to about 205 amino acid substitutions, about 15 amino acid substitutions to about 200 amino acid substitutions, about 15 amino acid substitutions to about 195 amino acid substitutions, about 15 amino acid substitutions to about 190 amino acid substitutions, about 15 amino acid substitutions to about 185 amino acid substitutions, about 15 amino acid substitutions to about 180 amino acid substitutions, about 15 amino acid substitutions to about 175 amino acid substitutions, about 15 amino acid substitutions to about 170 amino acid substitutions, about 15 amino acid substitutions to about 165 amino acid substitutions, about 15 amino acid substitutions to about 160 amino acids. Acid substitution, about 15 amino acid substitutions to about 155 amino acid substitutions, about 15 amino acid substitutions to about 150 amino acid substitutions, about 15 amino acid substitutions to about 145 amino acid substitutions, about 15 amino acid substitutions to about 140 amino acid substitutions, about 15 amino acid substitutions to about 135 amino acid substitutions, about 15 amino acid substitutions to about 130 amino acid substitutions, about 15 amino acid substitutions to about 125 amino acid substitutions, about 15 amino acid substitutions to about 120 amino acid substitutions, about 15 amino acid substitutions to about 115 amino acid substitutions, about 15 amino acid substitutions to about 110 amino acid substitutions, about 15 amino acid substitutions to about 105 amino acid substitutions, about 15 amino acid substitutions to about 100 15 amino acid substitutions to about 95 amino acid substitutions, about 15 amino acid substitutions to about 90 amino acid substitutions, about 15 amino acid substitutions to about 85 amino acid substitutions, about 15 amino acid substitutions to about 80 amino acid substitutions, about 15 amino acid substitutions to about 75 amino acid substitutions, about 15 amino acid substitutions 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 to about 55 amino acid substitutions, about 15 amino acid substitutions to about 50 amino acid substitutions, about 15 amino acid substitutions to about 45 amino acid substitutions, about 15 amino acid substitutions to about 40 amino acid substitutions, about The following amino acid substitutions are described: 15 amino acid substitutions to about 35 amino acid substitutions; about 15 amino acid substitutions to about 30 amino acid substitutions; about 15 amino acid substitutions to about 25 amino acid substitutions; about 15 amino acid substitutions to about 20 amino acid substitutions; about 20 amino acid substitutions to about 240 amino acid substitutions; about 20 amino acid substitutions to about 235 amino acid substitutions; about 20 amino acid substitutions to about 230 amino acid substitutions; about 20 amino acid substitutions to about 225 amino acid substitutions; about 20 amino acid substitutions to about 220 amino acid substitutions; about 20 amino acid substitutions to about 215 amino acid substitutions; about 20 amino acid substitutions to about 210 amino acid substitutions; about 20 amino acid substitutions to about 205 amino acid substitutions.Approximately 20 amino acid substitutions to approximately 200 amino acid substitutions, approximately 20 amino acid substitutions to approximately 195 amino acid substitutions, approximately 20 amino acid substitutions to approximately 190 amino acid substitutions, approximately 20 amino acid substitutions to approximately 185 amino acid substitutions, approximately 20 amino acid substitutions to approximately 180 amino acid substitutions, approximately 20 amino acid substitutions to approximately 175 amino acid substitutions, approximately 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 to approximately 160 amino acid substitutions, approximately 20 amino acid substitutions to approximately 155 amino acid substitutions, approximately 20 amino acid substitutions to approximately 150 amino acid substitutions, approximately 20 amino acid substitutions to approximately 145 amino acids. Acid substitution, about 20 amino acid substitutions to about 140 amino acid substitutions, about 20 amino acid substitutions to about 135 amino acid substitutions, about 20 amino acid substitutions to about 130 amino acid substitutions, about 20 amino acid substitutions to about 125 amino acid substitutions, about 20 amino acid substitutions to about 120 amino acid substitutions, about 20 amino acid substitutions to about 115 amino acid substitutions, about 20 amino acid substitutions to about 110 amino acid substitutions, about 20 amino acid substitutions to about 105 amino acid substitutions, about 20 amino acid substitutions to about 100 amino acid substitutions, about 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 to about 85 amino acids Acid substitution, about 20 amino acid substitutions to about 80 amino acid substitutions, about 20 amino acid substitutions to about 75 amino acid substitutions, about 20 amino acid substitutions to about 70 amino acid substitutions, about 20 amino acid substitutions to about 65 amino acid substitutions, about 20 amino acid substitutions to about 60 amino acid substitutions, about 20 amino acid substitutions to about 55 amino acid substitutions, about 20 amino acid substitutions to about 50 amino acid substitutions, about 20 amino acid substitutions to about 45 amino acid substitutions, about 20 amino acid substitutions to about 40 amino acid substitutions, about 20 amino acid substitutions to about 35 amino acid substitutions, about 20 amino acid substitutions to about 30 amino acid substitutions, about 20 amino acid substitutions to about 25 amino acid substitutions, about 25 amino acids The amino acid substitutions range from about 240 amino acid substitutions, from about 25 amino acid substitutions to about 235 amino acid substitutions, from about 25 amino acid substitutions to about 230 amino acid substitutions, from about 25 amino acid substitutions to about 225 amino acid substitutions, from about 25 amino acid substitutions to about 220 amino acid substitutions, from about 25 amino acid substitutions to about 215 amino acid substitutions, from about 25 amino acid substitutions to about 210 amino acid substitutions, from about 25 amino acid substitutions to about 205 amino acid substitutions, from about 25 amino acid substitutions to about 200 amino acid substitutions, from about 10 amino acid substitutions to about 195 amino acid substitutions, from about 25 amino acid substitutions to about 190 amino acid substitutions, and from about 25 amino acid substitutions to about 185 amino acid substitutions.Approximately 25 amino acid substitutions to approximately 180 amino acid substitutions, approximately 25 amino acid substitutions to approximately 175 amino acid substitutions, approximately 25 amino acid substitutions to approximately 170 amino acid substitutions, approximately 25 amino acid substitutions to approximately 165 amino acid substitutions, approximately 25 amino acid substitutions to approximately 160 amino acid substitutions, approximately 25 amino acid substitutions to approximately 155 amino acid substitutions, approximately 25 amino acid substitutions to approximately 150 amino acid substitutions, approximately 25 amino acid substitutions to approximately 145 amino acid substitutions, approximately 25 amino acid substitutions to approximately 140 amino acid substitutions, approximately 25 amino acid substitutions to approximately 135 amino acid substitutions, approximately 25 amino acid substitutions to approximately 130 amino acid substitutions, approximately 25 amino acid substitutions to approximately 125 amino acid substitutions. Substitution, about 25 amino acids substituted to about 120 amino acids substituted, about 25 amino acids substituted to about 115 amino acids substituted, about 25 amino acids substituted to about 110 amino acids substituted, about 25 amino acids substituted to about 105 amino acids substituted, about 25 amino acids substituted to about 100 amino acids substituted, about 25 amino acids substituted to about 95 amino acids substituted, about 25 amino acids substituted to about 90 amino acids substituted, about 25 amino acids substituted to about 85 amino acids substituted, about 25 amino acids substituted to about 80 amino acids substituted, about 25 amino acids substituted to about 75 amino acids substituted, about 25 amino acids substituted to about 70 amino acids substituted, about 25 amino acids substituted to about 65 amino acids substituted, about 25 amino acid substitutions to about 60 amino acid substitutions, about 25 amino acid substitutions to about 55 amino acid substitutions, about 25 amino acid substitutions to about 50 amino acid substitutions, about 25 amino acid substitutions to about 45 amino acid substitutions, about 25 amino acid substitutions to about 40 amino acid substitutions, about 25 amino acid substitutions to about 35 amino acid substitutions, about 25 amino acid substitutions to about 30 amino acid substitutions, about 30 amino acid substitutions to about 240 amino acid substitutions, about 30 amino acid substitutions to about 235 amino acid substitutions, about 30 amino acid substitutions to about 225 amino acid substitutions, about 30 amino acid substitutions to about 220 amino acid substitutions, about 30 The substitution patterns are: approximately 30 amino acid substitutions to approximately 215 amino acid substitutions, approximately 30 amino acid substitutions to approximately 210 amino acid substitutions, approximately 30 amino acid substitutions to approximately 205 amino acid substitutions, approximately 30 amino acid substitutions to approximately 200 amino acid substitutions, approximately 30 amino acid substitutions to approximately 195 amino acid substitutions, approximately 30 amino acid substitutions to approximately 190 amino acid substitutions, approximately 30 amino acid substitutions to approximately 185 amino acid substitutions, approximately 30 amino acid substitutions to approximately 180 amino acid substitutions, approximately 30 amino acid substitutions to approximately 175 amino acid substitutions, approximately 30 amino acid substitutions to approximately 170 amino acid substitutions, approximately 30 amino acid substitutions to approximately 165 amino acid substitutions, and approximately 30 amino acid substitutions to approximately 160 amino acid substitutions.Approximately 30 amino acid substitutions to approximately 155 amino acid substitutions, approximately 30 amino acid substitutions to approximately 150 amino acid substitutions, approximately 30 amino acid substitutions to approximately 145 amino acid substitutions, approximately 30 amino acid substitutions to approximately 140 amino acid substitutions, approximately 30 amino acid substitutions to approximately 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, approximately 30 amino acid substitutions to approximately 120 amino acid substitutions, approximately 30 amino acid substitutions to approximately 115 amino acid substitutions, approximately 30 amino acid substitutions to approximately 110 amino acid substitutions, approximately 30 amino acid substitutions to approximately 105 amino acid substitutions, approximately 30 amino acid substitutions to approximately 100 amino acids. Substitution, about 30 amino acids substituted to about 95 amino acids substituted, about 30 amino acids substituted to about 90 amino acids substituted, about 30 amino acids substituted to about 85 amino acids substituted, about 30 amino acids substituted to about 80 amino acids substituted, about 30 amino acids substituted to about 75 amino acids substituted, about 30 amino acids substituted to about 70 amino acids substituted, about 30 amino acids substituted to about 65 amino acids substituted, about 30 amino acids substituted to about 60 amino acids substituted, about 30 amino acids substituted to about 55 amino acids substituted, about 30 amino acids substituted to about 50 amino acids substituted, about 30 amino acids substituted to about 45 amino acids substituted, about 30 amino acids substituted to about 40 amino acids substituted, about 30 amino groups Acid substitution to about 35 amino acid substitutions, about 35 amino acid substitutions to about 240 amino acid substitutions, about 35 amino acid substitutions to about 235 amino acid substitutions, about 35 amino acid substitutions to about 230 amino acid substitutions, about 35 amino acid substitutions to about 225 amino acid substitutions, about 35 amino acid substitutions to about 220 amino acid substitutions, about 35 amino acid substitutions to about 215 amino acid substitutions, about 35 amino acid substitutions to about 210 amino acid substitutions, about 35 amino acid substitutions to about 205 amino acid substitutions, about 35 amino acid substitutions to about 200 amino acid substitutions, about 35 amino acid substitutions to about 195 amino acid substitutions, about 35 amino acid substitutions to about 190 amino acid substitutions, about 3 The substitutions are: 5 amino acids to about 185 amino acids; about 35 amino acids to about 180 amino acids; about 35 amino acids to about 175 amino acids; about 35 amino acids to about 170 amino acids; about 35 amino acids to about 165 amino acids; about 35 amino acids to about 160 amino acids; about 35 amino acids to about 155 amino acids; about 35 amino acids to about 150 amino acids; about 35 amino acids to about 145 amino acids; about 35 amino acids to about 140 amino acids; about 35 amino acids to about 135 amino acids; about 35 amino acids to about 130 amino acids.Approximately 35 amino acid substitutions to approximately 125 amino acid substitutions, approximately 35 amino acid substitutions to approximately 120 amino acid substitutions, approximately 35 amino acid substitutions to approximately 115 amino acid substitutions, approximately 35 amino acid substitutions to approximately 110 amino acid substitutions, approximately 35 amino acid substitutions to approximately 105 amino acid substitutions, approximately 35 amino acid substitutions to approximately 100 amino acid substitutions, approximately 35 amino acid substitutions to approximately 95 amino acid substitutions, approximately 35 amino acid substitutions to approximately 90 amino acid substitutions, approximately 35 amino acid substitutions to approximately 85 amino acid substitutions, approximately 35 amino acid substitutions to approximately 80 amino acid substitutions, approximately 35 amino acid substitutions to approximately 75 amino acid substitutions, approximately 35 amino acid substitutions to approximately 70 amino acid substitutions, approximately 35 The substitution patterns are as follows: approximately 35 amino acid substitutions to approximately 60 amino acid substitutions; approximately 35 amino acid substitutions to approximately 55 amino acid substitutions; approximately 35 amino acid substitutions to approximately 50 amino acid substitutions; approximately 35 amino acid substitutions to approximately 45 amino acid substitutions; approximately 35 amino acid substitutions to approximately 40 amino acid substitutions; approximately 40 amino acid substitutions to approximately 240 amino acid substitutions; approximately 40 amino acid substitutions to approximately 235 amino acid substitutions; approximately 40 amino acid substitutions to approximately 230 amino acid substitutions; approximately 40 amino acid substitutions to approximately 225 amino acid substitutions; approximately 40 amino acid substitutions to approximately 220 amino acid substitutions; approximately 40 amino acid substitutions to approximately 215 amino acid substitutions; approximately 40... Amino acid substitution to about 210 amino acid substitutions, about 40 amino acid substitutions to about 205 amino acid substitutions, about 40 amino acid substitutions to about 200 amino acid substitutions, about 40 amino acid substitutions to about 195 amino acid substitutions, about 40 amino acid substitutions to about 190 amino acid substitutions, about 40 amino acid substitutions to about 185 amino acid substitutions, about 40 amino acid substitutions to about 180 amino acid substitutions, about 40 amino acid substitutions to about 175 amino acid substitutions, about 40 amino acid substitutions to about 170 amino acid substitutions, about 40 amino acid substitutions to about 165 amino acid substitutions, about 40 amino acid substitutions to about 160 amino acid substitutions, about 40 amino acid substitutions to about 155 amino acid substitutions, about 40 amino acid substitutions to about 150 amino acid substitutions, about 40 amino acid substitutions to about 145 amino acid substitutions, about 40 amino acid substitutions to about 140 amino acid substitutions, about 40 amino acid substitutions to about 135 amino acid substitutions, about 40 amino acid substitutions to about 130 amino acid substitutions, about 40 amino acid 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, about 40 amino acid substitutions to about 110 amino acid substitutions, about 40 amino acid substitutions to about 105 amino acid substitutions, about 40 amino acid substitutions to about 100 amino acid substitutions, about 40 amino acid substitutions to about 95 amino acid substitutions.Approximately 40 amino acid substitutions to approximately 90 amino acid substitutions, approximately 40 amino acid substitutions to approximately 85 amino acid substitutions, approximately 40 amino acid substitutions to approximately 80 amino acid substitutions, approximately 40 amino acid substitutions to approximately 75 amino acid substitutions, approximately 40 amino acid substitutions to approximately 70 amino acid substitutions, approximately 40 amino acid substitutions to approximately 65 amino acid substitutions, approximately 40 amino acid substitutions to approximately 60 amino acid substitutions, approximately 40 amino acid substitutions to approximately 55 amino acid substitutions, approximately 40 amino acid substitutions to approximately 50 amino acid substitutions, approximately 40 amino acid substitutions to approximately 45 amino acid substitutions, approximately 45 amino acid substitutions to approximately 240 amino acid substitutions, approximately 45 amino acid substitutions to approximately 235 amino acid substitutions, approximately 45 The substitution of one amino acid 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 to about 215 amino acid substitutions, about 45 amino acid substitutions to about 210 amino acid substitutions, about 45 amino acid substitutions to about 205 amino acid substitutions, about 45 amino acid substitutions to about 200 amino acid substitutions, about 45 amino acid substitutions to about 195 amino acid substitutions, about 45 amino acid substitutions to about 190 amino acid substitutions, about 45 amino acid substitutions to about 185 amino acid substitutions, about 45 amino acid substitutions to about 180 amino acid substitutions, about 45 amino acid substitutions to about 175 amino acid substitutions. Approximately 45 amino acid substitutions to approximately 170 amino acid substitutions, approximately 45 amino acid substitutions to approximately 165 amino acid substitutions, approximately 45 amino acid substitutions to approximately 160 amino acid substitutions, approximately 45 amino acid substitutions to approximately 155 amino acid substitutions, approximately 45 amino acid substitutions to approximately 150 amino acid substitutions, approximately 45 amino acid substitutions to approximately 145 amino acid substitutions, approximately 45 amino acid substitutions to approximately 140 amino acid substitutions, approximately 45 amino acid substitutions to approximately 135 amino acid substitutions, approximately 45 amino acid substitutions to approximately 130 amino acid substitutions, approximately 45 amino acid substitutions to approximately 125 amino acid substitutions, approximately 45 amino acid substitutions to approximately 120 amino acid substitutions, approximately 45 amino acid substitutions to approximately 115 amino acid substitutions. Amino acid substitution, about 45 amino acid substitutions to about 110 amino acid substitutions, about 45 amino acid substitutions to about 105 amino acid substitutions, about 45 amino acid substitutions to about 100 amino acid substitutions, about 45 amino acid substitutions to about 95 amino acid substitutions, about 45 amino acid substitutions to about 90 amino acid substitutions, about 45 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 to about 75 amino acid substitutions, about 45 amino acid substitutions to about 70 amino acid substitutions, about 45 amino acid substitutions to about 65 amino acid substitutions, about 45 amino acid substitutions to about 60 amino acid substitutions, about 45 amino acid substitutions to about 55 amino acid substitutions.Approximately 45 amino acid substitutions to approximately 50 amino acid substitutions, approximately 50 amino acid substitutions to approximately 240 amino acid substitutions, approximately 50 amino acid substitutions to approximately 235 amino acid substitutions, approximately 50 amino acid substitutions to approximately 230 amino acid substitutions, approximately 50 amino acid substitutions to approximately 225 amino acid substitutions, approximately 50 amino acid substitutions to approximately 220 amino acid substitutions, approximately 50 amino acid substitutions to approximately 215 amino acid substitutions, approximately 50 amino acid substitutions to approximately 210 amino acid substitutions, approximately 50 amino acid substitutions to approximately 205 amino acid substitutions, approximately 50 amino acid substitutions to approximately 200 amino acid substitutions, approximately 50 amino acid substitutions to approximately 195 amino acid substitutions, approximately 50 amino acid substitutions to approximately 19... 0 amino acid substitutions, about 50 amino acid substitutions to about 185 amino acid substitutions, about 50 amino acid substitutions to about 180 amino acid substitutions, about 50 amino acid substitutions to about 175 amino acid substitutions, about 50 amino acid substitutions to about 170 amino acid substitutions, about 50 amino acid substitutions to about 165 amino acid substitutions, about 50 amino acid substitutions to about 160 amino acid substitutions, about 50 amino acid substitutions to about 155 amino acid substitutions, about 50 amino acid substitutions to about 150 amino acid substitutions, about 50 amino acid substitutions to about 145 amino acid substitutions, about 50 amino acid substitutions to about 140 amino acid substitutions, about 50 amino acid substitutions to about 135 amino acid substitutions, about 50 amino acids Acid substitution to about 130 amino acid substitutions, about 50 amino acid substitutions to about 125 amino acid substitutions, about 50 amino acid substitutions to about 120 amino acid substitutions, about 50 amino acid substitutions to about 115 amino acid substitutions, about 50 amino acid substitutions to about 110 amino acid substitutions, about 50 amino acid substitutions to about 105 amino acid substitutions, about 50 amino acid substitutions to about 100 amino acid substitutions, about 50 amino acid substitutions to about 95 amino acid substitutions, about 50 amino acid substitutions to about 90 amino acid substitutions, about 50 amino acid substitutions to about 85 amino acid substitutions, about 50 amino acid substitutions to about 80 amino acid substitutions, about 50 amino acid substitutions to about 75 amino acid substitutions, about 50 Amino acid substitutions of about 70 amino acid substitutions, about 50 amino acid substitutions of about 65 amino acid substitutions, about 50 amino acid substitutions of about 60 amino acid substitutions, about 50 amino acid substitutions of about 55 amino acid substitutions, about 60 amino acid substitutions of about 240 amino acid substitutions, about 60 amino acid substitutions of about 235 amino acid substitutions, about 60 amino acid substitutions of about 230 amino acid substitutions, about 60 amino acid substitutions of about 225 amino acid substitutions, about 60 amino acid substitutions of about 220 amino acid substitutions, about 60 amino acid substitutions of about 215 amino acid substitutions, about 60 amino acid substitutions of about 210 amino acid substitutions, about 60 amino acid substitutions of about 205 amino acid substitutions.Approximately 60 amino acid substitutions to approximately 200 amino acid substitutions, approximately 60 amino acid substitutions to approximately 195 amino acid substitutions, approximately 60 amino acid substitutions to approximately 190 amino acid substitutions, approximately 60 amino acid substitutions to approximately 185 amino acid substitutions, approximately 60 amino acid substitutions to approximately 180 amino acid substitutions, approximately 60 amino acid substitutions to approximately 175 amino acid substitutions, approximately 60 amino acid substitutions to approximately 170 amino acid substitutions, approximately 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 to approximately 155 amino acid substitutions, approximately 60 amino acid substitutions to approximately 150 amino acid substitutions, approximately 60 amino acid substitutions to approximately 145 60 amino acid substitutions to about 140 amino acid substitutions, about 60 amino acid substitutions to about 135 amino acid substitutions, about 60 amino acid substitutions to about 130 amino acid substitutions, about 60 amino acid substitutions to about 125 amino acid substitutions, about 60 amino acid substitutions to about 120 amino acid substitutions, about 60 amino acid substitutions to about 115 amino acid substitutions, about 60 amino acid substitutions to about 110 amino acid substitutions, about 60 amino acid substitutions to about 105 amino acid substitutions, about 60 amino acid substitutions to about 100 amino acid substitutions, about 60 amino acid substitutions to about 95 amino acid substitutions, about 60 amino acid substitutions to about 90 amino acid substitutions, about 60 amino acid substitutions to... Approximately 85 amino acid substitutions, approximately 60 amino acid substitutions to approximately 80 amino acid substitutions, approximately 60 amino acid substitutions to approximately 75 amino acid substitutions, approximately 60 amino acid substitutions to approximately 70 amino acid substitutions, approximately 60 amino acid substitutions to approximately 65 amino acid substitutions, approximately 70 amino acid substitutions to approximately 240 amino acid substitutions, approximately 70 amino acid substitutions to approximately 235 amino acid substitutions, approximately 70 amino acid substitutions to approximately 230 amino acid substitutions, approximately 70 amino acid substitutions to approximately 225 amino acid substitutions, approximately 70 amino acid substitutions to approximately 220 amino acid substitutions, approximately 70 amino acid substitutions to approximately 215 amino acid substitutions, approximately 70 amino acid substitutions to approximately 210 amino acid substitutions, approximately 10 amino acids Acid substitution to about 205 amino acid substitutions, about 70 amino acid substitutions to about 200 amino acid substitutions, about 70 amino acid substitutions to about 195 amino acid substitutions, about 70 amino acid substitutions to about 190 amino acid substitutions, about 70 amino acid substitutions to about 185 amino acid substitutions, about 70 amino acid substitutions to about 180 amino acid substitutions, about 70 amino acid substitutions to about 175 amino acid substitutions, about 70 amino acid substitutions to about 170 amino acid substitutions, about 70 amino acid substitutions to about 165 amino acid substitutions, about 70 amino acid substitutions to about 160 amino acid substitutions, about 70 amino acid substitutions to about 155 amino acid substitutions, about 70 amino acid substitutions to about 150 amino acid substitutions.Approximately 70 amino acid substitutions to approximately 145 amino acid substitutions, approximately 70 amino acid substitutions to approximately 140 amino acid substitutions, approximately 70 amino acid substitutions to approximately 135 amino acid substitutions, approximately 70 amino acid substitutions to approximately 130 amino acid substitutions, approximately 70 amino acid substitutions to approximately 125 amino acid substitutions, approximately 70 amino acid substitutions to approximately 120 amino acid substitutions, approximately 70 amino acid substitutions to approximately 115 amino acid substitutions, approximately 70 amino acid substitutions to approximately 110 amino acid substitutions, approximately 70 amino acid substitutions to approximately 105 amino acid substitutions, approximately 70 amino acid substitutions to approximately 100 amino acid substitutions, approximately 70 amino acid substitutions to approximately 95 amino acid substitutions, approximately 70 amino acid substitutions to approximately 90 amino acids. Amino acid substitution, approximately 70 amino acid substitutions to approximately 85 amino acid substitutions, approximately 70 amino acid substitutions to approximately 80 amino acid substitutions, approximately 70 amino acid substitutions to approximately 75 amino acid substitutions, approximately 80 amino acid substitutions to approximately 240 amino acid substitutions, approximately 80 amino acid substitutions to approximately 235 amino acid substitutions, approximately 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 to approximately 220 amino acid substitutions, approximately 80 amino acid substitutions to approximately 215 amino acid substitutions, approximately 80 amino acid substitutions to 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 amino acid substitutions to approximately 195 amino acid substitutions, approximately 80 amino acid substitutions to approximately 190 amino acid substitutions, approximately 80 amino acid substitutions to approximately 185 amino acid substitutions, approximately 80 amino acid substitutions to approximately 180 amino acid substitutions, approximately 80 amino acid substitutions to approximately 175 amino acid substitutions, approximately 80 amino acid substitutions to approximately 170 amino acid substitutions, approximately 80 amino acid substitutions to approximately 165 amino acid substitutions, approximately 80 amino acid substitutions to approximately 160 amino acid substitutions, approximately 80 amino acid substitutions to approximately 155 amino acid substitutions, approximately 80 amino acid substitutions to approximately 150 amino acid substitutions, approximately 80 amino acid substitutions to approximately 145 amino acid substitutions, approximately 80 The substitution of one amino acid to about 140 amino acid substitutions, about 80 amino acid substitutions to about 135 amino acid substitutions, about 80 amino acid substitutions to about 130 amino acid substitutions, about 80 amino acid substitutions to about 125 amino acid substitutions, about 80 amino acid substitutions to about 120 amino acid substitutions, about 80 amino acid substitutions to about 115 amino acid substitutions, about 80 amino acid substitutions to about 110 amino acid substitutions, about 80 amino acid substitutions to about 105 amino acid substitutions, about 80 amino acid substitutions to about 100 amino acid substitutions, about 80 amino acid substitutions to about 95 amino acid substitutions, about 80 amino acid substitutions to about 90 amino acid substitutions, about 80 amino acid substitutions to about 85 amino acid substitutions.Approximately 90 amino acid substitutions to approximately 240 amino acid substitutions; approximately 90 amino acid substitutions to approximately 235 amino acid substitutions; approximately 90 amino acid substitutions to approximately 230 amino acid substitutions; approximately 90 amino acid substitutions to approximately 225 amino acid substitutions; approximately 90 amino acid substitutions to approximately 220 amino acid substitutions; approximately 90 amino acid substitutions to approximately 215 amino acid substitutions; approximately 90 amino acid substitutions to approximately 210 amino acid substitutions; approximately 90 amino acid substitutions to approximately 205 amino acid substitutions; approximately 90 amino acid substitutions to approximately 200 amino acid substitutions; approximately 90 amino acid substitutions to approximately 195 amino acid substitutions; approximately 90 amino acid substitutions to approximately 190 amino acid substitutions; approximately 90 amino acid substitutions to approximately 18... 5 amino acid substitutions, approximately 90 amino acid substitutions to approximately 180 amino acid substitutions, approximately 90 amino acid substitutions to approximately 175 amino acid substitutions, approximately 90 amino acid substitutions to approximately 170 amino acid substitutions, approximately 90 amino acid substitutions to approximately 165 amino acid substitutions, approximately 90 amino acid substitutions to approximately 160 amino acid substitutions, approximately 90 amino acid substitutions to approximately 155 amino acid substitutions, approximately 90 amino acid substitutions to approximately 150 amino acid substitutions, approximately 90 amino acid substitutions to approximately 145 amino acid substitutions, approximately 90 amino acid substitutions to approximately 140 amino acid substitutions, approximately 90 amino acid substitutions to approximately 135 amino acid substitutions, approximately 90 amino acid substitutions to approximately 130 amino acid substitutions, approximately 90 amino acids Replacement to about 125 amino acid substitutions, about 90 amino acid substitutions to about 120 amino acid substitutions, about 90 amino acid substitutions to about 115 amino acid substitutions, about 90 amino acid substitutions to about 110 amino acid substitutions, about 90 amino acid substitutions to about 105 amino acid substitutions, about 90 amino acid 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, about 100 amino acid substitutions to about 235 amino acid substitutions, about 100 amino acid substitutions to about 230 amino acid substitutions, about 100 amino acid substitutions to about 225 amino acid substitutions, about 100 amino acid substitutions to about 220 amino acids. Amino acid substitution, about 100 amino acid substitutions to about 215 amino acid substitutions, about 100 amino acid substitutions to about 210 amino acid substitutions, about 100 amino acid substitutions to about 205 amino acid substitutions, about 100 amino acid substitutions to about 200 amino acid substitutions, about 100 amino acid substitutions to about 195 amino acid substitutions, about 100 amino acid substitutions to about 190 amino acid substitutions, about 100 amino acid substitutions to about 185 amino acid substitutions, about 100 amino acid substitutions to about 180 amino acid substitutions, about 100 amino acid substitutions to about 175 amino acid substitutions, about 100 amino acid substitutions to about 170 amino acid substitutions, about 100 amino acid substitutions to about 165 amino acid substitutions.Approximately 100 amino acid substitutions to approximately 160 amino acid substitutions, approximately 100 amino acid substitutions to approximately 155 amino acid substitutions, approximately 100 amino acid substitutions to approximately 150 amino acid substitutions, approximately 100 amino acid substitutions to approximately 145 amino acid substitutions, approximately 100 amino acid substitutions to approximately 140 amino acid substitutions, approximately 100 amino acid substitutions to approximately 135 amino acid substitutions, approximately 100 amino acid substitutions to approximately 130 amino acid substitutions, approximately 100 amino acid substitutions to approximately 125 amino acid substitutions, approximately 100 amino acid substitutions to approximately 120 amino acid substitutions, approximately 100 amino acid substitutions to approximately 115 amino acid substitutions, approximately 100 amino acid substitutions to approximately 110 amino acid substitutions, approximately 100 amino acids Amino acid substitution to about 105 amino acid substitutions, about 110 amino acid substitutions to about 240 amino acid substitutions, about 110 amino acid substitutions to about 235 amino acid substitutions, about 110 amino acid substitutions to about 230 amino acid substitutions, about 110 amino acid substitutions to about 225 amino acid substitutions, about 110 amino acid substitutions to about 220 amino acid substitutions, about 110 amino acid substitutions to about 215 amino acid substitutions, about 110 amino acid substitutions to about 210 amino acid substitutions, about 110 amino acid substitutions to about 205 amino acid substitutions, about 110 amino acid substitutions to about 200 amino acid substitutions, about 110 amino acid substitutions to about 195 amino acid substitutions, about 110 amino acid substitutions to... Approximately 190 amino acid substitutions, approximately 110 amino acid substitutions to approximately 185 amino acid substitutions, approximately 110 amino acid substitutions to approximately 180 amino acid substitutions, approximately 110 amino acid substitutions to approximately 175 amino acid substitutions, approximately 110 amino acid substitutions to approximately 170 amino acid substitutions, approximately 110 amino acid substitutions to approximately 165 amino acid substitutions, approximately 110 amino acid substitutions to approximately 160 amino acid substitutions, approximately 110 amino acid substitutions to approximately 155 amino acid substitutions, approximately 110 amino acid substitutions to approximately 150 amino acid substitutions, approximately 110 amino acid substitutions to approximately 145 amino acid substitutions, approximately 110 amino acid substitutions to approximately 140 amino acid substitutions, approximately 110 amino acid substitutions to approximately 135 amino acid substitutions. Amino acid substitution, approximately 110 amino acid substitutions to approximately 130 amino acid substitutions, approximately 110 amino acid substitutions to approximately 125 amino acid substitutions, approximately 110 amino acid substitutions to approximately 120 amino acid substitutions, approximately 110 amino acid substitutions to approximately 115 amino acid substitutions, approximately 120 amino acid substitutions to approximately 240 amino acid substitutions, approximately 120 amino acid substitutions to approximately 235 amino acid substitutions, approximately 120 amino acid substitutions to approximately 230 amino acid substitutions, approximately 120 amino acid substitutions to approximately 225 amino acid substitutions, approximately 120 amino acid substitutions to approximately 220 amino acid substitutions, approximately 120 amino acid substitutions to approximately 215 amino acid substitutions, approximately 120 amino acid substitutions to approximately 210 amino acid substitutions.Approximately 120 amino acid substitutions to approximately 205 amino acid substitutions, approximately 120 amino acid substitutions to approximately 200 amino acid substitutions, approximately 120 amino acid substitutions to approximately 195 amino acid substitutions, approximately 120 amino acid substitutions to approximately 190 amino acid substitutions, approximately 120 amino acid substitutions to approximately 185 amino acid substitutions, approximately 120 amino acid substitutions to approximately 180 amino acid substitutions, approximately 120 amino acid substitutions to approximately 175 amino acid substitutions, approximately 120 amino acid substitutions to approximately 170 amino acid substitutions, approximately 100 amino acid substitutions to approximately 165 amino acid substitutions, approximately 120 amino acid substitutions to approximately 160 amino acid substitutions, approximately 120 amino acid substitutions to approximately 155 amino acid substitutions, approximately 120 amino acids Amino acid substitution to about 150 amino acid substitutions, about 120 amino acid substitutions to about 145 amino acid substitutions, about 120 amino acid substitutions to about 140 amino acid substitutions, about 120 amino acid substitutions to about 135 amino acid substitutions, about 120 amino acid substitutions to about 130 amino acid substitutions, about 120 amino acid substitutions to about 125 amino acid substitutions, about 130 amino acid substitutions to about 240 amino acid substitutions, about 130 amino acid substitutions to about 235 amino acid substitutions, about 130 amino acid substitutions to about 225 amino acid substitutions, about 130 amino acid substitutions to about 220 amino acid substitutions, about 130 amino acid substitutions to... Approximately 215 amino acid substitutions, approximately 130 amino acid substitutions to approximately 210 amino acid substitutions, approximately 130 amino acid substitutions to approximately 205 amino acid substitutions, approximately 130 amino acid substitutions to approximately 200 amino acid substitutions, approximately 130 amino acid substitutions to approximately 195 amino acid substitutions, approximately 130 amino acid substitutions to approximately 190 amino acid substitutions, approximately 130 amino acid substitutions to approximately 185 amino acid substitutions, approximately 130 amino acid substitutions to approximately 180 amino acid substitutions, approximately 130 amino acid substitutions to approximately 175 amino acid substitutions, approximately 130 amino acid substitutions to approximately 170 amino acid substitutions, approximately 130 amino acid substitutions to approximately 165 amino acid substitutions, approximately 130 amino acid substitutions to approximately 160 amino acids. Amino acid substitution, approximately 130 amino acid substitutions to approximately 155 amino acid substitutions, approximately 130 amino acid substitutions to approximately 150 amino acid substitutions, approximately 130 amino acid substitutions to approximately 145 amino acid substitutions, approximately 130 amino acid substitutions to approximately 140 amino acid substitutions, approximately 130 amino acid substitutions to approximately 135 amino acid substitutions, approximately 140 amino acid substitutions to approximately 240 amino acid substitutions, approximately 140 amino acid substitutions to approximately 235 amino acid substitutions, approximately 140 amino acid substitutions to approximately 225 amino acid substitutions, approximately 140 amino acid substitutions to approximately 220 amino acid substitutions, approximately 140 amino acid substitutions to approximately 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 substitutions, approximately 140 amino acid substitutions to approximately 200 amino acid substitutions, approximately 140 amino acid substitutions to approximately 195 amino acid substitutions, approximately 140 amino acid substitutions to approximately 190 amino acid substitutions, approximately 140 amino acid substitutions to approximately 185 amino acid substitutions, approximately 140 amino acid substitutions to approximately 180 amino acid substitutions, approximately 140 amino acid substitutions to approximately 175 amino acid substitutions, approximately 140 amino acid substitutions to approximately 170 amino acid substitutions, approximately 140 amino acid substitutions to approximately 165 amino acid substitutions, approximately 140 amino acid substitutions to approximately 160 amino acid substitutions, approximately 140 amino... Acid substitution to about 155 amino acid substitutions, about 140 amino acid substitutions to about 150 amino acid substitutions, about 140 amino acid substitutions to about 145 amino acid substitutions, about 150 amino acid substitutions to about 240 amino acid substitutions, about 150 amino acid substitutions to about 235 amino acid substitutions, about 150 amino acid substitutions to about 230 amino acid substitutions, about 150 amino acid substitutions to about 225 amino acid substitutions, about 150 amino acid substitutions to about 220 amino acid substitutions, about 150 amino acid substitutions to about 215 amino acid substitutions, about 150 amino acid substitutions to about 210 amino acid substitutions, about 150 amino acid substitutions to about 205 amino acid substitutions, about 150 amino acid substitutions to about 200 amino acid substitutions, approximately 150 amino acid substitutions to approximately 195 amino acid substitutions, approximately 150 amino acid substitutions to approximately 190 amino acid substitutions, approximately 150 amino acid substitutions to approximately 185 amino acid substitutions, approximately 150 amino acid substitutions to approximately 180 amino acid substitutions, approximately 150 amino acid substitutions to approximately 175 amino acid substitutions, approximately 150 amino acid substitutions to approximately 170 amino acid substitutions, approximately 150 amino acid substitutions to approximately 165 amino acid substitutions, approximately 150 amino acid substitutions to approximately 160 amino acid substitutions, or approximately 150 amino acid substitutions to approximately 155 amino acid substitutions, approximately 160 amino acid substitutions to approximately 240 amino acid substitutions, approximately 160 amino acid substitutions to approximately 235 amino acid substitutions. 160 amino acid substitutions to about 230 amino acid substitutions, about 160 amino acid substitutions to about 225 amino acid substitutions, about 160 amino acid substitutions to about 220 amino acid substitutions, about 160 amino acid substitutions to about 215 amino acid substitutions, about 160 amino acid substitutions to about 210 amino acid substitutions, about 160 amino acid substitutions to about 205 amino acid substitutions, about 160 amino acid substitutions to about 200 amino acid substitutions, about 160 amino acid substitutions to about 195 amino acid substitutions, about 160 amino acid substitutions to about 190 amino acid substitutions, about 160 amino acid substitutions to about 185 amino acid substitutions, about 160 amino acid substitutions to about 180 amino acid substitutions.Approximately 160 amino acid substitutions to approximately 175 amino acid substitutions, approximately 160 amino acid substitutions to approximately 170 amino acid substitutions, approximately 160 amino acid substitutions to approximately 165 amino acid substitutions, approximately 170 amino acid substitutions to approximately 240 amino acid substitutions, approximately 170 amino acid substitutions to approximately 235 amino acid substitutions, approximately 170 amino acid substitutions to approximately 230 amino acid substitutions, approximately 170 amino acid substitutions to approximately 225 amino acid substitutions, approximately 170 amino acid substitutions to approximately 220 amino acid substitutions, approximately 170 amino acid substitutions to approximately 215 amino acid substitutions, approximately 170 amino acid substitutions to approximately 210 amino acid substitutions, approximately 170 amino acid substitutions to approximately 205 amino acid substitutions, approximately 170 Amino acid substitutions up to about 200 amino acid substitutions, about 170 amino acid substitutions up to about 195 amino acid substitutions, about 170 amino acid substitutions up to about 190 amino acid substitutions, about 170 amino acid substitutions up to about 185 amino acid substitutions, about 170 amino acid substitutions up to about 180 amino acid substitutions, about 170 amino acid substitutions up to about 175 amino acid substitutions, about 180 amino acid substitutions up to about 240 amino acid substitutions, about 180 amino acid substitutions up to about 235 amino acid substitutions, about 180 amino acid substitutions up to about 230 amino acid substitutions, about 180 amino acid substitutions up to about 225 amino acid substitutions, about 180 amino acid substitutions up to about 220 amino acid substitutions, about 180 amino acid substitutions to Approximately 215 amino acid substitutions, approximately 180 amino acid substitutions to approximately 210 amino acid substitutions, approximately 180 amino acid substitutions to approximately 205 amino acid substitutions, approximately 180 amino acid substitutions to approximately 200 amino acid substitutions, approximately 180 amino acid substitutions to approximately 195 amino acid substitutions, approximately 180 amino acid substitutions to approximately 190 amino acid substitutions, approximately 180 amino acid substitutions to approximately 185 amino acid substitutions, approximately 190 amino acid substitutions to approximately 240 amino acid substitutions, approximately 190 amino acid substitutions to approximately 235 amino acid substitutions, approximately 190 amino acid substitutions to approximately 230 amino acid substitutions, approximately 190 amino acid substitutions to approximately 225 amino acid substitutions, approximately 190 amino acid substitutions to approximately 220 amino acid substitutions. Amino acid substitution, approximately 190 amino acid substitutions to approximately 215 amino acid substitutions, approximately 190 amino acid substitutions to approximately 210 amino acid substitutions, approximately 190 amino acid substitutions to approximately 205 amino acid substitutions, approximately 190 amino acid substitutions to approximately 200 amino acid substitutions, approximately 190 amino acid substitutions to approximately 195 amino acid substitutions, approximately 200 amino acid substitutions to approximately 240 amino acid substitutions, approximately 200 amino acid substitutions to approximately 235 amino acid substitutions, approximately 200 amino acid substitutions to approximately 230 amino acid substitutions, approximately 200 amino acid substitutions to approximately 225 amino acid substitutions, approximately 200 amino acid substitutions to approximately 220 amino acid substitutions, approximately 200 amino acid substitutions to approximately 215 amino acid substitutions.Approximately 200 amino acid substitutions to approximately 210 amino acid substitutions, approximately 200 amino acid substitutions to approximately 205 amino acid substitutions, approximately 205 amino acid substitutions to approximately 240 amino acid substitutions, approximately 205 amino acid substitutions to approximately 235 amino acid substitutions, approximately 205 amino acid substitutions to approximately 230 amino acid substitutions, approximately 205 amino acid substitutions to approximately 225 amino acid substitutions, approximately 205 amino acid substitutions to approximately 220 amino acid substitutions, approximately 205 amino acid substitutions to approximately 215 amino acid substitutions, approximately 205 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 to approximately 235 amino acid substitutions, approximately 210 amino acid substitutions to approximately 230 amino acid substitutions, approximately 210 amino acid substitutions to approximately 225 amino acid substitutions, approximately 210 amino acid substitutions to approximately 220 amino acid substitutions, approximately 210 amino acid substitutions to approximately 2... 15 amino acid substitutions, between about 215 amino acid substitutions and about 240 amino acid substitutions, between about 215 amino acid substitutions and about 235 amino acid substitutions, between about 215 amino acid substitutions and about 230 amino acid substitutions, between about 215 amino acid substitutions and about 225 amino acid substitutions, between about 215 amino acid substitutions and about 220 amino acid substitutions, between about 220 amino acid substitutions and about 240 amino acid substitutions, between about 220 amino acid substitutions and about 235 amino acid substitutions, between about 225 amino acid substitutions and about 235 amino acid substitutions, between about 225 amino acid substitutions and about 230 amino acid substitutions, between about 230 amino acid substitutions and about 240 amino acid substitutions, between about 230 amino acid substitutions and about 235 amino acid substitutions. Those skilled in the art will understand that non-conserved amino acids among wild-type ototoxic proteins from different species can mutate without loss of activity, while conserved amino acids among wild-type ototoxic proteins from different species should not mutate because they are more likely (compared to non-conserved amino acids among different species) to participate in activity.

[0231] Active otopathic proteins may include, for example, those with 1 amino acid deleted to about 200 amino acids, 1 amino acid deleted to about 195 amino acids, 1 amino acid deleted to about 190 amino acids, 1 amino acid deleted to about 185 amino acids, 1 amino acid deleted to about 180 amino acids, 1 amino acid deleted to about 175 amino acids, 1 amino acid deleted to about 170 amino acids, 1 amino acid deleted to about 165 amino acids, 1 amino acid deleted to about 160 amino acids, 1 amino acid deleted to about 155 amino acids, 1 amino acid deleted to about 150 amino acids, 1 amino acid deleted to about 145 amino acids, 1 amino acid deleted to about 140 amino acids, 1 amino acid deleted to about 135 amino acids, 1 amino acid deleted to about 130 amino acids, 1 amino acid deleted to about 1... 25 amino acids, 1 amino acid to about 120 amino acids, 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 acid to about 85 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 75 amino acids, 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 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 40 amino groups. Acid, 1 amino acid to about 35 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, 1 amino acid to about 15 amino acids, 1 amino acid 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 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 to about 190 amino acids, about 2 amino acids to about 185 amino acids, about 2 amino acids to About 180 amino acids, about 2 amino acids to about 175 amino acids, about 2 amino acids to about 170 amino acids, about 2 amino acids to about 165 amino acids, about 2 amino acids to about 160 amino acids, about 2 amino acids to about 155 amino acids, about 2 amino acids to about 150 amino acids, about 2 amino acids to about 145 amino acids, about 2 amino acids to about 140 amino acids, about 2 amino acids to about 135 amino acids, about 2 amino acids to about 130 amino acids, about 2 amino acids to about 125 amino acids, about 2 amino acids to about 120 amino acids, about 2 amino acids to about 115 amino acids, about 2 amino acids to about 110 amino acids, about 2 amino acids to about 105 amino acids.About 2 amino acids 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 to about 85 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids 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 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids 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 About 20 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 9 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 7 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 3 amino acids to about 200 amino acids, about 3 amino acids to about 195 amino acids, about 3 amino acids to about 190 amino acids, about 3 amino acids to about 185 amino acids, about 3 amino acids to about 180 amino acids, about 3 amino acids to about 175 amino acids, about 3 amino acids to about 170 amino acids, about 3 amino acids to about 165 amino acids. Amino acids, about 3 amino acids to about 160 amino acids, about 3 amino acids to about 155 amino acids, about 3 amino acids to about 150 amino acids, about 3 amino acids to about 145 amino acids, about 3 amino acids to about 140 amino acids, about 3 amino acids to about 135 amino acids, about 3 amino acids to about 130 amino acids, about 3 amino acids to about 125 amino acids, about 3 amino acids to about 120 amino acids, about 3 amino acids to about 115 amino acids, about 3 amino acids to about 110 amino acids, about 3 amino acids to about 105 amino acids, about 3 amino acids to about 100 amino acids, about 3 amino acids to about 95 amino acids, about 3 amino acids to about 90 amino acids, about 3 amino acids From about 85 amino acids, from about 3 amino acids to about 80 amino acids, from about 3 amino acids to about 75 amino acids, from about 3 amino acids to about 70 amino acids, from about 3 amino acids to about 65 amino acids, from about 3 amino acids to about 60 amino acids, from about 3 amino acids to about 55 amino acids, from about 3 amino acids to about 50 amino acids, from about 3 amino acids to about 45 amino acids, from about 3 amino acids to about 40 amino acids, from about 3 amino acids to about 35 amino acids, from about 3 amino acids to about 30 amino acids, from about 3 amino acids to about 25 amino acids, from about 3 amino acids to about 20 amino acids, from about 3 amino acids to about 15 amino acids, from about 3 amino acids to about 10 amino acids, from about 3 amino acids to about 9 amino acids.About 3 amino acids to about 8 amino acids, about 3 amino acids to about 7 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 to about 170 amino acids, about 4 amino acids to about 165 amino acids, about 4 amino acids to about 160 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. Approximately 4 amino acids to approximately 140 amino acids, approximately 4 amino acids to approximately 135 amino acids, approximately 4 amino acids to approximately 130 amino acids, approximately 4 amino acids to approximately 125 amino acids, approximately 4 amino acids to approximately 120 amino acids, approximately 4 amino acids to approximately 115 amino acids, approximately 4 amino acids to approximately 110 amino acids, approximately 4 amino acids to approximately 105 amino acids, approximately 4 amino acids to approximately 100 amino acids, approximately 4 amino acids to approximately 95 amino acids, approximately 4 amino acids to approximately 90 amino acids, approximately 4 amino acids to approximately 85 amino acids, approximately 4 amino acids to approximately 80 amino acids, approximately 4 amino acids to approximately 75 amino acids, approximately 4 amino acids to approximately 70 amino acids, approximately 4 amino acids to approximately 65 amino acids. Acid, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 55 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 9 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 7 amino acids, about 4 amino acids to about 6 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 195 amino acids, about 5 amino acids to about 190 amino acids, about 5 amino acids to about 185 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 175 amino acids, about 5 amino acids to about 170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids.About 5 amino acids to about 115 amino acids, about 5 amino acids to 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 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 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 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids 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 groups Acids of about 35 amino acids, about 5 amino acids of about 30 amino acids, about 5 amino acids of about 25 amino acids, about 5 amino acids of about 20 amino acids, about 5 amino acids of about 15 amino acids, about 5 amino acids of about 10 amino acids, about 5 amino acids of about 9 amino acids, about 5 amino acids of about 8 amino acids, about 5 amino acids of about 7 amino acids, about 6 amino acids of about 200 amino acids, about 6 amino acids of about 195 amino acids, about 6 amino acids of about 190 amino acids, about 6 amino acids of about 185 amino acids, about 6 amino acids of about 180 amino acids, about 6 amino acids of about 175 amino acids, about 6 amino acids of about 170 amino acids, about 6 amino acids of about 16 amino acids. 5 amino acids, about 6 amino acids to about 160 amino acids, about 6 amino acids to about 155 amino acids, about 6 amino acids to about 150 amino acids, about 6 amino acids to about 145 amino acids, about 6 amino acids to about 140 amino acids, about 6 amino acids to about 135 amino acids, about 6 amino acids to about 130 amino acids, about 6 amino acids to about 125 amino acids, about 6 amino acids to about 120 amino acids, about 6 amino acids to about 115 amino acids, about 6 amino acids to about 110 amino acids, about 6 amino acids to about 105 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 95 amino acids, about 6 amino acids to about 90 amino acids, about 6 amino groups Acid to about 85 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids to about 75 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 65 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 55 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 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 to about 15 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 9 amino acids,Approximately 6 amino acids to approximately 8 amino acids, approximately 7 amino acids to approximately 200 amino acids, approximately 7 amino acids to approximately 195 amino acids, approximately 7 amino acids to approximately 190 amino acids, approximately 7 amino acids to approximately 185 amino acids, approximately 7 amino acids to approximately 180 amino acids, approximately 7 amino acids to approximately 175 amino acids, approximately 7 amino acids to approximately 170 amino acids, approximately 7 amino acids to approximately 165 amino acids, approximately 7 amino acids to approximately 160 amino acids, approximately 7 amino acids to approximately 155 amino acids, approximately 7 amino acids to approximately 150 amino acids, approximately 7 amino acids to approximately 145 amino acids, approximately 7 amino acids to approximately 140 amino acids, approximately 7 amino acids to approximately 135 amino acids, approximately 7 amino acids to approximately 130 amino acids. Amino acids, about 7 amino acids to about 125 amino acids, about 7 amino acids to about 120 amino acids, about 7 amino acids to about 115 amino acids, about 7 amino acids to about 110 amino acids, about 7 amino acids to about 105 amino acids, about 7 amino acids to about 100 amino acids, about 7 amino acids to about 95 amino acids, about 7 amino acids to about 90 amino acids, about 7 amino acids to about 85 amino acids, about 7 amino acids to about 80 amino acids, about 7 amino acids to about 75 amino acids, about 7 amino acids to about 70 amino acids, about 7 amino acids to about 65 amino acids, about 7 amino acids to about 60 amino acids, about 7 amino acids to about 55 amino acids, about 7 amino acids to about 50 amino acids. Approximately 7 amino acids to approximately 45 amino acids, approximately 7 amino acids to approximately 40 amino acids, approximately 7 amino acids to approximately 35 amino acids, approximately 7 amino acids to approximately 30 amino acids, approximately 7 amino acids to approximately 25 amino acids, approximately 7 amino acids to approximately 20 amino acids, approximately 7 amino acids to approximately 15 amino acids, approximately 7 amino acids to approximately 10 amino acids, approximately 7 amino acids to approximately 9 amino acids, approximately 8 amino acids to approximately 200 amino acids, approximately 8 amino acids to approximately 195 amino acids, approximately 8 amino acids to approximately 190 amino acids, approximately 8 amino acids to approximately 185 amino acids, approximately 8 amino acids to approximately 180 amino acids, approximately 8 amino acids to approximately 175 amino acids, approximately 8 amino acids to approximately 170 amino acids, approximately 8 amino acids The sequence is: approximately 165 amino acids, approximately 8 amino acids to approximately 160 amino acids, approximately 8 amino acids to approximately 155 amino acids, approximately 8 amino acids to approximately 150 amino acids, approximately 8 amino acids to approximately 145 amino acids, approximately 8 amino acids to approximately 140 amino acids, approximately 8 amino acids to approximately 135 amino acids, approximately 8 amino acids to approximately 130 amino acids, approximately 8 amino acids to approximately 125 amino acids, approximately 8 amino acids to approximately 120 amino acids, approximately 8 amino acids to approximately 115 amino acids, approximately 8 amino acids to approximately 110 amino acids, approximately 8 amino acids to approximately 105 amino acids, approximately 8 amino acids to approximately 100 amino acids, approximately 8 amino acids to approximately 95 amino acids, approximately 8 amino acids to approximately 90 amino acids.About 8 amino acids to about 85 amino acids, about 8 amino acids 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 amino acids to about 65 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 55 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids About 200 amino acids, about 10 amino acids to about 195 amino acids, about 10 amino acids to about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 155 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids. Approximately 10 amino acids to approximately 125 amino acids, approximately 10 amino acids to approximately 120 amino acids, approximately 10 amino acids to approximately 115 amino acids, approximately 10 amino acids to approximately 110 amino acids, approximately 10 amino acids to approximately 105 amino acids, approximately 10 amino acids to approximately 100 amino acids, approximately 10 amino acids to approximately 95 amino acids, approximately 10 amino acids to approximately 90 amino acids, approximately 10 amino acids to approximately 85 amino acids, approximately 10 amino acids to approximately 80 amino acids, approximately 10 amino acids to approximately 75 amino acids, approximately 10 amino acids to approximately 70 amino acids, approximately 10 amino acids to approximately 65 amino acids, approximately 10 amino acids to approximately 60 amino acids, approximately 10 amino acids to approximately 55 amino acids. About 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids.About 15 amino acids to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to about 145 amino acids, about 15 amino acids to about 140 amino acids, about 15 amino acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15 amino acids to about 125 amino acids, about 15 amino acids to about 120 amino acids, about 15 amino acids to about 115 amino acids, about 15 amino acids to about 110 amino acids, about 15 amino acids to about 105 amino acids, about 15 amino acids to about 100 amino acids, about 15 amino acids to about 9 5 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 85 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 75 amino acids, about 15 amino acids to about 70 amino acids, about 15 amino acids to about 65 amino acids, about 15 amino acids to about 60 amino acids, about 15 amino acids to about 55 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, about 20 From about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 160 amino acids, from about 20 amino acids to about 155 amino acids, from about 20 amino acids to about 150 amino acids, from about 20 amino acids to about 145 amino acids, from about 20 amino acids to about 140 amino acids, from about 20 amino acids to about 135 amino acids, from about 20 amino acids to about 130 amino acids. Amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 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 20 amino acids to about 100 amino acids, about 20 amino acids 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 to about 80 amino acids, about 20 amino acids to about 75 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.Approximately 20 amino acids to approximately 50 amino acids, approximately 20 amino acids to approximately 45 amino acids, approximately 20 amino acids to approximately 40 amino acids, approximately 20 amino acids to approximately 35 amino acids, approximately 20 amino acids to approximately 30 amino acids, approximately 20 amino acids to approximately 25 amino acids, approximately 25 amino acids to approximately 200 amino acids, approximately 25 amino acids to approximately 195 amino acids, approximately 25 amino acids to approximately 190 amino acids, approximately 25 amino acids to approximately 185 amino acids, approximately 25 amino acids to approximately 180 amino acids, approximately 25 amino acids to approximately 175 amino acids, approximately 25 amino acids to approximately 170 amino acids, approximately 25 amino acids to approximately 165 amino acids, approximately 25 amino acids to approximately 160 amino acids, approximately 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 25 amino acids to about 110 amino acids, about 25 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids Approximately 25 amino acids to approximately 80 amino acids, approximately 25 amino acids to approximately 75 amino acids, approximately 25 amino acids to approximately 70 amino acids, approximately 25 amino acids to approximately 65 amino acids, approximately 25 amino acids to approximately 60 amino acids, approximately 25 amino acids to approximately 55 amino acids, approximately 25 amino acids to approximately 50 amino acids, approximately 25 amino acids to approximately 45 amino acids, approximately 25 amino acids to approximately 40 amino acids, approximately 25 amino acids to approximately 35 amino acids, approximately 25 amino acids to approximately 30 amino acids, approximately 30 amino acids to approximately 200 amino acids, approximately 30 amino acids to approximately 195 amino acids, approximately 30 amino acids to approximately 190 amino acids, approximately 30 amino acids to approximately 185 amino acids, approximately 30 amino acids... The amino acids are approximately 180 amino acids, approximately 30 amino acids are approximately 175 amino acids, approximately 30 amino acids are approximately 170 amino acids, approximately 30 amino acids are approximately 165 amino acids, approximately 30 amino acids are approximately 160 amino acids, approximately 30 amino acids are approximately 155 amino acids, approximately 30 amino acids are approximately 150 amino acids, approximately 30 amino acids are approximately 145 amino acids, approximately 30 amino acids are approximately 140 amino acids, approximately 30 amino acids are approximately 135 amino acids, approximately 30 amino acids are approximately 130 amino acids, approximately 30 amino acids are approximately 125 amino acids, approximately 30 amino acids are approximately 120 amino acids, approximately 30 amino acids are approximately 115 amino acids, and approximately 30 amino acids are approximately 110 amino acids.Approximately 30 amino acids to approximately 105 amino acids, approximately 30 amino acids to approximately 100 amino acids, approximately 30 amino acids to approximately 95 amino acids, approximately 30 amino acids to approximately 90 amino acids, approximately 30 amino acids to approximately 85 amino acids, approximately 30 amino acids to approximately 80 amino acids, approximately 30 amino acids to approximately 75 amino acids, approximately 30 amino acids to approximately 70 amino acids, approximately 30 amino acids to approximately 65 amino acids, approximately 30 amino acids to approximately 60 amino acids, approximately 30 amino acids to approximately 55 amino acids, approximately 30 amino acids to approximately 50 amino acids, approximately 30 amino acids to approximately 45 amino acids, approximately 30 amino acids to approximately 40 amino acids, approximately 30 amino acids to approximately 35 amino acids, approximately 35 amino groups. Acids of about 200 amino acids, about 35 amino acids of about 195 amino acids, about 35 amino acids of about 190 amino acids, about 35 amino acids of about 185 amino acids, about 35 amino acids of about 180 amino acids, about 35 amino acids of about 175 amino acids, about 35 amino acids of about 170 amino acids, about 35 amino acids of about 165 amino acids, about 35 amino acids of about 160 amino acids, about 35 amino acids of about 155 amino acids, about 35 amino acids of about 150 amino acids, about 35 amino acids of about 145 amino acids, about 35 amino acids of about 140 amino acids, about 35 amino acids of about 135 amino acids, about 35 amino acids of about 130 amino acids. Approximately 35 amino acids to approximately 125 amino acids, approximately 35 amino acids to approximately 120 amino acids, approximately 35 amino acids to approximately 115 amino acids, approximately 35 amino acids to approximately 110 amino acids, approximately 35 amino acids to approximately 105 amino acids, approximately 35 amino acids to approximately 100 amino acids, approximately 35 amino acids to approximately 95 amino acids, approximately 35 amino acids to approximately 90 amino acids, approximately 35 amino acids to approximately 85 amino acids, approximately 35 amino acids to approximately 80 amino acids, approximately 35 amino acids to approximately 75 amino acids, approximately 35 amino acids to approximately 70 amino acids, approximately 35 amino acids to approximately 65 amino acids, approximately 35 amino acids to approximately 60 amino acids, approximately 35 amino acids to approximately 55 amino acids, approximately 35 The sequence of amino acids is: approximately 1 to approximately 50 amino acids, approximately 35 amino acids to approximately 45 amino acids, approximately 35 amino acids to approximately 40 amino acids, approximately 40 amino acids to approximately 200 amino acids, approximately 40 amino acids to approximately 195 amino acids, approximately 40 amino acids to approximately 190 amino acids, approximately 40 amino acids to approximately 185 amino acids, approximately 40 amino acids to approximately 180 amino acids, approximately 40 amino acids to approximately 175 amino acids, approximately 40 amino acids to approximately 170 amino acids, approximately 40 amino acids to approximately 165 amino acids, approximately 40 amino acids to approximately 160 amino acids, approximately 40 amino acids to approximately 155 amino acids, approximately 40 amino acids to approximately 150 amino acids, approximately 40 amino acids to approximately 145 amino acids.Approximately 40 amino acids to approximately 140 amino acids, approximately 40 amino acids to approximately 135 amino acids, approximately 40 amino acids to approximately 130 amino acids, approximately 40 amino acids to approximately 125 amino acids, approximately 40 amino acids to approximately 120 amino acids, approximately 40 amino acids to approximately 115 amino acids, approximately 40 amino acids to approximately 110 amino acids, approximately 40 amino acids to approximately 105 amino acids, approximately 40 amino acids to approximately 100 amino acids, approximately 40 amino acids to approximately 95 amino acids, approximately 40 amino acids to approximately 90 amino acids, approximately 40 amino acids to approximately 85 amino acids, approximately 40 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 75 amino acids, approximately 40 amino acids to approximately 70 amino acids. Approximately 40 amino acids to approximately 65 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 40 amino acids to approximately 55 amino acids, approximately 40 amino acids to approximately 50 amino acids, approximately 40 amino acids to approximately 45 amino acids, approximately 45 amino acids to approximately 200 amino acids, approximately 45 amino acids to approximately 195 amino acids, approximately 45 amino acids to approximately 190 amino acids, approximately 45 amino acids to approximately 185 amino acids, approximately 45 amino acids to approximately 180 amino acids, approximately 45 amino acids to approximately 175 amino acids, approximately 45 amino acids to approximately 170 amino acids, approximately 45 amino acids to approximately 165 amino acids, approximately 45 amino acids to approximately 160 amino acids, approximately 45 amino acids to approximately 155 amino acids. Approximately 45 amino acids to approximately 150 amino acids, approximately 45 amino acids to approximately 145 amino acids, approximately 45 amino acids to approximately 140 amino acids, approximately 45 amino acids to approximately 135 amino acids, approximately 45 amino acids to approximately 130 amino acids, approximately 45 amino acids to approximately 125 amino acids, approximately 45 amino acids to approximately 120 amino acids, approximately 45 amino acids to approximately 115 amino acids, approximately 45 amino acids to approximately 110 amino acids, approximately 45 amino acids to approximately 105 amino acids, approximately 45 amino acids to approximately 100 amino acids, approximately 45 amino acids to approximately 95 amino acids, approximately 45 amino acids to approximately 90 amino acids, approximately 45 amino acids to approximately 85 amino acids, approximately 45 amino acids to approximately 80 amino acids. Approximately 45 amino acids to approximately 75 amino acids, approximately 45 amino acids to approximately 70 amino acids, approximately 45 amino acids to approximately 65 amino acids, approximately 45 amino acids to approximately 60 amino acids, approximately 45 amino acids to approximately 55 amino acids, approximately 45 amino acids to approximately 50 amino acids, approximately 50 amino acids to approximately 200 amino acids, approximately 50 amino acids to approximately 195 amino acids, approximately 50 amino acids to approximately 190 amino acids, approximately 50 amino acids to approximately 185 amino acids, approximately 50 amino acids to approximately 180 amino acids, approximately 50 amino acids to approximately 175 amino acids, approximately 50 amino acids to approximately 170 amino acids, approximately 50 amino acids to approximately 165 amino acids, approximately 50 amino acids to approximately 160 amino acids.Approximately 50 amino acids to approximately 155 amino acids, approximately 50 amino acids to approximately 150 amino acids, approximately 50 amino acids to approximately 145 amino acids, approximately 50 amino acids to approximately 140 amino acids, approximately 50 amino acids to approximately 135 amino acids, approximately 50 amino acids to approximately 130 amino acids, approximately 50 amino acids to approximately 125 amino acids, approximately 50 amino acids to approximately 120 amino acids, approximately 50 amino acids to approximately 115 amino acids, approximately 50 amino acids to approximately 110 amino acids, approximately 50 amino acids to approximately 105 amino acids, approximately 50 amino acids to approximately 100 amino acids, approximately 50 amino acids to approximately 95 amino acids, approximately 50 amino acids to approximately 90 amino acids, approximately 50 amino acids to approximately 8 5 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids 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 55 amino acids to about 200 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 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids to about 175 amino acids, about 55 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 to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 5 5 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids.Approximately 60 amino acids to approximately 155 amino acids, approximately 60 amino acids to approximately 150 amino acids, approximately 60 amino acids to approximately 145 amino acids, approximately 60 amino acids to approximately 140 amino acids, approximately 60 amino acids to approximately 135 amino acids, approximately 60 amino acids to approximately 130 amino acids, approximately 60 amino acids to approximately 125 amino acids, approximately 60 amino acids to approximately 120 amino acids, approximately 60 amino acids to approximately 115 amino acids, approximately 60 amino acids to approximately 110 amino acids, approximately 60 amino acids to approximately 105 amino acids, approximately 60 amino acids to approximately 100 amino acids, approximately 60 amino acids to approximately 95 amino acids, approximately 60 amino acids to approximately 90 amino acids, approximately 60 amino acids to approximately 85 amino acids. 1 amino acid, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 65 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids The sequence of amino acids is approximately 75 amino acids, approximately 65 amino acids, approximately 70 amino acids, approximately 70 amino acids, approximately 200 amino acids, approximately 70 amino acids, approximately 195 amino acids, approximately 70 amino acids, approximately 190 amino acids, approximately 70 amino acids, approximately 185 amino acids, approximately 70 amino acids, approximately 180 amino acids, approximately 70 amino acids, approximately 175 amino acids, approximately 70 amino acids, approximately 170 amino acids, approximately 165 amino acids, approximately 70 amino acids, approximately 160 amino acids, approximately 155 amino acids, approximately 70 amino acids, approximately 145 amino acids, and approximately 140 amino acids.Approximately 70 amino acids to approximately 135 amino acids, approximately 70 amino acids to approximately 130 amino acids, approximately 70 amino acids to approximately 125 amino acids, approximately 70 amino acids to approximately 120 amino acids, approximately 70 amino acids to approximately 115 amino acids, approximately 70 amino acids to approximately 110 amino acids, approximately 70 amino acids to approximately 105 amino acids, approximately 70 amino acids to approximately 100 amino acids, approximately 70 amino acids to approximately 95 amino acids, approximately 70 amino acids to approximately 90 amino acids, approximately 70 amino acids to approximately 85 amino acids, approximately 70 amino acids to approximately 80 amino acids, approximately 70 amino acids to approximately 75 amino acids, approximately 75 amino acids to approximately 200 amino acids, approximately 75 amino acids to approximately 195 amino acids. Acid, approximately 75 amino acids to approximately 190 amino acids, approximately 75 amino acids to approximately 185 amino acids, approximately 75 amino acids to approximately 180 amino acids, approximately 75 amino acids to approximately 175 amino acids, approximately 75 amino acids to approximately 170 amino acids, approximately 75 amino acids to approximately 165 amino acids, approximately 75 amino acids to approximately 160 amino acids, approximately 75 amino acids to approximately 155 amino acids, approximately 75 amino acids to approximately 150 amino acids, approximately 75 amino acids to approximately 145 amino acids, approximately 75 amino acids to approximately 140 amino acids, approximately 75 amino acids to approximately 135 amino acids, approximately 75 amino acids to approximately 130 amino acids, approximately 75 amino acids to approximately 125 amino acids, approximately 75 amino acids to approximately 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino groups Acids of about 170 amino acids, about 80 amino acids of about 165 amino acids, about 80 amino acids of about 160 amino acids, about 80 amino acids of about 155 amino acids, about 80 amino acids of about 150 amino acids, about 80 amino acids of about 145 amino acids, about 80 amino acids of about 140 amino acids, about 80 amino acids of about 135 amino acids, about 80 amino acids of about 130 amino acids, about 80 amino acids of about 125 amino acids, about 80 amino acids of about 120 amino acids, about 80 amino acids of about 115 amino acids, about 80 amino acids of about 110 amino acids, about 80 amino acids of about 105 amino acids, about 80 amino acids of about 100 amino acids.Approximately 80 amino acids to approximately 95 amino acids, approximately 80 amino acids to approximately 90 amino acids, approximately 80 amino acids to approximately 85 amino acids, approximately 85 amino acids to approximately 200 amino acids, approximately 85 amino acids to approximately 195 amino acids, approximately 85 amino acids to approximately 190 amino acids, approximately 85 amino acids to approximately 185 amino acids, approximately 85 amino acids to approximately 180 amino acids, approximately 85 amino acids to approximately 175 amino acids, approximately 85 amino acids to approximately 170 amino acids, approximately 85 amino acids to approximately 165 amino acids, approximately 85 amino acids to approximately 160 amino acids, approximately 85 amino acids to approximately 155 amino acids, approximately 85 amino acids to approximately 150 amino acids, approximately 85 amino acids to approximately 145 amino acids. Amino acids, approximately 85 amino acids to approximately 140 amino acids, approximately 85 amino acids to approximately 135 amino acids, approximately 85 amino acids to approximately 130 amino acids, approximately 85 amino acids to approximately 125 amino acids, approximately 85 amino acids to approximately 120 amino acids, approximately 85 amino acids to approximately 115 amino acids, approximately 85 amino acids to approximately 110 amino acids, approximately 85 amino acids to approximately 105 amino acids, approximately 85 amino acids to approximately 100 amino acids, approximately 85 amino acids to approximately 95 amino acids, approximately 85 amino acids to approximately 90 amino acids, approximately 90 amino acids to approximately 200 amino acids, approximately 90 amino acids to approximately 195 amino acids, approximately 90 amino acids to approximately 190 amino acids, approximately 90 amino acids to approximately 1 85 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 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 to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 Amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185 amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95 amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids.Approximately 95 amino acids to approximately 145 amino acids, approximately 95 amino acids to approximately 140 amino acids, approximately 95 amino acids to approximately 135 amino acids, approximately 95 amino acids to approximately 130 amino acids, approximately 95 amino acids to approximately 125 amino acids, approximately 95 amino acids to approximately 120 amino acids, approximately 95 amino acids to approximately 115 amino acids, approximately 95 amino acids to approximately 110 amino acids, approximately 95 amino acids to approximately 105 amino acids, approximately 95 amino acids to approximately 100 amino acids, approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 195 amino acids, approximately 100 amino acids to approximately 190 amino acids, approximately 100 amino acids to approximately 185 amino acids, approximately 100 amino acids Acids up to 180 amino acids, about 100 amino acids up to 175 amino acids, about 100 amino acids up to 170 amino acids, about 100 amino acids up to 165 amino acids, about 100 amino acids up to 160 amino acids, about 100 amino acids up to 155 amino acids, about 100 amino acids up to 150 amino acids, about 100 amino acids up to 145 amino acids, about 100 amino acids up to 140 amino acids, about 100 amino acids up to 135 amino acids, about 100 amino acids up to 130 amino acids, about 100 amino acids up to 125 amino acids, about 100 amino acids up to 120 amino acids, about 100 amino acids up to 115 amino acids, about 100 Amino acids to about 110 amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to about 200 amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105 amino acids to about 180 amino acids, about 105 amino acids to about 175 amino acids, about 105 amino acids to about 170 amino acids, about 105 amino acids to about 165 amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105 amino acids to about 145 amino acids, about 1 0.5 amino acids to about 140 amino acids, about 105 amino acids to about 135 amino acids, about 105 amino acids to about 130 amino acids, about 105 amino acids to about 125 amino acids, about 105 amino acids to about 120 amino acids, about 105 amino acids to about 115 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids.Approximately 110 amino acids to approximately 165 amino acids, approximately 110 amino acids to approximately 160 amino acids, approximately 110 amino acids to approximately 155 amino acids, approximately 110 amino acids to approximately 150 amino acids, approximately 110 amino acids to approximately 145 amino acids, approximately 110 amino acids to approximately 140 amino acids, approximately 110 amino acids to approximately 135 amino acids, approximately 110 amino acids to approximately 130 amino acids, approximately 110 amino acids to approximately 125 amino acids, approximately 110 amino acids to approximately 120 amino acids, approximately 110 amino acids to approximately 115 amino acids, approximately 115 amino acids to approximately 200 amino acids, approximately 115 amino acids to approximately 195 amino acids, approximately 115 amino acids to approximately 190 amino acids. 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to Approximately 120 amino acids, approximately 120 amino acids to approximately 200 amino acids, approximately 120 amino acids to approximately 195 amino acids, approximately 120 amino acids to approximately 190 amino acids, approximately 120 amino acids to approximately 185 amino acids, approximately 120 amino acids to approximately 180 amino acids, approximately 120 amino acids to approximately 175 amino acids, approximately 120 amino acids to approximately 170 amino acids, approximately 120 amino acids to approximately 165 amino acids, approximately 120 amino acids to approximately 160 amino acids, approximately 120 amino acids to approximately 155 amino acids, approximately 120 amino acids to approximately 150 amino acids, approximately 120 amino acids to approximately 145 amino acids, approximately 120 amino acids to approximately 140 amino acids, approximately 120 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their components. A direct translation wouldn't be meaningful without further context.Approximately 125 amino acids to approximately 145 amino acids, approximately 125 amino acids to approximately 140 amino acids, approximately 125 amino acids to approximately 135 amino acids, approximately 125 amino acids to approximately 130 amino acids, approximately 130 amino acids to approximately 200 amino acids, approximately 130 amino acids to approximately 195 amino acids, approximately 130 amino acids to approximately 190 amino acids, approximately 130 amino acids to approximately 185 amino acids, approximately 130 amino acids to approximately 180 amino acids, approximately 130 amino acids to approximately 175 amino acids, approximately 130 amino acids to approximately 170 amino acids, approximately 130 amino acids to approximately 165 amino acids, approximately 130 amino acids to approximately 160 amino acids, approximately 130 amino acids to approximately 155 amino acids. 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids From about 155 amino acids, from about 135 amino acids to about 150 amino acids, from about 135 amino acids to about 145 amino acids, from about 135 amino acids to about 140 amino acids, from about 140 amino acids to about 200 amino acids, from about 140 amino acids to about 195 amino acids, from about 140 amino acids to about 190 amino acids, from about 140 amino acids to about 185 amino acids, from about 140 amino acids to about 180 amino acids, from about 140 amino acids to about 175 amino acids, from about 140 amino acids to about 170 amino acids, from about 140 amino acids to about 165 amino acids, from about 140 amino acids to about 160 amino acids, from about 140 amino acids to about 155 amino acids, from about 14 0 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 200 amino acids.Approximately 150 amino acids to approximately 195 amino acids, approximately 150 amino acids to approximately 190 amino acids, approximately 150 amino acids to approximately 185 amino acids, approximately 150 amino acids to approximately 180 amino acids, approximately 150 amino acids to approximately 175 amino acids, approximately 150 amino acids to approximately 170 amino acids, approximately 150 amino acids to approximately 165 amino acids, approximately 150 amino acids to approximately 160 amino acids, approximately 150 amino acids to approximately 155 amino acids, approximately 155 amino acids to approximately 200 amino acids, approximately 155 amino acids to approximately 195 amino acids, approximately 155 amino acids to approximately 190 amino acids, approximately 155 amino acids to approximately 185 amino acids, approximately 155 From approximately 155 amino acids to approximately 175 amino acids, from approximately 155 amino acids to approximately 170 amino acids, from approximately 155 amino acids to approximately 165 amino acids, from approximately 155 amino acids to approximately 160 amino acids, from approximately 160 amino acids to approximately 200 amino acids, from approximately 160 amino acids to approximately 195 amino acids, from approximately 160 amino acids to approximately 190 amino acids, from approximately 160 amino acids to approximately 185 amino acids, from approximately 160 amino acids to approximately 180 amino acids, from approximately 160 amino acids to approximately 175 amino acids, from approximately 160 amino acids to approximately 170 amino acids, from approximately 160 amino acids to approximately 165 amino acids, from approximately 165 amino acids to... Approximately 200 amino acids, approximately 165 amino acids to approximately 195 amino acids, approximately 165 amino acids to approximately 190 amino acids, approximately 165 amino acids to approximately 185 amino acids, approximately 165 amino acids to approximately 180 amino acids, approximately 165 amino acids to approximately 175 amino acids, approximately 165 amino acids to approximately 170 amino acids, approximately 170 amino acids to approximately 200 amino acids, approximately 170 amino acids to approximately 195 amino acids, approximately 170 amino acids to approximately 190 amino acids, approximately 170 amino acids to approximately 185 amino acids, approximately 170 amino acids to approximately 180 amino acids, approximately 170 amino acids to approximately 175 amino acids, approximately 175 amino acids to approximately 200 175 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, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids.The sequence of a wild-type full-length auricular protein (e.g., wild-type human full-length auricular protein) is approximately 195 to approximately 200 amino acids. In some embodiments, two or more amino acids are deleted from the sequence of the wild-type full-length auricular protein, the two or more deleted amino acids may be continuous in the sequence of the wild-type full-length protein. In other instances, two or more amino acids are deleted from the sequence of the wild-type full-length auricular protein, the two or more deleted amino acids are not continuous in the sequence of the wild-type full-length protein. Those skilled in the art will understand that non-conserved amino acids between wild-type full-length auricular proteins from different species can be deleted without loss of activity, while those conserved amino acids between wild-type full-length auricular proteins from different species should not be deleted because they are more likely (compared to non-conserved amino acids between different species) to participate in activity.

[0232] In some instances, the active otopathic protein may, for example, comprise the sequence of the wild-type full-length otopathic protein, having removed 1 amino acid from its N-terminus and / or from its C-terminus to about 100 amino acids, 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 acid to about 80 amino acids, 1 amino acid to about 75 amino acids, 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 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 40 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, 1 amino acid to about 20 amino acids, 1 amino acid to about 15 amino acids, 1 amino acid 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 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 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 to about 85 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids to about 75 amino acids, about 2 amino acids The sequence is: approximately 70 amino acids, approximately 2 amino acids to approximately 65 amino acids, approximately 2 amino acids to approximately 60 amino acids, approximately 2 amino acids to approximately 55 amino acids, approximately 2 amino acids to approximately 50 amino acids, approximately 2 amino acids to approximately 45 amino acids, approximately 2 amino acids to approximately 40 amino acids, approximately 2 amino acids to approximately 35 amino acids, approximately 2 amino acids to approximately 30 amino acids, approximately 2 amino acids to approximately 25 amino acids, approximately 2 amino acids to approximately 20 amino acids, approximately 2 amino acids to approximately 15 amino acids, approximately 2 amino acids to approximately 10 amino acids, approximately 2 amino acids to approximately 9 amino acids, approximately 2 amino acids to approximately 8 amino acids, approximately 2 amino acids to approximately 7 amino acids, approximately 2 amino acids to approximately 6 amino acids, approximately 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 100 amino acids, about 3 amino acids to about 95 amino acids, about 3 amino acids to about 90 amino acids, about 3 amino acids to about 85 amino acids, about 3 amino acids to about 80 amino acids, about 3 amino acids to about 75 amino acids, about 3 amino acids to about 70 amino acids, about 3 amino acids to about 65 amino acids, about 3 amino acids to about 60 amino acids, about 3 amino acids to about 55 amino acids, about 3 amino acids to about 50 amino acids, about 3 amino acids to about 45 amino acids, about 3 amino acids to about 40 amino acids, about 3 amino acids to about 35 amino acids, about 3 amino acids to about 30 amino acids.About 3 amino acids to about 25 amino acids, about 3 amino acids to about 20 amino acids, about 3 amino acids to about 15 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 9 amino acids, about 3 amino acids to about 8 amino acids, about 3 amino acids to about 7 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 100 amino acids, about 4 amino acids to about 95 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 85 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 75 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 65 amino acids. Acid, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 55 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 9 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 7 amino acids, about 4 amino acids to about 6 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 95 amino acids 1 amino acid, about 5 amino acids 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 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids 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 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 amino acids to about 15 amino acids, about 5 amino acids The amino acids are approximately 10 amino acids, approximately 5 amino acids are approximately 9 amino acids, approximately 5 amino acids are approximately 8 amino acids, approximately 5 amino acids are approximately 7 amino acids, approximately 6 amino acids are approximately 100 amino acids, approximately 6 amino acids are approximately 95 amino acids, approximately 6 amino acids are approximately 90 amino acids, approximately 6 amino acids are approximately 85 amino acids, approximately 6 amino acids are approximately 80 amino acids, approximately 6 amino acids are approximately 75 amino acids, approximately 6 amino acids are approximately 70 amino acids, approximately 6 amino acids are approximately 65 amino acids, approximately 6 amino acids are approximately 60 amino acids, approximately 6 amino acids are approximately 55 amino acids, approximately 6 amino acids are approximately 50 amino acids, approximately 6 amino acids are approximately 45 amino acids, approximately 6 amino acids are approximately 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 to about 15 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, about 7 amino acids to about 100 amino acids, about 7 amino acids to about 95 amino acids, about 7 amino acids to about 90 amino acids, about 7 amino acids to about 85 amino acids, about 7 amino acids to about 80 amino acids, about 7 amino acids to about 75 amino acids, about 7 amino acids to about 70 amino acids, about 7 amino acids to about 65 amino acids, about 7 amino acids to about 6 0 amino acids, about 7 amino acids to about 55 amino acids, about 7 amino acids to about 50 amino acids, about 7 amino acids 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 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 10 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 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 to about 80 amino acids, about 8 From about 75 amino acids, from about 8 amino acids to about 70 amino acids, from about 8 amino acids to about 65 amino acids, from about 8 amino acids to about 60 amino acids, from about 8 amino acids to about 55 amino acids, from about 8 amino acids to about 50 amino acids, from about 8 amino acids to about 45 amino acids, from about 8 amino acids to about 40 amino acids, from about 8 amino acids to about 35 amino acids, from about 8 amino acids to about 30 amino acids, from about 8 amino acids to about 25 amino acids, from about 8 amino acids to about 20 amino acids, from about 8 amino acids to about 15 amino acids, from about 8 amino acids to about 10 amino acids, from about 10 amino acids to about 100 amino acids, from about 10 amino acids to about 95 amino acids, from about 10 amino acids From about 90 amino acids, from about 10 amino acids to about 85 amino acids, from about 10 amino acids to about 80 amino acids, from about 10 amino acids to about 75 amino acids, from about 10 amino acids to about 70 amino acids, from about 10 amino acids to about 65 amino acids, from about 10 amino acids to about 60 amino acids, from about 10 amino acids to about 55 amino acids, from about 10 amino acids to about 50 amino acids, from about 10 amino acids to about 45 amino acids, from about 10 amino acids to about 40 amino acids, from about 10 amino acids to about 35 amino acids, from about 10 amino acids to about 30 amino acids, from about 10 amino acids to about 25 amino acids, from about 10 amino acids to about 20 amino acids, from about 10 amino acids to about 15 amino acids.Approximately 20 amino acids to approximately 100 amino acids, approximately 20 amino acids to approximately 95 amino acids, approximately 20 amino acids to approximately 90 amino acids, approximately 20 amino acids to approximately 85 amino acids, approximately 20 amino acids to approximately 80 amino acids, approximately 20 amino acids to approximately 75 amino acids, approximately 20 amino acids to approximately 70 amino acids, approximately 20 amino acids to approximately 65 amino acids, approximately 20 amino acids to approximately 60 amino acids, approximately 20 amino acids to approximately 55 amino acids, approximately 20 amino acids to approximately 50 amino acids, approximately 20 amino acids to approximately 45 amino acids, approximately 20 amino acids to approximately 40 amino acids, approximately 20 amino acids to approximately 35 amino acids, approximately 20 amino acids to approximately 30 amino acids, approximately 20 amino acids to Approximately 25 amino acids, approximately 30 amino acids to approximately 100 amino acids, approximately 30 amino acids to approximately 95 amino acids, approximately 30 amino acids to approximately 90 amino acids, approximately 30 amino acids to approximately 85 amino acids, approximately 30 amino acids to approximately 80 amino acids, approximately 30 amino acids to approximately 75 amino acids, approximately 30 amino acids to approximately 70 amino acids, approximately 30 amino acids to approximately 65 amino acids, approximately 30 amino acids to approximately 60 amino acids, approximately 30 amino acids to approximately 55 amino acids, approximately 30 amino acids to approximately 50 amino acids, approximately 30 amino acids to approximately 45 amino acids, approximately 30 amino acids to approximately 40 amino acids, approximately 30 amino acids to approximately 35 amino acids, approximately 40 amino acids to approximately 100 amino acids. Approximately 40 amino acids to approximately 95 amino acids, approximately 40 amino acids to approximately 90 amino acids, approximately 40 amino acids to approximately 85 amino acids, approximately 40 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 75 amino acids, approximately 40 amino acids to approximately 70 amino acids, approximately 40 amino acids to approximately 65 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 40 amino acids to approximately 55 amino acids, approximately 40 amino acids to approximately 50 amino acids, approximately 40 amino acids to approximately 45 amino acids, approximately 50 amino acids to approximately 100 amino acids, approximately 50 amino acids to approximately 95 amino acids, approximately 50 amino acids to approximately 90 amino acids, approximately 50 amino acids to approximately 85 amino acids, approximately 50 amino acids to Approximately 80 amino acids, approximately 50 amino acids to approximately 75 amino acids, approximately 50 amino acids to approximately 70 amino acids, approximately 50 amino acids to approximately 65 amino acids, approximately 50 amino acids to approximately 60 amino acids, approximately 50 amino acids to approximately 55 amino acids, approximately 60 amino acids to approximately 100 amino acids, approximately 60 amino acids to approximately 95 amino acids, approximately 60 amino acids to approximately 90 amino acids, approximately 60 amino acids to approximately 85 amino acids, approximately 60 amino acids to approximately 80 amino acids, approximately 60 amino acids to approximately 75 amino acids, approximately 60 amino acids to approximately 70 amino acids, approximately 60 amino acids to approximately 65 amino acids, approximately 70 amino acids to approximately 100 amino acids, approximately 70 amino acids to approximately 95 amino acids.Approximately 70 amino acids to approximately 90 amino acids, approximately 70 amino acids to approximately 85 amino acids, approximately 70 amino acids to approximately 80 amino acids, approximately 70 amino acids to approximately 75 amino acids, approximately 80 amino acids to approximately 100 amino acids, approximately 80 amino acids to approximately 95 amino acids, approximately 80 amino acids to approximately 90 amino acids, approximately 80 amino acids to approximately 85 amino acids, approximately 90 amino acids to approximately 100 amino acids, approximately 90 amino acids to approximately 95 amino acids, or approximately 95 amino acids to approximately 100 amino acids.

[0233] In some embodiments, the active otopathic protein may, for example, comprise the sequence of wild-type full-length otopathic protein, wherein an insertion of 1 to 50 amino acids, 1 to 45 amino acids, 1 to 40 amino acids, 1 to 35 amino acids, 1 to 30 amino acids, 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids, or about 2 to 5 amino acids is permitted. 0 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 to 25 amino acids, approximately 2 to 20 amino acids, approximately 2 to 15 amino acids, approximately 2 to 10 amino acids, approximately 2 to 9 amino acids, approximately 2 to 8 amino acids, approximately 2 to 7 amino acids, approximately 2 to 6 amino acids, approximately 2 to 5 amino acids, approximately 2 to 4 amino acids, approximately 3 to 50 amino acids, approximately 3 to 45 amino acids, approximately 3 to 40 amino acids, approximately 3 to 35 amino acids Amino acids, approximately 3 to 30 amino acids, approximately 3 to 25 amino acids, approximately 3 to 20 amino acids, approximately 3 to 15 amino acids, approximately 3 to 10 amino acids, approximately 3 to 9 amino acids, approximately 3 to 8 amino acids, approximately 3 to 7 amino acids, approximately 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 to 40 amino acids, approximately 4 to 35 amino acids, approximately 4 to 30 amino acids, approximately 4 to 25 amino acids, approximately 4 to 20 amino acids, approximately 4 to 15 amino acids Amino acids, approximately 4 to 10 amino acids, approximately 4 to 9 amino acids, approximately 4 to 8 amino acids, approximately 4 to 7 amino acids, approximately 4 to 6 amino acids, approximately 5 to 50 amino acids, approximately 5 to 45 amino acids, approximately 5 to 40 amino acids, approximately 5 to 35 amino acids, approximately 5 to 30 amino acids, approximately 5 to 25 amino acids, approximately 5 to 20 amino acids, approximately 5 to 15 amino acids, approximately 5 to 10 amino acids, approximately 5 to 9 amino acids, approximately 5 to 8 amino acids, approximately 5 to 7 amino acids, approximately 6 to 50 amino acids.Approximately 6 to 45 amino acids, approximately 6 to 40 amino acids, approximately 6 to 35 amino acids, approximately 6 to 30 amino acids, approximately 6 to 25 amino acids, approximately 6 to 20 amino acids, approximately 6 to 15 amino acids, approximately 6 to 10 amino acids, approximately 6 to 9 amino acids, approximately 6 to 8 amino acids, approximately 7 to 50 amino acids, approximately 7 to 45 amino acids, approximately 7 to 40 amino acids, approximately 7 to 35 amino acids, approximately 7 to 30 amino acids, approximately 7 to 25 amino acids, approximately 7 amino acids Up to 20 amino acids, about 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 to 45 amino acids, about 8 to 40 amino acids, about 8 to 35 amino acids, about 8 to 30 amino acids, about 8 to 25 amino acids, about 8 to 20 amino acids, about 8 to 15 amino acids, about 8 to 10 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to... 35 amino acids, approximately 10 to 30 amino acids, approximately 10 to 25 amino acids, approximately 10 to 20 amino acids, approximately 10 to 15 amino acids, approximately 15 to 50 amino acids, approximately 15 to 45 amino acids, approximately 15 to 40 amino acids, approximately 15 to 35 amino acids, approximately 15 to 30 amino acids, approximately 15 to 25 amino acids, approximately 15 to 20 amino acids, approximately 20 to 50 amino acids, approximately 20 to 45 amino acids, approximately 20 to 40 amino acids, approximately 20 to 35 amino acids Amino acids, approximately 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 to 40 amino acids, approximately 25 to 35 amino acids, approximately 25 to 30 amino acids, approximately 30 to 50 amino acids, approximately 30 to 45 amino acids, approximately 30 to 40 amino acids, approximately 30 to 35 amino acids, approximately 35 to 50 amino acids, approximately 35 to 45 amino acids, approximately 35 to 40 amino acids, approximately 40 to 50 amino acids.Approximately 40 to 45 amino acids, or approximately 45 to approximately 50 amino acids. In some instances, the inserted amino acid may be inserted as a continuous sequence into the sequence of the wild-type full-length protein. In some instances, the amino acid is not inserted as a continuous sequence into the sequence of the wild-type full-length protein. As will be understood in the art, amino acids may be inserted into a portion of the sequence of a wild-type full-length protein that is not well conserved across species.

[0234] Scope: Throughout this disclosure, various aspects of the present disclosure may be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the description of the scope should be considered as having specifically disclosed all possible sub-scopes and individual numerical values ​​within that scope. For example, a description of a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope such as 95-99% identity includes substances having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-scopes such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the scope.

[0235] In one aspect, this disclosure provides a recombinant AAV vector of SEQ ID NO:96. In another aspect, this disclosure provides a recombinant AAV vector of SEQ ID NO:105.

[0236] In one aspect, this disclosure provides a recombinant AAV vector comprising, in 5' to 3' order: the 5' ITR sequence of SEQ ID NO:97; a CAG promoter comprising the CMV early enhancer element of SEQ ID NO:98, the chicken β-actin gene sequence of SEQ ID NO:99, and the chimeric intron of SEQ ID NO:100; a 5' OTOF coding region comprising exons 1 to (and through) 21 of the OTOF cDNA; the SD intron sequence of SEQ ID NO:102; the AK recombination sequence of SEQ ID NO:103; and the 3' ITR sequence of SEQ ID NO:104. In some embodiments, the 5' OTOF coding region is SEQ ID NO:101. In some embodiments, the 5'OTOF coding region is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to SEQ ID NO:101, and encodes the same amino acid sequence as encoded by SEQ ID NO:101.

[0237] In one aspect, this disclosure provides a recombinant AAV vector comprising, in 5' to 3' order: the 5' ITR sequence of SEQ ID NO:97; the AK recombination sequence of SEQ ID NO:103; the SA intron sequence of SEQ ID NO:106; a 3' OTOF coding region comprising exons 22 to (and through) exon 48 of the OTOF cDNA; the bgH polyA sequence of SEQ ID NO:108; and the 3' ITR sequence of SEQ ID NO:104. In some embodiments, the 3' OTOF coding region is SEQ ID NO:107. In some embodiments, the 3'OTOF coding region is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to SEQ ID NO:107, and encodes the same amino acid sequence as SEQ ID NO:107.

[0238] In one aspect, this disclosure provides rAAV particles comprising one of the aforementioned recombinant AAV vectors capped with an Anc80 capsid. In some embodiments, the Anc80 capsid comprises the polypeptide of SEQ ID NO:109.

[0239] In one aspect, this disclosure provides a composition comprising a first rAAV particle comprising a recombinant AAV vector comprising SEQ ID NO:96 and a second rAAV particle comprising a recombinant AAV vector comprising SEQ ID NO:105. In some embodiments, the recombinant AAV vector of the first rAAV particle is capped with an Anc80 capsid. In some embodiments, the recombinant AAV vector of the second rAAV particle is capped with an Anc80 capsid. In some embodiments, the recombinant AAV vectors of the first and second rAAV particles are each independently capped with an Anc80 capsid. In some embodiments, the Anc80 capsid comprises a polypeptide of SEQ ID NO:109.

[0240] In one aspect, this disclosure provides compositions comprising (a) a first rAAV particle comprising a recombinant AAV vector, the recombinant AAV vector comprising, in 5' to 3' order: the 5' ITR sequence of SEQ ID NO:97; a CAG promoter comprising the CMV early enhancer element of SEQ ID NO:98, the chicken β-actin gene sequence of SEQ ID NO:99, and the chimeric intron of SEQ ID NO:100; a 5' OTOF coding region comprising exons 1 to (and through) 21 of OTOF cDNA; the SD intron sequence of SEQ ID NO:102; the AK recombination sequence of SEQ ID NO:103; and the 3' ITR sequence of SEQ ID NO:104; and (b) a second rAAV particle comprising a recombinant AAV vector comprising the 5' ITR sequence of SEQ ID NO:97; the AK recombination sequence of SEQ ID NO:103; and the SEQ ID NO:104. The SA intron sequence of SEQ ID NO:106; the 3' OTOF coding region containing exons 22 to (and through) exon 48 of the OTOF cDNA; the bgH polyA sequence of SEQ ID NO:108; and the 3' ITR sequence of SEQ ID NO:104. In some embodiments, the 5' OTOF coding region is SEQ ID NO:101. In some embodiments, the 5' OTOF coding region is at least 70% identical to SEQ ID NO:101 and encodes the same amino acid sequence as SEQ ID NO:101. In some embodiments, the 3' OTOF coding region is SEQ ID NO:107. In some embodiments, the 3'OTOF coding region is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to SEQ ID NO:107, and encodes the same amino acid sequence as encoded by SEQ ID NO:107. The first and second rAAV particles are each independently capsidated by an Anc80 capsid. In some embodiments, the Anc80 capsid comprises the polypeptide of SEQ ID NO:109.

[0241] In one aspect, when the above composition is introduced into human cells, the recombinant AAV vectors of the first and second rAAV particles undergo polymerization or homologous recombination with each other to form a recombinant nucleic acid encoding full-length otorrhea protein in the cell.

[0242] In one aspect, this disclosure provides a method comprising introducing a therapeutically effective amount of any of the above-described compositions into the cochlea of ​​a mammal (e.g., a human). In some embodiments, the mammal has been previously identified as having a defective ototoxicity protein gene.

[0243] In one aspect, this disclosure provides a method for increasing the expression of full-length ototoxicity protein in mammalian cells, the method comprising introducing any of the above-described compositions into mammalian cells, such as inner hair cells, for example, human cells. In some embodiments, the mammalian cells have been previously identified as having a defective ototoxicity protein gene.

[0244] In one aspect, this disclosure provides a method for increasing the expression of full-length ototoxicity protein in the inner hair cells of a mammal, such as the human cochlea, the method comprising introducing a therapeutically effective amount of any of the above-described compositions into the cochlea of ​​the mammal. In some embodiments, the mammal has previously been identified as having a defective ototoxicity protein gene.

[0245] In one aspect, this disclosure provides a method for treating asymptomatic sensorineural hearing loss in a subject, such as a person identified as having a defective cochlear gene, the method comprising administering a therapeutically effective amount of any of the above-described compositions into the cochlea of ​​the subject. In some embodiments, the method further comprises, prior to the administration step, identifying that the subject has a defective ototoxicity protein gene. In some embodiments, a microcatheter is used to administer the composition to the cochlea. In some embodiments, the microcatheter is shaped such that it can pass through the external auditory canal into the middle ear cavity and the tip of the microcatheter is contacted with the RWM (Round-Wide Microvascular Mass). In some embodiments, the distal end of the microcatheter includes at least one microneedle with a diameter between 10 and 1,000 micrometers.

[0246] In one aspect, this disclosure provides a kit comprising any of the above-described compositions. In some embodiments, the composition is pre-loaded in a device such as a microcatheter. In some embodiments, the microcatheter is shaped such that it can pass through the external auditory canal into the middle ear cavity and the tip of the microcatheter contacts the RWM. In some embodiments, the distal end of the microcatheter includes at least one microneedle with a diameter between 10 and 1,000 micrometers.

[0247] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Methods and materials used in this disclosure are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative rather than limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Invention Details

[0249] Mutations in OTOF result in DFNB9, a recessive, non-syndromic prelingual hearing disorder. A deficiency of otoblastin, a protein encoded by OTOF, eliminates rapid exocytosis of the auditory inner hair cells (IHC). Because neurotransmission at the first auditory synapse is impaired, no sound signals are transmitted to the brain, explaining profound hearing loss.

[0250] The currently claimed method has been found to result in the expression of full-length ototoxicin in the inner ear hair cells and to successfully restore hearing in ototoxicin knockout mice, with ABR thresholds of 30 dB to 70 dB for short stimuli and 50 dB to 90 dB for pure tones. In light of this finding, this article provides compositions and methods for treating asymptomatic sensorineural hearing loss in subjects (e.g., humans) identified as having a defective ototoxicin gene.

[0251] This document provides compositions comprising at least two different nucleic acid vectors, wherein: each of the at least two different vectors contains a coding sequence encoding a different portion of an otopathic protein, each of the encoded portions being at least 30 amino acid residues in length, wherein the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; none of the at least two different vectors encodes an active otopathic protein (e.g., full-length otopathic protein); at least one of the coding sequences contains a nucleotide sequence spanning two adjacent exons of the otopathic protein genomic DNA and lacks an intron sequence between the two adjacent exons; and when introduced into mammalian cells, the at least two different vectors undergo homologous recombination with each other to form a recombinant nucleic acid, wherein the recombinant nucleic acid encodes an active otopathic protein (e.g., full-length otopathic protein). In some instances, the recombinant nucleic acid encoding an active otopathic protein (e.g., full-length otopathic protein) is present as an episome in mammalian cells (e.g., any type of mammalian cell described herein). Kits comprising any of the compositions described herein are also provided. This document also provides methods comprising introducing a therapeutically effective amount of any of the compositions described herein into the mammalian cochlea.

[0252] This article also provides methods for increasing the expression of active otopathic proteins (e.g., full-length otopathic proteins) in mammalian cells, comprising introducing any of the compositions described herein into mammalian cells. This article also provides methods for increasing the expression of active otopathic proteins (e.g., full-length otopathic proteins) in the inner hair cells of the mammalian cochlea, comprising introducing a therapeutically effective amount of any of the compositions described herein into the mammalian cochlea. This article also provides methods for treating asymptomatic sensorineural hearing loss in subjects identified as having a defective otopathic protein gene, comprising: administering a therapeutically effective amount of any of the compositions described herein into the cochlea of ​​the subject.

[0253] Other non-limiting aspects of the compositions, kits, and methods are described herein and may be used in any combination without limitation.

[0254] Otoferlin

[0255] The human OTOF gene encodes ototoxicin, a protein that plays a crucial role in the initiation, fusion, and / or supplementation of synaptic vesicles in the inner hair cell synapse during sound encoding in some embodiments. To date, hundreds of mutations in the human OTOF gene have been identified that result in profound prelingual hearing loss (DFNB9). In different populations, this type of mutation is responsible for 2–8% of people being born with autosomal recessive nonsyndromic hearing loss (Rodríguez-Ballesteros et al. (2008) Hum.Mutat. 29 823–831; Choi et al. (2009) Clinical Genetics 75 237–243; Duman et al. (2011) Genet Test Mol Biomarkers 15 29–33; Varga et al. (2006) J MedGenet 43 576–581; Iwasa et al. (2013) BMC Med.Genet. 14 95). Biallelic otomorphin gene mutations result in localized synaptic transmission defects between hair cells and the auditory nerve. Otomorphin enables sensory cells to release neurotransmitters in response to sound stimuli to activate auditory neurons, which then carry electronically encoded auditory information to the brain to produce “hearing.” When biallelic mutations are present in OTOF, this transmission is impaired, resulting in most subjects having congenital, severe to profound sensorineural hearing loss. For example, two substitutions in exon 15 at positions 490 and 515 of the conserved C2C domain of otodermal protein lead to DFNB9 (Mirqhomizadeh et al. (2002) Neurobiol. Dis. 10(2): 157-164). Migliosi et al. discovered a novel mutation Q829X in the OTOF of Spanish subjects with prelingual nonsyndromic hearing loss (Migliosi et al. (2002) J. Med. Genet. 39(7): 502-506).

[0256] Other exemplary mutations of the ototoxicin gene detected in subjects with hearing loss and methods for sequencing the nucleic acid encoding the ototoxicin are described, for example, in Rodriguez-Ballesteros et al. (2003) Hum Mutat. 22:451-456; Wang et al. (2010) BMC Med Genet. 11:79; Yildirim-Baylan et al. (2014) Int. J. Pediatr. Otorhinolaryngol 78:950-953; Choi et al. (2009) Clin. Genet. 75(3):237-243; and Marlin et al. (2010) Biochem Biophys Res Commun 394:737-742.

[0257] Otoacoustic emissions from DFNB9 subjects were normal, at least during the first decade of life, indicating the morphological integrity of the inner ear and normal function of the outer hair cells. Apart from the lack of synaptic transmission and subsequent synaptic loss, the morphology and physiology of the inner ear were preserved in DFNB9, at least during the first decade of life. Therefore, in some implementations, restoration of OTOF and / or otodermal protein function can be used to mitigate or prevent secondary degeneration of one or more cochlear structures.

[0258] Studies in mouse models revealed normal synaptic structure, and the IHC maintained a normal number of synapses in mice during the first week after birth. Between P6 and P15, approximately half of the synapses were lost (Roux et al. (2006) Cell 127 277–289). Animal models allow for the study of the effects of otoblastin mutations on synaptic transmission by recording changes in plasma membrane capacitance after vesicle fusion and auditory nerve activity. In otoblastin knockout (Otof- / -) mice, exocytosis was barely triggered in the IHC due to depolarization-induced Ca2+ flow voltage-gated Ca2+ channels (Roux et al. (2006) Cell 127 277–289). In pachanga (OtofPga / Pga) mice with profound hearing loss and random point mutations in the C2F domain, short (<10 ms) depolarization of the IHC elicits vesicle fusions of similar size to those in wild-type mice; however, sustained stimulation reveals a strong defect in replenishing vesicles to readily release pools (Pangrsic et al. (2010) Nat. Neurosci. 13 869–876). The p.Ile515Thr mutation, found in human subjects, presents with only a slight increase in hearing threshold but severe impairment of speech comprehension and temperature-dependent deafness (Varga et al. (2006) J Med Genet 43 576–581), revealing an intermediate phenotype when studied in mouse models (Strenzke et al. (2016) EMBO J. 35:2519–2535). When assessed by ABR, these OtofI515T / I515T mice showed moderately elevated hearing thresholds and reduced I-wave amplitude, but normal hearing thresholds in behavioral tests and recordings of individual auditory neurons. Exocytosis of the RRP was intact again, but sustained exocytosis was reduced, although not as severely as in OtofPga / Pga. In wild-type mice at room temperature, 750 vesicles per active area could fuse during sustained stimulation, a rate that decreased to 350 vesicles / s / active area in OtofI515T / I515T mice and further to 200 vesicles / s / active area in OtofPga / PgaIHC (Pangrsic et al. (2010) Nat. Neurosci. 13 869–876; Strenzke et al. (2016) EMBO J. 35 2519–2535). This is associated with lower levels of otopathic protein on the IHC plasma membrane, suggesting that the amount of otopathic protein is proportional to exocytosis and hearing (Strenzke et al. (2016) EMBO J.35 2519–2535).

[0259] Methods for detecting gene mutations are well known in the art. Non-limiting examples of such techniques include: real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.

[0260] The OTOF gene encodes otoblastin, a protein involved in the exocytosis of synaptic vesicles in cochlear hair cells (see, for example, Johnson and Chapman (2010) J. Cell Biol. 191(1):187-198; and Heidrych et al. (2008) Hum. Mol. Genet. 17:3814-3821).

[0261] The human OTOF gene is located on chromosome 2p23.3. It contains 48 exons, covering approximately 132 kilobases (kb) (NCBI accession number NG009937.1). The mRNA encoding the long form of ototoxicity protein expressed in the brain comprises 48 exons (Yasunaga et al., Am.J.Hum.Genet.67:591-600, 2000). Forward and reverse primers that can be used to amplify each of the 48 exons in the OTOF gene are 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 the full-length wild-type OTOF protein. The full-length wild-type OTOF protein expressed from the human OTOF gene is 1997 residues in length.

[0262] Exemplary human wild-type otopathic protein is or includes the sequence of any one of SEQ ID NO:1-5. The isotype e of human otopathic protein (SEQ ID NO:5) is encoded by an mRNA containing exon 48 of the otopathic protein gene but not exon 47 (Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000). In some embodiments, the active otopathic protein has the sequence of SEQ ID NO:5 but lacks the 20 amino acids included in the RXR motif identified in Strenzke et al., EMBO J. 35(23):2499-2615, 2016. Non-limiting examples of nucleic acids encoding wild-type otopathic protein are or include any one of SEQ ID NO:7-11. As will be appreciated in the art, at least some or all of the codons in SEQ ID NO:7-11 can be codon-optimized to allow optimal expression in non-human mammals or humans. Orthologs of human ear malformation protein are known in the art.

[0263] Human ear malformation protein cDNA sequence:

[0264] Human typical (long) isotype sequence (otoxin) (SEQ ID NO:1) (also known as otoxin isotype a) (NCBI accession number AAD26117.1)

[0265] Human isotype 2 (short 1) (Otocin) (SEQ ID NO:2) (also known as otocin isotype d) (NCBI accession number NP_919304.1)

[0266] Human isotype 3 (short 2) (otoxin) (SEQ ID NO:3) (also known as otoxin isotype c) (NCBI accession number NP_919303.1)

[0267] Human isotype 4 (short 3) (otoxin) (SEQ ID NO:4) (also known as otoxin isotype b) (NCBI accession number NP_004793.2)

[0268] Human isotype 5 (short 4) (otoxin) (SEQ ID NO:5) (also known as otoxin isotype e) (NCBI accession number NP_001274418.1)

[0269] Complete cds (ear-degeneration protein cDNA) (www.ncbi.nlm.nih.gov / nuccore / AF107403.1) (SEQ ID NO:6) (encoding the protein of SEQ ID NO:1)

[0270] Human ear malformation protein transcript variant 1 (www.ncbi.nlm.nih.gov / nuccore / NM_194248.2) (SEQ ID NO:7) (encoding the protein of SEQ ID NO:1)

[0271] Human ear malformation protein transcript variant 2 (www.ncbi.nlm.nih.gov / nuccore / NM_004802.3) (SEQ ID NO:8) (encoding the protein of SEQ ID NO:4)

[0272] Human ear malformation protein transcript variant 3 (www.ncbi.nlm.nih.gov / nuccore / NM_194322.2) (SEQ ID NO:9) (encoding the protein of SEQ ID NO:3)

[0273] Human ear malformation protein transcript variant 4 (www.ncbi.nlm.nih.gov / nuccore / NM_194323.2) (SEQ ID NO:10) (encoding the protein of SEQ ID NO:2)

[0274] Human ear malformation protein transcript variant 5 (www.ncbi.nlm.nih.gov / nuccore / NM_001287489.1) (SEQ ID NO:11) (encoding the protein of SEQ ID NO:5)

[0275] A non-limiting example of the genomic DNA sequence of human wild-type ototoxicity protein is SEQ ID NO:12. The exons in SEQ ID NO:12 are: nucleotide positions 5001-5206 (exon 1), nucleotide positions 25925-25983 (exon 2), nucleotide positions 35779-35867 (exon 3), nucleotide positions 44590-44689 (exon 4), nucleotide positions 47100-47281 (exon 5), nucleotide positions 59854-59927 (exon 6), nucleotide positions 61273-61399 (exon 7), nucleotide positions 61891-61945 (exon 8), nucleotide positions 68626-68757 (exon 9), and nucleotide positions 73959-74021 (exon 2). Exon 10), nucleotide positions 74404-74488 (exon 11), nucleotide positions 79066-79225 (exon 12), nucleotide positions 80051-80237 (exon 13), nucleotide positions 81107-81293 (exon 14), nucleotide positions 82690-82913 (exon 15), nucleotide positions 83388-83496 (exon 16), nucleotide positions 84046-84226 (exon 17), nucleotide positions 84315-84435 (exon 18), nucleotide positions 85950-86050 (exon 19), nucleotide positions 86193- Nucleotide positions: 86283 (exon 20), 86411-86527 (exon 21), 86656-86808 (exon 22), 87382-87571 (exon 23), 87661-87785 (exon 24), 88206-88340 (exon 25), 89025-89186 (exon 26), 89589-89708 (exon 27), 90132-90293 (exon 28), 90405-90567 (exon 29). Nucleotide positions 91050-91180 (exon 30), 92549-92578 (exon 31), 92978-93106 (exon 32), 95225-95291 (exon 33), 96198-96334 (exon 34), 96466-96600 (exon 35), 96848-96985 (exon 36), 97623-97750 (exon 37), 97857-98027 (exon 38), and 98670-98830 (exon 39).Nucleotide positions 99593-99735 (exon 40), 100128-100216 (exon 41), 101518-101616 (exon 42), 101762-102003 (exon 43), 102669-102847 (exon 44), 102952-103052 (exon 45), 103494-103691 (exon 46), 105479-106496 (exon 47), and exon 48 (a sequence beginning with CCGGCCCGAC; see also the description of this exon in Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000).

[0276] Introns are located between each pair of adjacent exons in SEQ ID NO:12, specifically at nucleotide positions 100-5001 (intron 1), 5207-25924 (intron 2), 25984-35778 (intron 3), 35868-44589 (intron 4), 44690-47099 (intron 5), 47282-59853 (intron 6), 59928-61272 (intron 7), 61400-61890 (intron 8), 61946-68625 (intron 9), and 68758-73958 (intron 10). Nucleotide positions 74022-74403 (intron 11), 74489-79065 (intron 12), 79226-80050 (intron 13), 80238-81106 (intron 14), 81294-82689 (intron 15), 82914-83387 (intron 16), 83497-84045 (intron 17), 84227-84314 (intron 18), 84436-85949 (intron 19), 86051-86192 (intron 20), and 86284. -86410 (intron 21), nucleotide positions 86528-86655 (intron 22), nucleotide positions 86809-87381 (intron 23), nucleotide positions 87572-87660 (intron 24), nucleotide positions 87786-88205 (intron 25), nucleotide positions 88341-89024 (intron 26), nucleotide positions 89187-89588 (intron 27), nucleotide positions 89709-90131 (intron 28), nucleotide positions 90294-90404 (intron 29), nucleotide positions 90568-91049 (intron 30), nucleotide positions 91181-92548 ( Intron 31), nucleotide positions 92579-92977 (intron 32), nucleotide positions 93107-95224 (intron 33), nucleotide positions 95292-96197 (intron 34), nucleotide positions 96335-96465 (intron 35), nucleotide positions 96601-96847 (intron 36), nucleotide positions 96986-97622 (intron 37), nucleotide positions 97751-97856 (intron 38), nucleotide positions 98028-98669 (intron 39), nucleotide positions 98831-99592 (intron 40), nucleotide positions 99736-100127 (intron 41).Nucleotide positions 100217-101517 (intron 42), 101617-101761 (intron 43), 102004-102668 (intron 44), 102848-102951 (intron 45), 103053-103494 (intron 46), 103692-105478 (intron 47), and 106497-108496 (intron 48).

[0277] In some embodiments, the ototoxicin gene may split into two or more segments between or within any suitable exons and / or introns, wherein each segment is included in a different vector of this disclosure. In some embodiments, the ototoxicin gene splits at exon 21, i.e., having exons 1 to (and through) 21 in the first vector and exons 22 to (and through) 48 in the second vector. In some such embodiments, the ototoxicin segments in the first and second vectors are derived from the ototoxicin cDNA sequence and lack introns, i.e., having exons 1 to (and through) 21 in the first vector and exons 22 to (and through) 48 in the second vector, each vector lacking ototoxicin introns. In some embodiments, the ototoxicin gene may split at one or more other exons and / or introns, provided that when combined with all other components of the vector, it does not exceed the packaging capacity of the vector.

[0278] Human ear malformation protein gene sequence (ncbi.nlm.nih.gov / nuccore / 224465243) (SEQ ID NO:12)

[0279] Mouse otomorphin (SEQ ID NO:13) (NCBI accession number NP_001300696.1)

[0280] Mouse otomorphin cDNA (SEQ ID NO:14) (NCBI accession number NM_001313767.1)

[0281] Mouse ototoxicity protein gene sequence (www.ncbi.nlm.nih.gov / gene / 83762) (SEQ ID NO:15) accession: NC_000071 region: complement (30367066..30462730) GPC_000000778; NCBI reference sequence: NC_000071.6

[0282] Zebrafish auricula-judae protein A gene sequence (www.ncbi.nlm.nih.gov / gene / 557476) (SEQ ID NO:16) ACCESSION: NC_007131 REGION: 31173357..31310109 GPC_000001574 NCBI reference sequence: NC_007131.7

[0283] Rhesus monkey ear malformation protein gene sequence (www.ncbi.nlm.nih.gov / gene / 696717) (SEQ ID NO:17) ACCESSION NC_027905 region: complement (26723411..26826586) GPC_000002105 NCBI reference sequence: NC_027905.1

[0284] The canine ear malformation protein gene sequence (www.ncbi.nlm.nih.gov / gene / 607961) (SEQ ID NO:18) is accessed in region NC_006599: complement (20518502..20619461) GPC_000000676. NCBI reference sequence: NC_006599.3

[0285] The chimpanzee ear malformation protein gene sequence (www.ncbi.nlm.nih.gov / gene / 459083) (SEQ ID NO:19) is mapped to region NC_006469: complement (27006052..27107747) GPC_000002338. NCBI reference sequence: NC_006469.4

[0286] Rat otomorphin (SEQ ID NO:20)

[0287] Zebrafish ear deformity protein (SEQ ID NO:21)

[0288] Bovine ear malformation protein (SEQ ID NO:22)

[0289] Baboon ear deformity protein (SEQ ID NO:23)

[0290] In some embodiments, the first vector contains the 5' portion of OTOF cDNA, for example, as shown in SEQ ID NO:94. In some embodiments, the second vector contains the 3' portion of OTOF cDNA, for example, as shown in SEQ ID NO:95.

[0291] 5'mOTOF DNA sequence (SEQ ID NO:94)

[0292]

[0293] 3'mOTOF DNA sequence (SEQ ID NO:95)

[0294]

[0295] Some embodiments of any of the compositions described herein may include a first vector comprising the coding sequence of SEQ ID NO:94 (or comprising at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:94). Some embodiments of any of the compositions described herein may include a second vector comprising the coding sequence of SEQ ID NO:95 (or comprising at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the same sequence as SEQ ID NO:95).

[0296] Some embodiments of any of the compositions described herein may include a first vector having a 5' OTOF coding region comprising exons 1 to (and through) 21 of an OTOF cDNA. Some embodiments of any of the compositions described herein may include a first vector comprising the nucleotide sequence of SEQ ID NO:101 (or a sequence identical to at least 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% of the nucleotide sequence of SEQ ID NO:101). Some embodiments of any of the compositions described herein may include a first vector comprising the same nucleotide sequence as SEQ ID NO:101 in at least 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% of the nucleotide sequence of SEQ ID NO:101, and encoding the same amino acid sequence as SEQ ID NO:101. In some embodiments, the compositions of this disclosure include a first vector comprising a codon-optimized form of SEQ ID NO:101, i.e., a nucleotide sequence encoding the same amino acid sequence as SEQ ID NO:101, but the codons have been optimized for expression in a specific cell type (e.g., mammalian cells, such as human cells). In some embodiments, the first vector does not include any other portion of the OTOF gene. In some embodiments, the first vector does not include any other portion of the OTOF cDNA.

[0297] Some embodiments of any of the compositions described herein may include a second vector having a 3' OTOF coding region comprising exons 22 to (and through) exon 48 of the OTOF cDNA. Some embodiments of any of the compositions described herein may include a second vector comprising the nucleotide sequence of SEQ ID NO:108 (or a sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:108). Some embodiments of any of the compositions described herein may include a second vector comprising at least 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% of the nucleotide sequence identical to that of SEQ ID NO:108, and encoding the same amino acid sequence as SEQ ID NO:108. In some embodiments, the compositions of this disclosure include a second vector comprising a codon-optimized form of SEQ ID NO:108, i.e., a nucleotide sequence encoding the same amino acid sequence as SEQ ID NO:108, but the codons have been optimized for expression in a specific cell type (e.g., mammalian cells, such as human cells). In some embodiments, the second vector does not include any other portion of the OTOF gene. In some embodiments, the second vector does not include any other portion of the OTOF cDNA.

[0298] carrier

[0299] This article provides material compositions and methods of use for treating diseases such as nonsyndromic hearing loss using nucleic acid therapeutic agents such as auditory polypeptide messenger RNA. Preferably, the auditory polypeptide nucleic acid is contained in a viral vector, such as an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, and a retroviral vector.

[0300] The methods described above for packaging recombinant vectors in a desired AAV capsid to produce the rAAV (or rAAV particles) of this disclosure are not intended to be limiting, and other suitable methods will be apparent to those skilled in the art.

[0301] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that, when desired, enhance the secretion of the encoded product. A large number of expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and are available for use.

[0302] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are referred to as "operably linked" when they are covalently linked in such a way that the expression or transcription of the nucleic acid sequence is influenced or controlled by the regulatory sequence. Two DNA sequences are considered operably linked if the translation of the nucleic acid sequence into a functional protein is desired, if the induction of a promoter in the 5' regulatory sequence leads to transcription of the coding sequence, and if the nature of the link between the two DNA sequences does not (1) lead to the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region will be operably linked to a nucleic acid sequence if the promoter region is able to influence the transcription of that DNA sequence, resulting in a transcript that may be translated into the desired protein or polypeptide. Similarly, two or more coding regions are operably linked when their transcription from a common promoter leads to the expression of two or more proteins already translated within a reading frame. In some embodiments, operably linked coding sequences produce fusion proteins. In some implementations, the operatively linked coding sequences produce functional RNAs (e.g., shRNAs, miRNAs, miRNA inhibitors).

[0303] For nucleic acids encoding proteins, a polyadenylated sequence is typically inserted after the transgene sequence and before the 3' AAV ITR sequence. The rAAV constructs useful in this disclosure may also contain introns, ideally located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40T intron sequence. Another possible intron sequence has the nucleotide sequence of SEQ ID NO:100. Another vector element that can be used is the internal ribosome entry site (IRES). The IRES sequence is used to generate more than one polypeptide from a single gene transcript. The IRES sequence will be used to generate proteins containing more than one polypeptide chain. The selection of these and other common vector elements is routine, and many such sequences are available [see, for example, Sambrook et al. "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and references cited therein, for example, pp. 3.18, 3.26 and 16.17, 16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some implementations, the foot-and-mouth disease virus 2A sequence is contained in a polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of the polyprotein (Ryan, MD et al., EMBO, 1994; 4:928-933; Mattion, NM et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459).The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931; and Klump, H et al., Gene Therapy, 2001; 8: 811-817).

[0304] The exact nature of the regulatory sequences required for gene expression in host cells may vary depending on species, tissue, or cell type, but generally should include, as needed, 5' non-transcriptional and 5' non-translational sequences, such as TATA boxes, capping sequences, CAAT sequences, enhancer elements, etc. In particular, such 5' non-transcriptional regulatory sequences will include promoter regions containing promoter sequences for transcriptional control of operatively linked genes. Depending on the desired outcome, the regulatory sequences may also include enhancer sequences or upstream activator sequences. Vectors disclosed herein may optionally include 5' leader or signal sequences. The selection and design of appropriate vectors is within the competence and judgment of those skilled in the art.

[0305] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, for example, Boshartet et al. (1985) Cell, 41:521-530], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter (Gilham et al., J. Gene Med. 12(2):129-136, 2010), and the EF1.alpha promoter [Invitrogen].

[0306] Inducible promoters allow for the regulation of gene expression and can be modulated by exogenously supplied compounds, environmental factors (such as temperature), or the presence of specific physiological states (e.g., acute phase, specific differentiation state of the cell, or only in replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al. (1996) Proc. Natl. Acad. Sci. USA, 93:3346-3351), the tetracycline inhibitory system (Gossen et al. Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), and the tetracycline inducible system (Gossen et al. Science, 268:1766-1769 (1995), see also Harveyet... The inducible promoters include those described in Wang et al. Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486 inducible system (Wang et al. Nat. Biotech., 15:239-243 (1997) and Wang et al. Gene Ther., 4:432-441 (1997)), and the rapamycin inducible system (Magari et al. J. Clin. Invest., 100:2865-2872 (1997)). Other types of inducible promoters that may be useful in this paper are those that are regulated by specific physiological states, such as temperature, acute phase, specific differentiation state of the...

Claims

1. Multiple recombinant adeno-associated virus (rAAV) vectors, including: a) A first rAAV vector containing the nucleic acid sequence of SEQ ID NO: 96; and b) A second rAAV vector containing the nucleic acid sequence of SEQ ID NO:

105. The first rAAV vector is encapsulated by an Anc80 capsid, and the second rAAV vector is encapsulated by an Anc80 capsid, wherein the Anc80 capsid contains the polypeptide sequence of SEQ ID NO:

109.

2. A composition comprising the various rAAV carriers of claim 1, wherein the composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

3. The composition of claim 2, wherein the composition is formulated for intracochlear administration.

4. The composition of claim 3, wherein the composition is formulated to contain a synthetic perilymph solution.

5. Use of multiple recombinant adeno-associated virus (rAAV) vectors in a method for preparing a medicament for expressing recombinant full-length otopathic protein in mammalian cells, said multiple rAAV vectors including: a) A first rAAV vector containing the nucleic acid sequence of SEQ ID NO: 96; and b) A second rAAV vector containing the nucleic acid sequence of SEQ ID NO: 105; The first rAAV vector is encapsulated by an Anc80 capsid, and the second rAAV vector is encapsulated by an Anc80 capsid, wherein the Anc80 capsid contains the polypeptide sequence of SEQ ID NO: 109; The method includes administering multiple rAAV vectors to the mammalian cells, wherein the mammalian cells have reduced, absent, or impaired expression of ototoxic proteins.

6. The use according to claim 5, wherein the mammalian cell is an inner hair cell.

7. The use as described in claim 5 or 6, wherein the mammalian cell is a human cell.

8. The use according to claim 5 or 6, wherein the mammalian cell contains a defective otopathic protein gene.

9. Use of multiple recombinant adeno-associated virus (rAAV) vectors in a method for preparing a medicament for treating hearing loss in a subject suffering from ototoxicin-related hearing loss, said multiple rAAV vectors comprising: a) A first rAAV vector containing the nucleic acid sequence of SEQ ID NO: 96; and b) A second rAAV vector containing the nucleic acid sequence of SEQ ID NO: 105; The first rAAV vector is encapsulated by an Anc80 capsid, and the second rAAV vector is encapsulated by an Anc80 capsid, wherein the Anc80 capsid contains the polypeptide sequence of SEQ ID NO: 109; The method includes administering multiple rAAV vectors to the cochlea of ​​the subject, wherein the subject has reduced, absent, or impaired expression of ototoxic proteins.

10. The use as claimed in claim 9, wherein the subject is a human.

11. The use as described in claim 9 or 10, wherein the plurality of rAAV carriers are administered as a single dose.

12. The use as described in claim 9 or 10, wherein the plurality of rAAV carriers are administered in two or more doses.

13. The use according to claim 9 or 10, wherein the subject has a defective otopathic protein gene.

14. An in vitro method for expressing recombinant full-length auricularia protein in mammalian cells, said in vitro method comprising administering a variety of recombinant adeno-associated virus (rAAV) vectors to said mammalian cells, said variety of rAAV vectors including: a) A first rAAV vector containing the nucleic acid sequence of SEQ ID NO: 96; and b) A second rAAV vector containing the nucleic acid sequence of SEQ ID NO: 105; The first rAAV vector is encapsulated by an Anc80 capsid, and the second rAAV vector is encapsulated by an Anc80 capsid, wherein the Anc80 capsid contains the polypeptide sequence of SEQ ID NO:

109.

15. The in vitro method of claim 14, wherein the mammalian cell is an inner hair cell.

16. The in vitro method of claim 14 or 15, wherein the mammalian cell is a human cell.

17. The in vitro method of claim 14 or 15, wherein the mammalian cell contains a defective otopathic protein gene.

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