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

Abstract

This invention provides drugs and methods for preventing or reversing hearing loss. [Solution] A composition comprising at least two different nucleic acid vectors, each of which comprises a coding sequence encoding a different portion of the otoferrin protein, and the use of these compositions for treating hearing loss in a subject are provided herein. This disclosure is based on the discovery that a composition comprising at least two different nucleic acid vectors, each of which comprises a coding sequence encoding a different portion of the otoferrin protein, can be used to generate a sequence encoding an active otoferrin protein (e.g., full-length otoferrin protein) in mammalian cells, thereby enabling the treatment of asymptomatic sensorineural hearing loss in a subject in need.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 979,792, filed on February 21, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. A copy of the said ASC II created on February 21, 2020, is named 2013615 - 0002_SL.txt and has a size of 1,298,432 bytes.

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

Background Art

[0004] Background of the Present Disclosure The ear is a complex organ and is classically described as including the outer ear, middle ear, inner ear, the hearing (acoustic) nerve, and the auditory system (which processes sound as it moves from the ear to the brain). In addition to detecting sound, the ear also helps maintain a sense of balance. Thus, disorders of the inner ear can cause hearing loss, tinnitus, spatial disorientation, and imbalance. Spatial disorientation is a hallucination of movement and is a major symptom of vestibular system disorders. Spatial disorientation can be caused by problems in the inner ear or the central nervous system. Common inner ear causes of spatial disorientation include vestibular neuritis (sudden unilateral vestibular loss), Ménière's disease (episodic spatial disorientation), benign paroxysmal positional vertigo (BPPV), and both ​ One example is lateral vestibular loss. A common central nervous system cause of spatial disorientation is post-concussion syndrome. These include vertigo syndrome, cervical vertigo, vestibular migraine, cerebrovascular disease, and acoustic neuroma.

[0005] Hearing loss is one of the most common sensory impairments in humans and can occur for many reasons. Some people are born with hearing loss, while others lose their hearing slowly over time. Hearing loss (presbycusis) (also written as age-related hearing loss (presbyacusis)) This is age-related hearing loss. Approximately 36 million adult Americans experience some degree of hearing loss. They have reported hearing loss in several cases, with one in three people over 60 and half of those over 85 experiencing hearing loss. I have experienced that.

[0006] Hearing loss can be the result of environmental factors, or a combination of genetic and environmental factors. Tinnitus - Auditory hallucinations in the auditory system (bell sounds, buzzer sounds, cries, humming, or beat sounds) Approximately half of all people who have ) also have a preference for certain audible frequency and volume ranges. Therefore, they have excessive sensitivity / reduced tolerance, which is hyperacusis (hyperacus). It is known as hyperacoussis (also written as "hearing hypersensitivity"). Ams syndrome (also known as Williams-Buren syndrome) is caused by LIM kinase 1 ( 7q11.23 contains approximately 26 genes, including the gene encoding LIMK1. This is a multi-system failure caused by a hemijunctional deletion in a 1.6 Mb region. Individuals with Iliams syndrome exhibit hyperacusis and progressive hearing loss. The early onset of the disease suggests that it may be associated with one of the genes that are deleted.

[0007] Environmental causes of hearing loss include certain drugs, certain prenatal or postnatal infections, and This includes exposure to loud noises over a long period of time. Hearing loss can be caused by noise, ototoxic agents, Caused by age-related hearing loss, disease, infection, or cancer affecting a specific part of the ear. Ischemic injury can lead to hearing loss through pathophysiological mechanisms initiated by it. It can happen. Another example is autoimmune inner ear disease (AIED), in which the body's immune system attacks the inner ear. A rapidly progressing bilateral disease that occurs when cells are mistakenly attacked as if they were viruses or bacteria. It is characterized by sensorineural hearing loss.

[0008] Approximately 1.5 children out of every 1,000 are born with severe hearing loss, and 1, Two to three out of every 1,000 children are born with partial hearing loss (Smith et al.). al., 2005, Lancet 365:879-890). Half of these cases More often, the cause is attributed to genetic principles (Di Domenico, et al., 20 11, J. Cell. Physiol. 226:2494-2499).

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

[0010] Most cases of genetic hearing loss (70-80 percent) are asymptomatic. The remaining cases are caused by specific genetic syndromes.

[0011] Hearing loss can be conductive (originating from the external auditory canal or middle ear) or sensorineural. It can be (originating from the inner ear or auditory nerve) or a mixed type. Many forms of asymptomatic hearing loss. The condition is associated with permanent hearing loss caused by damage to the structures within the inner ear. (Sensorineural hearing loss). The majority of human sensorineural hearing loss is due to the hair cells of the organ of Corti in the cochlea. It is caused by an abnormality in the eighth cranial nerve (vestibulocochlear nerve) or the auditory part of the brain. There are also extremely rare sensorineural hearing impairments. The rarest of these types of hearing loss In some cases, only the auditory center of the brain is affected. In this situation, sounds are heard at a normal threshold. It can be heard, but the quality of the perceived sound is so poor that it is impossible to understand the speech. Sensorineural hearing loss can occur. However, most sensorineural hearing loss is due to a failure of hair cell function. It is caused by a condition where hair cells are abnormal at birth or damaged during the individual's lifespan. This can occur. Exogenous injuries such as noise-induced trauma and infections, as well as cochlear anatomy. or essential mutations such as congenital mutations in genes that play an important role in physiology Both abnormal and abnormal conditions exist.

[0012] Hearing loss resulting from changes in the middle ear is called conductive hearing loss. Some forms of hearing loss involve changes in both the inner and middle ear and are called mixed hearing loss. Hearing loss that exists before a child learns to speak is classified as pre-language acquisition or congenital. They are classified as follows. Hearing loss that occurs after the development of speech is classified as post-language acquisition. Autosomal recessive loci can cause severe to severe hearing loss before language acquisition.

[0013] Non-symptomatic hearing loss can have different genetic patterns and can occur at any age. The types of hearing loss are named according to their genetic patterns. The autosomal dominant form is called DFN. It is denoted as A, and the autosomal recessive form is DFNB, while the X-linked form is DFN. Each type is also numbered in the order in which it is described. For example, DFNA1 It was initially described as autosomal dominant non-symptomatic hearing loss.

[0014] 75 to 80 percent of cases are inherited in an autosomal recessive pattern. This means that both copies of the gene in each cell have mutations. Typically, each parent of an individual with autosomal recessive hearing loss has one of the mutated genes. He possesses a copy of it, but does not suffer from this form of hearing loss.

[0015] Another 20-25 percent of non-symptomatic hearing loss cases are autosomal dominant. Therefore, this is because one copy of the altered gene in each cell causes hearing loss. This means that it is sufficient to cause hearing loss. People with autosomal dominant hearing loss experience hearing loss. Inheriting a modified copy of the gene from one of the parents who possesses it is more likely There are many.

[0016] One to two percent of the cases show an X-linked inheritance pattern, which is The mutated gene involved in this condition is on the X chromosome (one of the two sex chromosomes). This means that it is located in [location]. Men with X-linked non-symptomatic hearing loss have the same gene mutation. Women who inherit a different copy of the gene experience earlier and more severe hearing loss throughout their lives. There is a tendency to develop the disease. A characteristic of X-linked inheritance is that the father is unable to pass on the X-linked trait to his son. That's a good thing.

[0017] Mitochondrial non-symptomatic hearing loss caused by changes in mitochondrial DNA This occurs in less than 1 percent of cases in the United States. Modified mitochondrial DNA A is inherited from the mother to all of her sons and daughters. This type of hearing loss is inherited from the father or It is not hereditary.

[0018] Characterized by normal outer hair cell function and abnormal auditory brainstem responses or their absence. Auditory neuropathy spectrum disorder (ANSD), a type of hearing impairment, affects infants and young children. It is one of the most common diseases that causes hearing and speech transmission barriers in young children. Yes. Approximately 10 percent of children with permanent hearing loss may have ANSD. The OTOF gene was the first gene identified for autosomal recessive non-syndromic ANSD. Therefore, mutations in OTOF are autosomal recessive non-syndromic (no (Nsydromic) accounts for approximately 5% of all cases of hearing loss (Rodri guez-Ballesteros et al. 2008 Human Mut 2 9(6):823-831).

[0019] The causes of non-symptomatic hearing loss are complex. Researchers suggest that non-symptomatic hearing loss can be altered. They have identified more than 30 genes associated with this, but how many of these genes are there? This is not fully characterized. Different mutations in the same gene are different species. It may be associated with a type of hearing loss, and some genes are associated with symptomatic and asymptomatic hearing loss. It is associated with both types of hearing loss.

[0020] For example, genes associated with non-symptomatic hearing loss include, but are not limited to, ATP2 B2, ACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5 , DFNB31, DFNB59, ESPN, EYA4, GJB3, KCNQ4, LHFP L5, MYO1A, MYO15A, MYO6, MYO7A, OTOF, PCDH15, S LC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIO Examples include BP, USH1C, and WFS1.

[0021] The most common causes of hearing loss are asymptomatic hearing loss and hearing impairment, DFNB1 (GJ B2-related DFNB1 non-syndromic hearing loss and hearing impairment, autosomal recessive hearing loss 1, sensory neurological non-syndromic hearing loss This is also called symptomatic inferior hearing loss (DFNB1). Non-symptomatic hearing loss and hearing impairment (DFNB1) is It is characterized by congenital, non-progressive, and mild to severe sensorineural hearing impairment. JB2 (encodes protein connexin 26) and GJB6 (encodes connexin 30) It is caused by a mutation in (the gene). The diagnosis of DFNB1 is GJB2 Upstream of the gap junction beta-2 protein (connexin 26) Molecular genetic testing to identify mutations in cis regulatory elements that cause hearing loss It exists. Molecular genetic testing for GJB2 reveals that these genes have mutations that cause hearing loss. It detects abnormalities with over 99% accuracy. Unlike some other forms of hearing loss, DFNB1 non- Symptomatic hearing loss and hearing impairment do not affect balance or movement. Degree of hearing loss It is difficult to predict based on which gene mutations are present. Even if a member has DFNB1 non-symptomatic hearing loss and hearing impairment, hearing The degree of loss can vary among them.

[0022] A mutation in the genes encoding connexin 26 (Cx26) and / or Cx30 The mutation is associated with approximately half of all cases of human autosomal non-syndromic pre-language acquisition hearing loss. They are related. Cx26 and Cx30 are two major Cx eyes found in the cochlea. They are homoforms, and they are co-assembled into hybrids (heteromers and heteromers). It forms a gap junction (GJ) of the oval type (Ahmad, et al.). , Proc. Natl. Acad. Sci., 2007, 104(4):1 337-1341). Non-symptomatic hearing loss and hearing impairment, DFNA3, are proteins. Change either Connexin 26 (Cx26) or Connexin 30 (Cx30). Dominant-negative pathogenicity variants in the GJB2 or GJB6 genes This can cause mild to severe, progressive high-frequency sensorineural hearing loss, either before or after language acquisition. Therefore, it can be characterized.

[0023] OTOF-related hearing loss (DFNB9 non-symptomatic hearing loss) is non-symptomatic hearing loss that occurs before language acquisition. , and two less common, temperature-sensitive asymptomatic auditory neuropathy (TS-NSAN). It is characterized by two phenotypes.

[0024] Another form of progressive hearing impairment is associated with mutations in the otoferrin gene. (For example, the I1573T mutation or the P1987R mutation, and / or E (1700Q mutation) or not temperature sensitive.

[0025] Pendred syndrome / DFNB4 (hearing loss with goiter) is an autosomal recessive genetic disorder. It is a harmful condition and accounts for 7.5% of all cases of congenital hearing loss. Pendred syndrome is PDS, located on the long arm of chromosome 7 (7q31), encodes the pendrin protein. Genes (DFNB4, EVA, PDS, TDH2B, and solute transporter family 26) This mutation is associated with member 4 (also known as SLC26A4). Mutations in the gene are also a cause of hearing loss, including vestibular aqueduct dilation syndrome (E). It can also cause VA or EVAS, and certain mutations can cause EVAS. The higher the level, the more the other factors are associated with Pendred syndrome. (Aza iez, et al. (December 2007), Hum. Genet. 122 (5): 451-7).

[0026] The transmembrane protease serine 3 is produced by the TMPRSS3 gene (DFNB10, DFNB8 It is an enzyme encoded by ECHOS1 and TADG12 (also known as TADG12). This gene was identified through its association with both congenital and childhood-onset autosomal recessive hearing loss. The mutation in TMPRSS3 is associated with hearing impairment that occurs after language acquisition and progresses rapidly. It is related to the protein encoded by the TMPRSS3 gene, serine pro Thease domain, transmembrane domain, LDL receptor-like domain, and scavenger receptor It contains a cysteine-rich domain. Serine proteases are various biological agents. It is publicly known that Seth is involved, and its dysfunction causes human diseases and disorders. This is often the case. This gene is expressed in the cochlea and many other tissues during the fetal stage, and internally It is thought to be involved in the development and maintenance of the contents of the ear, or the perilymph and endolymph. This gene has also been identified as a tumor-associated gene that is overexpressed in ovarian tumors. Four alternative splicing variants are described, two of which are identical. To code the product.

[0027] DFN3 hearing loss is caused by a mutation in the POU3F4 gene located on the X chromosome. It is caused by one of the small bones in the middle ear. The stapes bone cannot move normally, which interferes with hearing. This characteristic of DFN3 A characteristic sign is called stapes fixation. At least four other regions of the X chromosome are affected by hearing loss. Although involved in the loss, the genes involved have not been identified. Pejvakin or DFNB59 (hearing loss, autosomal recessive 59), also known as PJVK, is a vertebrate that It is a 352-amino acid protein belonging to the gasdermin family. (DFNB59) This is human chromosome 2q31, which is essential for the proper function of auditory pathway neurons and outer hair cell function. Encoded by the gene that maps to 2. Mutations in DFNB59 result in hearing The absence or severe abnormality of the brainstem response, while otoacoustic emissions are normal. This is a form of sensorineural hearing impairment characterized by the autosomal recessive type 5 non-symptomatic sensorineural hearing loss. It is thought to cause 9 (auditory neuropathy or auditory dyssynchrony). DF NB59 shares significant similarities with DFNA5, suggesting that these genes share a common origin. This indicates sharing.

[0028] Alport syndrome involves COL4A3, COL4A4, and other proteins involved in collagen biosynthesis. This is caused by mutations in the COL4A5 gene. Mutations in any of these areas affect important structural components of the basement membrane in the kidney, inner ear, and eye. It prevents the proper production or assembly of the type IV collagen network. One of the criteria used in diagnosing rheumatoid arthritis is that the frequency range is between 2000 and 8000 Hz. This is bilateral sensorineural hearing loss. The hearing loss develops gradually and is not present in early infancy. Generally, it appears before the age of 30.

[0029] Deficiencies in ion channels are associated with hearing loss, and DFNA2 non-symptomatic hearing loss. The loss is inherited as an autosomal dominant mutation in the KCNQ4 gene, which is voltage-dependent. Potassium voltage-gated channel, also known as the saturating potassium channel subunit Kv7.4 This codes for member 4 of the Rusub family KQT. DFNA2 non-symptomatic hearing loss is a Symmetrical, primarily high-frequency sensorineural hearing loss (SNHL) that progresses across a wide range of frequencies. It is characterized by the following: In young people, hearing loss is mild at low frequencies, and at high frequencies. In terms of wavenumber, the degree is moderate, and in elderly individuals, hearing loss is moderate at low frequencies. Furthermore, at high frequencies, hearing impairment ranges from severe to severe. Hearing impairment is a common symptom in school-aged children. Although often detected during force assessment, hearing impairment can occur from birth, particularly at high frequencies. It is highly likely that most affected individuals first experience sound amplification between the ages of 10 and 40. Hearing aids are needed to assist with hearing loss. All individuals with DFNA2 hearing loss by age 70. These individuals have severe to serious hearing impairment.

[0030] Mutations in the KCNE1 and KCNQ1 genes are associated with severe bilateral hearing loss. This can cause Jerbel-Langenilsen syndrome (JLNS), a type of long QT syndrome. This condition is an autosomal recessive disorder that affects an estimated 1.6 to 6 out of every 1 million children. It is harmful and is involved in less than 10 percent of all cases of QT prolongation syndrome. In Sweden and the West, the number of cases is remarkably high, up to 1:200,000. The proteins produced by the KCNE1 and KCNQ1 genes function together. They form potassium channels that transport positively charged potassium ions from the cell. The movement of potassium ions through the channels is crucial for maintaining the normal function of the inner ear and myocardium. It is essential.

[0031] Characterized by intellectual disability, ataxia, seizures, hearing loss, and renal salt loss, E AST / SeSAME syndrome is caused by a prolapse in the KCNJ10 inward rectifying potassium channel. It is thought to be caused by natural mutation.

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

[0033] Mutations in the ATP6V1B1 gene, which are expressed in both the kidney and the cochlea, It is associated with distal tubular acidosis (DRTA). It has autosomal recessive DRTA. A significant percentage of children experience progressive bilateral sensorineural hearing loss. It was served.

[0034] Usher syndrome (Hargren's syndrome, Usher-Hargren syndrome, retinitis pigmentosa) -Auditory impairment syndrome (also known as retinal malnutrition auditory impairment syndrome) is at least A rare condition caused by a mutation in any one of the 10 genes. It is a disability that causes a combination of hearing loss and gradual vision impairment, and is the main cause of deafblindness. Hearing loss is caused by a damaged inner ear, while vision loss is caused by changes in retinal cells. It is caused by retinitis pigmentosa (RP), which is a sexually transmitted disease. Usher syndrome is classified into type I, type II, and It has three clinical subtypes, designated as type III. Subjects with type I Usher are He was born with severe hearing loss, began losing his sight within the first 10 years of life, and had problems with the vestibular system. This can cause children to be late in starting to walk and to have difficulty with balance. (Type II Usher syndrome) The subjects are those who do not have congenital hearing loss, but who have hearing loss, and whose sense of balance is clearly impaired. There seemed to be no major problems, and although he later began to lose his eyesight (by the age of 20), he was still able to see in middle age. Some individuals may retain some degree of vision. Individuals with Usher syndrome type III are born with this condition. While not hearing loss, the individual experiences a gradual loss of hearing and vision, and has difficulty with balance. They may not always be present.

[0035] A mouse model of congenital hearing loss uses vesicular glutamate transporter-3 (VGLUT3). It is produced by causing a null mutation in the gene that codes for ). In recent years, In VGLUT3 knockout mice, hearing was improved using virus-mediated gene therapy. (Akil, et al., 2012, Neuron 75:283-) 293).

[0036] Math1 (mouse homolog of ATH1) is an atonal version of HATH1 or (ATOH1). It is also known as a Drosophila homolog and is essential for the development of hair cells in the inner ear. Therefore, Math1 is a “hair cell-promoting gene” in the developing sensory epithelium. It was proposed that it would function as follows (Bermingham et al., 1999, Sc (Ethnicity 284:1837-1841). Several studies now suggest that the virus is not transmitted by The delivery of Math1 leads to the regeneration of hair cells in the traumatized mouse cochlea. Furthermore, it shows improvements in both hearing and balance (Baker et al., 20 09, Adv. Otorhinolaryngol. 66:52-63, Huss eman and Raphael, 2009, Adv. Yngol. 66:37-51, Izumikawa et al., 2008, Hear. Res. 240:52-56, Kawamoto et al., 20 03, J. Neurosci. 23:4395-4400, Praetorius et al., 2010, Acta Otolaryngol. 130:215 -222, Staecker et al., 2007, Otol. (ol. 28:223-231).

[0037] Mutations in the WFS1 gene account for 90 percent of cases of Wolfram syndrome type 1. Wolfram syndrome, which causes more problems than other conditions, is caused by a deficiency in the hormone insulin. This results in high blood sugar levels (diabetes melanin) and degeneration of nerves that carry information from the eyes to the brain. It is most often characterized by progressive vision loss (optic nerve atrophy), It is a condition that affects many of the systems. However, Wolfram syndrome These individuals may experience excessive urine excretion (diabetes insipidus) and hearing loss due to changes in the inner ear. Loss of hearing (sensorineural hearing loss), ureteral problems, low levels of testosterone, a sex hormone in men. Hypogonadism (low gonadal function), or pituitary dysfunction resulting in neurological or psychiatric disorders. They also often have it. About 65 percent of people with Wolfram syndrome feel Hearing loss is caused by sound, and its severity ranges from hearing loss that begins at birth to hearing loss that begins in adolescence and worsens over time. It can progress to mild hearing loss. Furthermore, among people with Wolfram syndrome... Approximately 60 percent of these cases involve neurological or psychiatric disorders, most commonly occurring in early adulthood. Problems with balance and coordination (ataxia) begin during this period.

[0038] The WFS1 gene is thought to regulate the amount of calcium in cells. It codes for a protein called WFS1. Wolfram syndrome is caused by an abnormality in the WFS1 gene. When caused by a natural mutation, it is inherited in an autosomal recessive pattern, and Wolff Lamin proteins have reduced or absent function. As a result, intracellular The calcium levels are not regulated, and the endoplasmic reticulum does not function properly. Without wolframin, cells trigger their own cell death (apoptosis). The death of cells in the pancreas, especially insulin-producing cells (beta cells), is a major factor in the Wolf Diabetes mellitus develops in people with Flamm's syndrome. (Cellular stage along the optic nerve) This loss ultimately leads to blindness in the affected individual. Cell death in this condition can cause various signs and symptoms of Wolfram syndrome type 1. It is highly likely.

[0039] Mutations in the mitochondrial genes MT-TS1 and MT-RNR1 are non-symptomatic. It has been found to increase the risk of developing non-symptomatic mitochondrial hearing loss. Hearing loss and hearing impairment are characterized by moderate to severe hearing loss. MT-TS The pathogenic variant in 1 is usually associated with the childhood onset of sensorineural hearing loss. The pathogenic variant in MT-RNR1 is called an aminoglycoside (acoustic toxicity). Hearing loss and / or late-onset sensorineural hearing loss that can occur upon exposure to certain antibiotics. Although associated with a predisposition to sexual hearing loss, it involves a mutation in the MT-RNR1 gene. Some people develop hearing loss even without exposure to these antibiotics. Hearing loss associated with aminoglycoside ototoxicity is bilateral and can range from severe to severe. Any amount (even a single dose) of an aminoglycoside antibiotic, such as gentamicin, After administration of bramycin, amikacin, kanamycin, or streptomycin, for several days It will occur within a few weeks.

[0040] Treatment for hearing loss currently involves hearing amplification for mild to severe hearing loss, and for severe hearing loss... For serious loss, the treatment consists of cochlear implant surgery (Kral and O'Donog hue, 2010, N. Engl. J. Med. 363:1438-145 0). To date, most research in this field has focused on age-related hearing loss, noise-induced injury, infections, Cochlear hair cell regeneration applicable to the most common forms of hearing loss, including ototoxicity. We have focused on this.

[0041] In animal models of cochlear ischemia, ischemic injury is associated with insulin-like growth factor (IGF-1). AM-111 (apoptosis inhibitor), edaravone (free radical scavenger) Ginsenoside RB1 (Kappo), glial cell-derived neurotrophic factor (GDNF), B DNF, CNTF, SOD1, SOD2, necrostatin-1, DFNA5, and MS It can be prevented by compounds such as RB3. However, the combination of substances is simple It appears that it may be more effective than one compound (for example, oxidative stress, exotoxicity, blood flow, etc.) Lucium and stimulation overload, apoptotic pathways, neurotrophic or hormonal regulatory mechanisms Modulating complementary therapies).

[0042] Inhibition of JNK-1-induced apoptosis (mitochondrial-induced) is dominant. This is prevented by compounds such as the negative JNK-1 and the d-stereoisomer JNK-1. (Mol. Pharmacol. 2007 March; 71(3):65) 4-66. This content is incorporated herein by reference in its entirety.

[0043] There remains a long-standing need for drugs and methods to prevent or reverse hearing loss. . [Prior art documents] [Non-patent literature]

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

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

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

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

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

[0049] [Figure 5] Figure 5 is an exemplary graph showing the ABR threshold (dB SPL) against frequency (in kHz) in response to a pure tone or click stimulus. The minimum sound pressure level (SPL) at which a wave can be detected in response to a pure tone or click stimulus is shown for individual Otof- / - animals expressing otoferrin mediated by dual AAV (white circles represent individual animals, and circles filled with diagonal lines represent the average threshold across all animals), untransduced Otof- / - animals (black circles), and Otofwt / wt animals transduced with dual AAV and possessing eGFP (circles filled with grid patterns).

[0050] [Figure 6] Figure 6 is a graph showing the transduction rates of IHC along the cochlea (e.g., whole corticotropy, apical rotation, and mesobasal / basal rotation) as determined by immunohistochemistry using two different antibodies (one bound to the N-terminal otoferrin fragment and the other to the very C-terminal portion of otoferrin).

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

[0052] [Figure 8] Figure 8 is a graph showing the Ca2+ current in response to depolarization stimulation for Otof- / - IHC (white; n=10), Otof- / - IHC after otoferrin expression mediated by dual AAV (shaded; n=8), and wild-type IHC from control animals adapted to the background (black; n=6).

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

[0054] [Figure 10]Figure 10 is a representative graph showing Qreal against the duration (ms) of depolarization in Otof- / - IHC (white diamonds), Otof- / - mice transduced by dual AAV (black circles), and wild-type controls adapted to the background (black diamonds).

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

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

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

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

[0059] [Figure 15] Figure 15 is a graph showing the IHC (medium shade; thick line) of Otof- / - mice transduced using the two vectors shown in Figure 2, the IHC (dark; medium thick line) of wild-type mice, and the plasma membrane capacitance (ΔCm) as a function of the duration of depolarization in the IHC of Otof- / - mice.

[0060] [Figure 16] Figure 16 is a representative graph showing the Qreal against the duration (ms) of depolarization in Otof- / - IHC (white diamonds), Otof- / - mice transduced using the two vectors shown in Figure 2 (gray diamonds), and wild-type control IHC adapted to the background (black diamonds).

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

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

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

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

[0065] [Figure 21] Figure 21 is a representative schematic diagram of a part of pAKOS102 (sequence number 43).

[0066] [Figure 22] Figure 22 is a representative schematic diagram of a part of pAKOS103 (sequence number 44).

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

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

[0069] [Figure 25] Figure 25 is a representative schematic diagram of a part of pAKOS104-DHFR (Sequence ID 46).

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

[0071] [Figure 27] Figure 27 is a representative schematic diagram of a part of pAKOS105 (sequence number 47).

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

[0073] [Figure 29] Figure 29 is a representative schematic diagram of a portion of pAKOS105_GFP (sequence number 48).

[0074] [Figure 30] Figure 30 is a representative schematic diagram of a part of pAKOS106 (sequence number 49).

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

[0076] [Figure 32] Figure 32 is a representative schematic diagram of a part of pAKOS107 (sequence number 50).

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

[0078] [Figure 34] Figure 34 is a representative schematic diagram of a part of pAKOS108 (sequence number 51).

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

[0080] [Figure 36]Figure 36 is a representative schematic diagram of a part of pAKOS109 (sequence number 52).

[0081] [Figure 37] Figure 37 is a representative schematic diagram of Factor VIII, Staffor (Sequence IDs 54-57).

[0082] [Figure 38-1] Figure 38 is a representative schematic diagram of p109 (sequence number 84). [Figure 38-2] Figure 38 is a representative schematic diagram of p109 (sequence number 84).

[0083] [Figure 39-1] Figure 39 is a representative schematic diagram of p105 (sequence number 85). [Figure 39-2] Figure 39 is a representative schematic diagram of p105 (sequence number 85).

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

[0085] [Figure 41] Figure 41 is a representative schematic diagram of page 108.

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

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

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

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

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

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

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

[0093] [Figure 49] Figure 49 shows immunoblots illustrating the expression of full-length human otoferrin in HEK293FT cells transfected with the various plasmid pairs shown.

[0094] [Figure 50] Figure 50 is a table showing the quantification of full-length human otoferrin expression from three replicas of the experiment described in Figure 49.

[0095] [Figure 51] Figure 51 is a graph of click ABR thresholds in wild-type mice not treated with a vector, or Otof- / - mice not treated with a vector or treated with dual AAV Anc80.hOtof vectors (p105 vector and p109 vector). Hearing was measured in Otof- / - mice administered with dual AAV Anc80.hOtof vectors (p105 vector and p109 vector) 26-28 days and 91 days after treatment.

[0096] [Figure 52]Figure 52 is a graph of burst sound ABR thresholds in wild-type mice not treated with a vector, or Otof- / - mice that were not treated with a vector or were treated with the dual AAV Anc80.hOtof vector (p105 vector and p109 vector). Hearing was measured in Otof- / - mice administered with the DualAAV Anc80.hOtof vector (p105 vector and p109 vector) 26-28 days and 91 days after treatment.

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

[0098] [Figure 54-1] Figure 54 is a representative schematic diagram of a part of pAAV-SA-3'mOTOF.WPRE (Sequence ID 88). [Figure 54-2] Figure 54 is a representative schematic diagram of a part of pAAV-SA-3'mOTOF.WPRE (Sequence ID 88).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0113] [Figure 69]Figure 69 is a representative immunoblot showing the expression of full-length human otoferrin in HEK293FT cells transfected with the DNA transfection reagent jetPRIME® along with 600 ng of each of the various plasmids shown. Lane 1 contained the protein ladder before staining. Lane 2 contained protein samples from HEK293FT cells transfected with vector pAKOS104 (shown in Figures 24 and 59) and vector pAKOS105 (shown in Figures 27, 28, and 39). Lane 3 contained protein samples from HEK293FT cells transfected with vector pAKOS108 (shown in Figures 34, 35, 41, and 57) and vector pAKOS105 (shown in Figures 27, 28, and 39). Lane 4 contained protein samples of HEK293FT cells transfected with vector pAKOS109 (shown in Figures 36 and 38) and vector pAKOS105 (shown in Figures 27, 28, and 39). Lane 5 contained protein samples of HEK293FT cells transfected with vector pAAV-HBA-5'hOTOFcodop-SD-AK (shown in Figure 60) and vector pAAV-AK-SA-3'OTOFcodop (shown in Figure 68). Lane 6 contained protein samples of HEK293FT cells transfected with vector pAAV-CMV-5'hOTOFcodop-SD-AK (shown in Figure 63) and vector pAAV-AK-SA-3'OTOFcodop (shown in Figure 68). Lane 7 contained protein samples from HEK293FT cells transfected with the vectors pAAV_CBA-5'hOTOFcodop-SD-AK (shown in Figure 64) and pAAV-AK-SA-3'OTOFcodop (shown in Figure 68). Lane 8 contained protein samples from HEK293FT cells transfected with the vectors pAKOS102 (shown in Figure 21) and pAKOS103 (shown in Figures 22, 23, and 66).Lane 9 contained protein samples of HEK293FT cells transfected with the CBA.TS vector and vector pAKOS103 (shown in Figures 22, 23, and 66). Lane 10 contained protein samples of HEK293FT cells transfected with vector pAKOS106 (shown in Figures 30, 31, and 58) and vector pAKOS107 (shown in Figures 32, 33, and 67). 96 hours after transfection, cells were harvested, lysed with RIPA buffer, analyzed in a 4–12% Bolt protein gel, and then transferred to a nitrocellulose membrane. Human otoferrin was detected using an anti-OTOF polyclonal antibody (Thermo PA5-52935). Human beta-actin was used as the primary antibody for internal loading control between lanes. The experiment was repeated in triplicate. Relative quantitative measurements for each experiment, along with mean measurements and standard deviation (STDEV), are provided by immunoblotting.

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

[0115] [Figure 71] A set of immunohistochemical images of one organ of Corti from a 17-day-old Otof- / - mouse after unilateral intracochlear administration of a dual AAV vector (p105 vector and 109 vector) expressing CBA.hOTOF(AK). The ipsilateral cochlea was dissected and protein expression was analyzed using immunohistochemistry in three different frequency regions (basal-apical).

[0116] [Figure 72]Figure 72 is a graph showing the percentage of N- and C-terminal otoferrin-labeled inner hair cells (IHCs) in CD1B6F1-Otof- / - mice (ages P18-P30) injected with dual AAV-TS (n=10 mice) and dual AAV-Hyb (n=9 mice). Individual animals are indicated by white symbols. Data are presented as mean ± standard error (sem) of the mean, with ns P>0.05;*P≦0.05;**P≦0.01;***P≦0.001, Wilcoxon matched pair signed-rank test, and Welch-corrected unpaired t-test.

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

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

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

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

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

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

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

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

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

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

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

[0128] [Figure 84] Figure 84 shows a perspective view of a curved needle subassembly connected to the distal end of a device according to an aspect of the present disclosure. [Overview of the Initiative] [Means for solving the problem]

[0129] overview This disclosure relates to a composition comprising at least two different nucleic acid vectors, wherein at least two Each of the different vectors encodes a different part of the otoferrin protein. Using a composition containing a sequence, active otoferrin protein in mammalian cells (For example, a sequence encoding the full-length otoferrin protein) is generated, thereby, non This means that treatment for symptomatic sensorineural hearing loss can be provided to those who need it. Based on discovery.

[0130] A composition comprising at least two different nucleic acid vectors, wherein at least two different Each vector contains a coding sequence that encodes a different part of the otoferrin protein. Each of the parts containing and encoding has a length of at least 30 amino acid residues. Each amino acid sequence of the encoded portion is, as needed, different from the encoded portion. The amino acid sequence may partially overlap with that of the vector, and at least two different vectors may be used. None of these single vectors encode the full-length otoferrin protein, At least one of the coding sequences is located in two adjacent exotypes of the otoferrin genomic DNA. It contains a nucleotide sequence that spans a nucleotide, and the intron sequence between two adjacent exons is It is lacking, and when introduced into mammalian cells, at least two different vectors are con Catemer formation or homologous recombination occurs, thereby transforming the full-length otoferrin protein. A composition for forming recombinant nucleic acids that encode a substance is provided herein. In some embodiments of the compositions described herein, at least two Each of the different vectors is a plasmid, transposon, cosmid, artificial chromosome, or a viral vector. Any part of any of the compositions described herein. In the embodiment, each of at least two different vectors is a human artificial chromosome (HAC). ), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC) is used in any of the embodiments of the compositions described herein. Each of at least two different vectors is an adeno-associated virus (AAV) vector. - Adenovirus vectors, lentivirus vectors, or retrovirus vectors A viral vector selected from any of the compositions described herein. In some embodiments, each of at least two different vectors is an AAV vector That is the case.

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

[0132] In some embodiments of any of the compositions described herein, the vector contains two different vectors, each of which contains a different segment of an intron, and the intron contains the nucleotide sequence of an intron present in otoferlin genomic DNA, and the two different segments have at least 100 nucleotides overlapping within the sequence. In some embodiments of any of the compositions described herein, the two different [[ID=2X]] segments have from about 100 nucleotides to about 800 nucleotides overlapping within the sequence. In some embodiments of any of the compositions described herein, each nucleotide sequence of at least two different vectors is from about 500 nucleotides to about 10,000 nucleotides in length. In some embodiments of any of the compositions described herein, each nucleotide sequence of at least two different vectors is from 500 nucleotides to 5,000 nucleotides in length. [[ID=X2]]

[0133] ​​​​​​In some embodiments of the compositions described herein, the composition The number of different vectors is two. Any one of the compositions described herein In one embodiment, the first of two different vectors is otoferrin protein Includes a coding sequence that codes for the N-terminal portion of the substance. Among the compositions described herein In some embodiments, the N-terminal portion of the otoferrin protein consists of 30 amino acids. The length of an acid is approximately 1600 amino acids. Any of the compositions described herein. In some embodiments, the N-terminal portion of the otoferrin protein consists of 200 amino acids. This is the length of 1500 amino acids. Any one of the compositions described herein. In one embodiment, the first vector is one of the promoter and the Kozak sequence. or further including both. Implementations of any part of any of the compositions described herein. In this state, the first vector is an inducible promoter, a constitutive promoter, or tissue-specific It is a promoter, and includes promoters.

[0134] In some embodiments of the compositions described herein, two vectors One of them contains (or has at least 80% of) SEQ ID NO: 39 At least 82%, at least 84%, at least 86%, at least 88%, and at least 90%, at least 92%, at least 94%, at least 96%, at least 98% (or containing sequences that are at least 99% identical), the second of the two vectors This includes (or at least 80% of) the sequence number 40, 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 (Containing sequences that are at least 99% identical). Any of the compositions described herein. In some embodiments, one of the two vectors includes sequence number 41 (or For sequence number 41, at least 80%, at least 82%, at least 84%, and less 86%, at least 88%, at least 90%, at least 92%, at least 94% Sequences that are identical by at least 96%, at least 98%, or at least 99% (including), the second of the two vectors includes sequence number 42 (or sequence number 4 2 to at least 80%, at least 82%, at least 84%, at least 86% , at least 88%, at least 90%, at least 92%, at least 94%, less (Containing sequences that are 96%, at least 98%, or at least 99% identical.) In some embodiments of the compositions described in the specification, two vectors One of these includes (or contains at least 80% of) the sequence number 84. At least 82%, at least 84%, at least 86%, at least 88%, at least 9 0%, at least 92%, at least 94%, at least 96%, at least 98%, also (or containing sequences that are at least 99% identical), the second of the two vectors is , including sequence number 85 (or at least 80% of sequence number 85, at least 82 %, at least 84%, at least 86%, at least 88%, at least 90%, less At least 92%, at least 94%, at least 96%, at least 98%, or less (Including sequences that are 99% identical).

[0135] In some embodiments of the compositions described herein, at least two One of the different vectors contains a sequence encoding the NTF3 protein.

[0136] In some embodiments of the compositions described herein, NTF3 tamper The sequence encoding the substance is at least 80%, at least 82%, for sequence number 78. At least 84%, at least 86%, at least 88%, at least 90%, and 92%, at least 94%, at least 96%, at least 98%, or at least They are 99% identical.

[0137] In some embodiments of the compositions described herein, the first vector It further includes a coding sequence that encodes a destabilization domain, and the destabilization domain is Oto It is located at 3' relative to the coding sequence that encodes the N-terminal portion of the ferrin protein.

[0138] In some embodiments of the compositions described herein, Otoferinta The coding sequence that encodes the N-terminal portion of the protein is an isophon of the human otoferrin gene. Includes exons 1-21 of 5.

[0139] In some embodiments of the compositions described herein, two different types of ve The second of the two codes encodes the C-terminal portion of the otoferrin protein. Includes an array. In some embodiments of the compositions described herein, oto The C-terminal portion of ferrin protein is between 30 and 1600 amino acids in length. Yes. In some embodiments of the compositions described herein, Otofeli The C-terminus of a protein is typically between 200 and 1500 amino acids long. In some embodiments of the compositions described herein, a second vector It further comprises a poly(dA) signal sequence. Any of the compositions described herein In some embodiments, the coding code that encodes the C-terminal portion of the otoferrin protein The column contains exons 22-48 of isoform 5 of the human otoferrin gene. In some embodiments of the compositions described in this book, the second vector is mR Further comprising sequences for NA stabilization. Any of the compositions described herein. Some embodiments further include pharmaceutically acceptable excipients.

[0140] A kit containing any of the compositions described herein is also described herein. Provided. Any of the embodiments of any of the kits described herein are compositions Further includes a pre-filled syringe containing [the specified substance].

[0141] A therapeutically effective amount of any of the compositions described herein is introduced into the cochlea of ​​a mammal. Methods including the step of doing the following are also provided herein. In some embodiments of the product, the mammal is a human. In some embodiments of the compositions, mammals are deficient in Otoferii It has been previously identified as possessing the n gene.

[0142] Active otoferrin proteins in mammalian cells, e.g., full-length otoferrin protein A method for increasing the expression of a substance, comprising any of the compositions described herein. Methods including the step of introducing into mammalian cells are also provided herein. In some embodiments of the methods described in the specification, mammalian cells are cochlea These are inner hair cells. In some embodiments of the methods described herein, Mammalian cells are human cells. Any part of the methods described herein In this embodiment, mammalian cells were previously determined to have a missing otoferrin gene. It is being done.

[0143] Active otoferrin protein in the inner hair cells of mammalian cochlea, e.g., full-length otoferrin protein. A method for increasing the expression of ferrin protein, wherein a therapeutically effective amount is described herein. A method comprising the step of introducing one of the compositions into the cochlea of ​​a mammal is also considered a method of this invention. Provided in the specification. Implementations of any part of the methods described herein. In this context, mammals have been previously identified as having a missing otoferrin gene. In some embodiments of the methods described herein, the mammal is a human. That is the case.

[0144] In subjects identified as having a deficient otoferrin gene, non-symptomatic sensorineural A method for treating hearing loss, comprising a therapeutically effective amount of one of the compositions described herein. A method comprising the step of administering either one of the compositions to a target cochlea is also described herein. Provided as follows. In some embodiments of any of the methods described herein, An elephant is a human. Any of the embodiments of the methods described herein are Before the step in which the subject is given, a step is taken to determine whether the subject has a missing otoferrin gene. Includes more.

[0145] A composition comprising two different nucleic acid vectors, of which The first nucleic acid vector is a promoter, located at 3' of the promoter, otoferrinta A first coding sequence that codes for the N-terminal portion of the protein, and the 3' terminal portion of the first coding sequence. It contains a splicing donor signal sequence located at the end, and two different nucleic acid vectors The second nucleic acid vector is a splicing acceptor signal sequence, splicing The C-terminal portion of the otoferrin protein, located at the 3' end of the acceptor signal sequence. A second coding sequence that encodes, and a polyadenylated sequence at the 3' end of the second coding sequence Each of the encoded portions, including the column, is at least 30 amino acid residues long. Furthermore, the amino acid sequences of the encoded portion do not overlap, and one of the two different vectors This single vector does not encode the full-length otoferrin protein, but rather the coding vector When a sequence is transcribed in mammalian cells and an RNA transcript is produced, the sequence on one of the transcripts The pricing donor signal sequence and the splicing acceptor signal sequence on the other transcript. Splicing occurs between the null sequence and the full-length otoferrin protein. Compositions that form recombinant RNA molecules encoding the molecule are also provided herein. In some embodiments of the compositions described herein, among the vectors At least one coding sequence is two adjacent exons of the otoferrin genomic DNA. It contains a nucleotide sequence spanning across two adjacent exons, and lacks an intron sequence between two adjacent exons. They are doing it.

[0146] A composition comprising a promoter, and an otoferrin protein located at the 3' position of the promoter. A first coding sequence that codes for the N-terminal portion of the substance, and located at the 3' end of the first coding sequence. The splicing donor signal sequence, and the 3' of the splicing donor signal sequence A first nucleic acid vector containing a first detectable marker gene located at [location], and a second detection The splice located at 3' of the possible marker gene, the second detectable marker gene. The acceptor signal sequence and the splicing acceptor signal sequence are located at the 3' end. The second coding sequence, which encodes the C-terminal portion of the otoferrin protein, and the A first nucleic acid vector containing a polyadenylated sequence located at the 3' end of the coding sequence 2, and It includes a different second nucleic acid vector, and each of the encoded portions is at least 30 The length of each amino acid, where the amino acid sequence of each coded portion overlaps with each other. Without using any single vector from the two different vectors, the full-length otoferintan Rather than encoding a protein, the coding sequence is transcribed in mammalian cells and then transfused into RNA. When a copy is produced, the splicing donor signal on one copy and the other copy Splicing occurs between the above splicing acceptor signal, and as a result A recombinant RNA molecule encoding the full-length otoferrin protein is formed, and the composition is also Furthermore, any part of the compositions described herein is provided herein. In this embodiment, at least one coding sequence of the vector is the otoferrin genome. It contains a nucleotide sequence that spans two adjacent exons of DNA, and two adjacent exons Inter-xon intron sequences are absent. Any of the compositions described herein In some embodiments, the first or second detectable marker gene is alkaline phosphatidylose. Encoding a phatase. An embodiment of any part of the compositions described herein. In this state, the first and second detectable marker genes are the same.

[0147] A composition comprising a promoter and an otoferin located at 3' relative to the promoter. The first coding sequence that encodes the N-terminal portion of the protein, and the 3' end of the first coding sequence The splicing donor signal sequence located there, and the splicing donor signal sequence A highly recombination-inducible sequence located at 3' relative to it (e.g., the F1 phage recombination-inducible region) For example, a first nucleic acid vector containing sequence number 66, and a second highly recombination-inducible sequence (For example, the F1 phage recombination-inducible region, e.g., SEQ ID NO: 67, or alkaline phosphate) Fatase recombination-inducible region, e.g., SEQ ID NO: 89), second highly recombination-inducible sequence ( For example, F1 phage recombination-inducible region or alkaline phosphatase recombination-inducible region The splicing acceptor signal sequence located at 3' of ) The 3' end of the ter signaling sequence encodes the C-terminal portion of the otoferrin protein. A second code sequence, and a polyadenylated sequence located at the 3' end of the second code sequence A second nucleic acid vector, different from the first nucleic acid vector, which includes a column, and the encoded part Each of the minutes is the length of at least 30 amino acids, and each of the coded parts These amino acid sequences do not overlap with each other, and either single vector of the two different vectors Kutar also does not encode the full-length otoferrin protein, but rather the coding sequence is mammalian. When transcribed in a cell and an RNA transcript is produced, splicing occurs on one of the transcripts. Between the donor signal and the splicing acceptor signal on the other transcript, Rising occurs, thereby recombinant RN encoding full-length otoferrin protein. A composition in which molecule A is formed is also provided herein.

[0148] In some embodiments of the compositions described herein, among the vectors At least one coding sequence is two adjacent exons of the otoferrin genomic DNA. It contains a nucleotide sequence spanning across two adjacent exons, and lacks an intron sequence between two adjacent exons. They are doing it.

[0149] A kit containing any of the compositions described herein is also described herein. Provided. Any of the embodiments of any of the kits described herein are compositions Further includes a pre-filled syringe containing [the specified substance].

[0150] A therapeutically effective amount of any of the compositions described herein is introduced into the cochlea of ​​a mammal. Methods including the step of doing so are also provided herein. In some embodiments of the Act, mammal is human. In some embodiments of the method, mammals are deficient in otoferrin It has been previously identified as having the gene.

[0151] A method for increasing the expression of full-length otoferrin protein in mammalian cells, The method includes the step of introducing one of the compositions described herein into mammalian cells. Methods for doing so are also provided herein. Any of the methods described herein In some embodiments, the mammalian cells are cochlear hair cells. In some embodiments of the method, the mammalian cells are human cells. In some embodiment of the methods described in the details, mammalian cells are deficient. It has been previously determined that the individual possesses the otoferrin gene.

[0152] Increases the expression of full-length otoferrin protein in the inner hair cells of the mammalian cochlea. A method comprising introducing a therapeutically effective amount of any of the compositions described herein into the cochlea. Methods including the step of entering are also provided herein. In some embodiment of the method, mammals have a missing otoferrin gene. It has been previously identified as having any one of the methods described herein. In this embodiment, the mammal is a human.

[0153] In subjects identified as having a deficiency in the otoferrin gene, asymptomatic sensorineural A method for treating hearing loss, comprising a therapeutically effective amount of one of the compositions described herein. A method comprising the step of administering either to the target cochlea is also provided herein. In some embodiments of the methods described herein, the subject is human. Yes. Some embodiments of any of the methods described herein involve administering steps Before proceeding, there is a further step of determining whether the subject has a missing otoferrin gene. include.

[0154] A therapeutic composition comprising multiple adeno-associated virus (AAV) vectors, wherein multiple A AV vectors, in target cells of human subjects to which the therapeutic composition is administered, auditory polypeptides A therapeutic composition capable of constituting cidomestic messenger RNA is presented herein. To be served.

[0155] In some embodiments of the therapeutic compositions described herein, a plurality of A AV vectors, in target cells of human subjects to which the therapeutic composition is administered, produce full-length auditory polypropylene It can be used to construct peptide messenger RNA.

[0156] Some embodiments of any of the therapeutic compositions described herein are first AA Further comprising a V vector and a second AAV vector, and the first and second AAV vectors It independently contains a packaging volume of less than approximately 6kb.

[0157] In some embodiments of the therapeutic compositions described herein, auditory polyphony Peptide messenger RNA is otoferrin and its ortholog or homolog. Encodes an auditory polypeptide selected from a group.

[0158] Some embodiments of any of the therapeutic compositions described herein contain Ca v 1. 3. A scaffold selected from bassoon, piccolo, ribeye, and harmonin. Protein, Vglut3, synaptotagmin, vesicle-binding / docking protein, vesicle Priming protein, vesicle fusion protein, GluA2 / 3, and GluA4 Auditory polypeptide messengers that encode auditory polypeptides selected from a group. It may further include nucleic acids (e.g., vectors) containing nucleic acid sequences that encode RNA.

[0159] In some embodiments of the therapeutic compositions described herein, the first A The AV vector is selected from at least one of the following promoters: CBA, CMV, or CB7. It further includes one promoter sequence.

[0160] In some embodiments of the therapeutic compositions described herein, the first A AV vectors are at least one promoter selected from cochlear-specific promoters. Includes further arrays.

[0161] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The product is formulated for intracochlear administration. Among the therapeutic compositions described herein In some embodiment, the therapeutic composition is formulated to include lipid nanoparticles. In some embodiments of the therapeutic compositions described herein, therapeutic The composition is formulated to contain polymer nanoparticles. The therapeutic combination described herein In some embodiment of the product, the therapeutic composition contains minicircle DNA. It is formulated to be used in any part of any of the therapeutic compositions described herein. In the application form, the therapeutic composition is formulated to contain CELiD DNA. In some embodiments of the compositions described, the therapeutic composition is synthetic extracellular Formulated to contain Npa liquid. Any of the therapeutic compositions described herein. In some embodiments, the therapeutic composition is 20-200 mM NaCl, 1-5 mM K Cl, 0.1-10 mM CaCl2, 1-10 mM glucose, and 2-50 mM The formulation contains HEPES and is formulated to include a synthetic extralymphatic fluid with a pH of approximately 6 to 9. .

[0162] Therapeutic compositions containing auditory polypeptide messenger RNA are also described herein. It will be provided.

[0163] A therapeutic composition comprising one or more adenovirus (AV) vectors, one Alternatively, multiple AV vectors may be used in human target cells to which the therapeutic composition is administered. A therapeutic composition that can constitute auditory polypeptide messenger RNA is also, Provided herein.

[0164] In some embodiments of the therapeutic compositions described herein, one or Multiple AV vectors are used to fully transform target cells in human subjects to which the therapeutic composition is administered. It can be used to construct auditory polypeptide messenger RNA.

[0165] A therapeutic composition comprising one or more lentiviral vectors, wherein one or more A number of lentiviral vectors were found in human target cells to which the therapeutic composition was administered. Therapeutic compositions that can constitute auditory polypeptide messenger RNA are also available. , provided herein.

[0166] In some embodiments of the therapeutic compositions described herein, one or Multiple lentiviral vectors are used to target human cells to which the therapeutic composition is administered. It can then be activated, for example, to form full-length auditory polypeptide messenger RNA. ru.

[0167] i) A step of introducing the first incision into the cochlea of ​​a human subject at the first incision point, ii ) an effective dose of a therapeutic composition (for example, any of the therapeutic compositions described herein) A surgical method including the step of intracochlear administration of (k) is also provided herein. .

[0168] In some embodiments of the methods described herein, the therapeutic composition is It is administered to the subject at the first incision site. Any of the methods described herein. In some embodiments, the therapeutic composition is injected into or through a first incision into the target. To be given.

[0169] In some embodiments of the methods described herein, the therapeutic composition is The drug is administered to the subject via or through the oval window membrane of the cochlea. In some of our embodiments, the therapeutic composition is placed in or within the round window membrane of the cochlea. It is administered to the target through this medium.

[0170] In some embodiments of the methods described herein, the therapeutic composition is The drug is administered using a medical device that can create multiple incisions within the round window membrane. ru.

[0171] In some embodiments of the methods described herein, the medical device , including multiple microneedles. Any part of any of the methods described herein. In the embodiment, the medical device includes a plurality of microneedles, including a first substantially circular form Each microneedle has a diameter of at least approximately 10 microns.

[0172] In some embodiments of the methods described herein, the medical device , comprising a base and / or reservoir capable of holding a therapeutic composition. In some embodiment of the methods described in this document, the medical device is therapeutic It contains multiple hollow microneedles, each containing a lumen capable of transporting the composition. nothing.

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

[0174] i) Multiple incisions can be made in the round window membrane of the inner ear of a human subject who requires it. ii) a medical device containing multiple adeno-associated virus (AAV) vectors A therapeutic composition in which multiple AAV vectors are activated in target cells of the inner ear. For example, a therapeutic bloc that can construct full-length auditory polypeptide messenger RNA. A therapeutic delivery system including the product is also provided herein.

[0175] i) To a human subject who requires it, an effective dose of the therapeutic composition (e.g., as described herein) A surgical procedure is performed which includes the step of intracochlear administration of one of the therapeutic compositions of the following: A means for which a therapeutic composition a) a means for creating multiple incisions in a round window membrane b) by using a medical device containing an effective dose of the therapeutic composition. Means that can be provided herein are available.

[0176] Implementation of any part of the means for performing the surgical procedures described herein In this context, the medical device includes multiple microneedles.

[0177] A therapeutic composition comprising a single adeno-associated virus (AAV) vector, wherein the AAV The actor observed that in target cells of human subjects to which the therapeutic composition is administered, auditory polypeptide molecules Therapeutic compositions that can constitute messenger RNA are also presented herein. To be served.

[0178] In some embodiments of the therapeutic compositions described herein, a single A AV vectors, in target cells of human subjects to which the therapeutic composition is administered, produce full-length auditory polypropylene Peptide messenger RNA can be constructed using the method described herein. In some of the embodiments of this, active otoferrin protein (for example, as specified herein) The code encodes either the full-length or truncated active otoferrin protein described. A single vector containing the sequence (for example, any of the vectors described herein) (k) can be administered to the target.

[0179] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The product further contains a second vector other than the AAV vector, and a single AAV vector and The second vector independently contains a packaging volume of less than approximately 6kb.

[0180] In some embodiments of the therapeutic compositions described herein, auditory polyphony Peptide messenger RNA is otoferrin and its ortholog or homolog. Encodes an auditory polypeptide selected from a group.

[0181] In some embodiments of the therapeutic compositions described herein, auditory polyphony Peptide messenger RNAs are selected from otoferrin and its truncated mutants. It codes for the auditory polypeptide.

[0182] In some embodiments of the therapeutic compositions described herein, Otofe The phosphorus truncated mutant contains at least the following single C2 domain: [ka]

[0183] In some embodiments of the therapeutic compositions described herein, Otofe Phosphorus truncated mutants lack the C-terminal region of the endogenous otoferrin polypeptide.

[0184] In some embodiments of the therapeutic compositions described herein, auditory polyphony Peptide messenger RNA is Ca v 1.3, bassoon, piccolo, ribeye, oyo Scaffold proteins selected from biharmonins, Vglut3, synaptotag Min, vesicle binding / docking protein, vesicle priming protein, vesicle fusion protein Auditory polypeptides selected from the group consisting of GluA2 / 3 and GluA4 Code it.

[0185] In some embodiments of the therapeutic compositions described herein, a single A The AV vector is selected from at least one of the following promoters: CBA, CMV, or CB7. It further includes one promoter sequence.

[0186] In some embodiments of the therapeutic compositions described herein, a single A AV vectors are at least one promoter selected from cochlear-specific promoters. Includes further arrays.

[0187] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The finished product is formulated for intracochlear administration.

[0188] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The product is formulated to contain lipid nanoparticles.

[0189] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The product is formulated to contain polymer nanoparticles.

[0190] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The product is formulated to contain minicircle DNA.

[0191] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The product is formulated to contain CELiD DNA.

[0192] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The product is formulated to contain synthetic perilymphatic fluid.

[0193] In some embodiments of the therapeutic compositions described herein, the therapeutic combination The resulting products are 20-200 mM NaCl, 1-5 mM KCl, and 0.1-10 mM CaC. It contains l2, 1-10 mM glucose, and 2-50 mM HEPES, with a pH of approximately 6-9. It is formulated to contain synthetic extralymphatic fluid.

[0194] Contains auditory polypeptide messenger RNA encoding an otoferrin truncated mutant. Therapeutic compositions are also provided herein.

[0195] Unless otherwise defined, all technical and scientific terms used herein are defined in this document. The disclosure has the same meaning as that widely understood by those skilled in the art in which the disclosure pertains.

[0196] The terms "one (a)" and "one (an)" mean one or more than one. It refers to the grammatical object of that article (that is, at least one). For example, "one The term "element" encompasses one element and more than one element.

[0197] The term "approximately" when referring to measurable values, such as quantities or time ranges, means "maximum" This means that it includes a variation of ±20%. These variations are, for example, a given value or The range is up to ±10%, or in some cases ±5%, or in some cases ±1%, and In some cases, this may be ±0.1%, and such fluctuations may be handled in the manner disclosed. It is appropriate for that purpose.

[0198] The term "signaling domain" refers to the second domain that transmits information within a cell. By creating a messenger or responding to such a messenger By functioning as an effector, it controls cell activity via a predetermined signaling pathway. It refers to the functional part of a protein that acts in a certain way. Otoferrin is used in inner hair cells. It is required for exocytosis and neurotransmitter release in immature outer hair cells. It is thought to be a calcium sensor. The six C2 dormancies of the otoferrin protein Five of the ions (C2B~F) are calcium carbonate in solution with moderate and low affinity constants. Binds to mu ions (Kd = 25-95 μM and 400-700 μM respectively) (P admanarayana et al. 2014 Biochem 53:5023 -5033). In the presence of phosphatidylserine (PS), 10 μM calcium The concentration is such that the C2 liposome interaction is significant for the C2C-C2E domains of otoferrin. Therefore, otoferrin is related to the "electrostatic switch" mechanism and calcium. It has a domain that appears to operate using a binding domain. The signaling molecule PI(4,5)P2 is a calcium-independent signaling molecule. It has been shown to interact with the C2C and C2F domains of phosphorus (Padmana Rayana et al. 2014 Biochem 53:5023-5033) .

[0199] As used herein, the term "antibody" refers to an immune agent that specifically binds to an antigen. This refers to a protein or polypeptide sequence derived from a robulin molecule. Antibodies are polyclonal proteins. monoclonal or monoclonal, multi-stranded or single-stranded, or intact immunoglobulins It can be phosphorus, and may originate from natural or recombinant sources. Antibodies are immunoglobulins. It may also be a tetramer of 1 molecule. The term "antibody fragment" refers to a small amount of intact antibody. It also refers to a part of it, or a recombinant variant thereof, for example, the target of an antibody fragment, for example, anti An intact antibody antigen sufficient to confer recognition and specific binding to the original antigen. This refers to the binding domain, for example, the antigenicity-determining variable region. Examples of antibody fragments are limited to these. Undetermined, but includes Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments. Linear antibodies, single-domain antibodies, e.g., sdAb (either VL or VH), lac Examples include multispecific antibodies formed from the VHH domain and antibody fragments. The term "cFv" refers to at least one antibody fragment containing a variable region of the light chain and a variable region of the heavy chain. This refers to a fusion protein containing at least one antibody fragment that includes a variable region, where the light chain is variable. The regions and heavy chain variable regions are continuously linked via short, mobile polypeptide linkers. It can be expressed as a single-stranded polypeptide, and scFv is the intact from which it is derived. It retains the specificity of the antibody. Unless otherwise specified, when used herein, scFv is For example, with respect to the N-terminus and C-terminus of a polypeptide, the VL variable region and the VH variable region The domains may be in any order, and scFv may include VL-linker-VH. Alternatively, it may contain VH-linker-VL.

[0200] The term "recombinant polypeptide" is used, for example, in viral vector expression systems. This refers to polypeptides produced using recombinant DNA technology, such as recombinant polypeptides. This term also refers to the polypeptides produced by the synthesis of DNA molecules that encode polypeptides. A peptide whose DNA molecule identifies a protein or polypeptide. Express an acid sequence, and its DNA or amino acid sequence is available in the art and Polypeptide obtained using well-known recombinant DNA or amino acid sequencing techniques. It should be interpreted as meaning "do".

[0201] The term "mutation in the otoferrin gene" refers to the wild-type otoferrin protein. Compared to quality, one or more amino acid deletions, one or more amino acid substitutions, and mammals that do not have one or more amino acid insertions and / or mutations. Compared to the expression levels of the encoded otoferrin protein in cells, mammalian cells One of the following is a decrease in the expression level of the encoded otoferrin protein in cells or The wild-type otoferrin gene, which produces multiple otoferrin proteins, This refers to a modification. In some embodiments, a mutation is one or more amino acids (e.g., , 2 pieces, 3 pieces, 4 pieces, 5 pieces, 6 pieces, 7 pieces, 8 pieces, 9 pieces, 10 pieces, 11 pieces, 12 pieces, 13 pieces Deletion of 14, 15, 16, 17, 18, 19, or 20 amino acids This can result in the production of an otoferrin protein having a mutation. In some embodiments, mutations This can lead to a frameshift in the otoferrin gene. The term refers to a shift in the reading frame of the code sequence, in the code sequence. It is known in the art that any mutation can be included. Some embodiments Therefore, frameshift can result in non-functional proteins. In some embodiments, point Mutations can be nonsense mutations (i.e., immaturity in the exons of a gene). (resulting in a stop codon). Nonsense mutations may or may not be functional. This also leads to the production of shortened proteins (compared to the corresponding wild-type proteins). In some embodiments, the mutation occurs in the otoferrin mRNA or otoferrinta. Loss of expression of protein, or both mRNA and protein (or a reduction in their levels) (Slight) may result. In some embodiments, the mutation may result in the wild-type otoferrin protein Compared to, modified which has a loss or reduction of one or more biological activities (functions) This can lead to the production of otoferrin protein.

[0202] In some embodiments, the mutation results in one or more nucleos in the otoferrin gene. This is a cydos insertion. In some embodiments, the mutation is in the regulatory sequence of the otoferrin gene. In other words, it is in the part of the gene that is not the coding sequence. In some embodiments, Mutations in regulatory sequences are in promoter or enhancer regions. This may prevent or reduce the proper transcription of the otoferrin gene.

[0203] Modifications include site-directed mutagenesis and PCR-mediated mutagenesis, and other technologies in the relevant field. It can be introduced into the nucleotide sequence using known standard techniques.

[0204] The term "conservative sequence modification" refers to the binding of an antibody or antibody fragment containing an amino acid sequence. This refers to amino acid modifications that do not significantly affect or alter the characteristics. Conservative modifications include amino acid substitutions, additions, and deletions. Modifications are site-directed. Mutagenesis and PCR-mediated mutagenesis, etc., are standard methods known in the relevant technical field. Conservative amino acids can be introduced into the antibody or antibody fragment of this disclosure by the following techniques. A substitution is when an amino acid residue is replaced by an amino acid residue that has a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include basic side chains (e.g., lysine, arginine, and hist). Tizidine), acidic side chain (e.g., aspartic acid and glutamic acid), non-charged side chain ( For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine (Lysine and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, ylyl) (Soloisin, proline, phenylalanine, and methionine), beta branched side chain (for example) (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine) Examples of amino acids containing phenylalanine, tryptophan, and histidine include those containing phenylalanine, tryptophan, and histidine. ru.

[0205] The term "coding" refers to the synthesis of amino acid sequences defined according to the genetic code. Polynucleotides, such as genes, cDNA, or m, serve as templates. This refers to the unique characteristics of a specific nucleotide sequence in RNA. Therefore, genes, c DNA, or RNA, is the mRNA that corresponds to its gene, cDNA, or RNA. When a protein is produced through copying and translation, it codes for that protein. The ocide sequence is identical to the mRNA sequence, and the coding strand is usually provided in the sequence listing. Both the protein product and the non-coding strand used as a template for transcription are used to produce the protein product. This can be referred to as coding.

[0206] The terms "homology" or "identity" refer to the relationship between two polymer molecules, for example, two nucleic acids. Intermolecular relationships, for example, between two DNA molecules or two RNA molecules, or between two polypeptides This refers to the sequence identity of subunits within a molecule. If the position of a unit is occupied by the same monomer subunit, for example, two If a certain position in each DNA molecule is occupied by adenine, then those positions are... They are homologous or identical at that position. Homologousity between two sequences means they are either identical or identical. It is a direct function of the number of identical positions, for example, half of the positions in two arrays (for example, If the lengths of the 10 subunits in a polymer are homologous at five positions, then these two The arrays are 50% homologous, meaning 90% of the positions (for example, 9 out of 10) match. If they are homologous, then these two sequences are 90% homologous.

[0207] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" are abbreviated relative to each other. It is a duplicate version, and therefore all nucleotides encode the same amino acid sequence. The sequence includes nucleotides. The nucleotide sequence that codes for proteins also includes introns. obtain.

[0208] The term “intrinsic” originates from or is internal to an organism, cell, tissue, or system. This refers to any material produced by [the process].

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

[0210] The term "isolated" means that something has been altered or taken out from its natural state. This means that nucleic acids or peptides that are naturally present in living animals are "isolated". Although not "derived," the same nucleic acid or peptide is partially derived from the naturally occurring coexisting material. Alternatively, if completely separated, it is considered "isolated".

[0211] Isolated nucleic acids or proteins may exist in a substantially purified form, or For example, it can exist in unnatural environments such as host cells.

[0212] The term "substantially purified cells" refers to cells that essentially do not contain other cell types. The substantially purified cells are also associated with the state in which the cells normally exist in nature. This refers to cells isolated from a specific cell type. In some cases, it refers to a collection of essentially purified cells. A group refers to a homogeneous collection of cells. In other contexts, the term simply means that cells are in their natural state. This refers to cells that are isolated from naturally occurring cells in their natural state. In some embodiments, the cells are They are cultured in vitro. In other embodiments, the cells are cultured in vitro. It hasn't been done.

[0213] "Transfected," "transformed," or "transformed" The term refers to the process by which foreign nucleic acids are transferred or introduced into host cells. Cells that have been "transformed" or "transformed" are exogenous nucleic acids It has been transfected, transformed, or transduced. The cell contains the primary target cell and its offspring.

[0214] The term "expression" refers to the transformation of a specific nucleotide sequence driven by a promoter. Refers to copies and / or translations.

[0215] As used herein, “transient” means a period of time of several hours, several days, or several weeks. This refers to the expression of non-integrated transgenes, where the expression period is integrated into the genome. or gene expression when contained within a stable plasmid replicon in the host cell It's shorter than the duration.

[0216] The term "target" includes organisms in which an immune response can be induced (e.g., mammals, humans). It is intended to do so. In some embodiments, the subject is a rodent (e.g., a rat or This includes mice, rabbits, sheep, dogs, cats, horses, non-human primates, or humans. In this embodiment, the subject has or is at risk of developing non-symptomatic hearing loss. In some embodiments, the subject is assumed to have a mutation in the otoferrin gene. Previously identified. In some embodiments, the target is a mutation in the otoferrin gene. The patient has been identified as having a hearing impairment and has been diagnosed with asymptomatic sensorineural hearing loss.

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

[0218] The term "therapeutic," as used herein, means a treatment. The therapeutic effect is, This is achieved by reducing, suppressing, relieving, or eradicating the disease state.

[0219] When used herein, the term “preventive” means the prevention of a disease or disease condition. "T" means protective measures. In this context, "prevention" means the possibility that the subject will experience the disease. This includes reducing the risk of infection.

[0220] The terms “effective dose” or “therapeutic effective dose” are used interchangeably herein. , compounds, formulations, and materials described herein that are effective in achieving specific biological outcomes. , or the amount of the composition. In some embodiments, a therapeutically effective amount of the composition is active otophe Phosphoproteins (e.g., full-length wild-type otoferrin protein, or desired activity) Increased expression level of an otoferrin protein variant having (for example, in a composition) This may result in (compared to the expression level before treatment). In some embodiments, a therapeutically effective dose The composition activates otoferrin protein in target cells (e.g., cochlear hair cells). Expression levels of (e.g., full-length wild-type otoferrin protein or active variant) This can lead to an increase. In some embodiments, a therapeutically effective amount of the composition is used to target cells (e.g., Active otoferrin protein in cochlear hair cells (e.g., full-length wild-type otoferrin) This can result in different cellular localization of phosphoproteins or active variants. In terms of form, the therapeutically effective amount of composition is active otoferrin protein (e.g., full-length wild-type protein). Increased expression levels of otoferrin protein or active variant, and / or Increased activity of one or more otoferrin proteins in target cells (e.g., Irradiation level, for example, the level in the subject before treatment, mutation in the otoferrin gene The level in subjects with differences, or subjects or individuals with asymptomatic sensorineural hearing loss. This could result in a level compared to that in elephant populations.

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

[0222] The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or This refers to ribonucleic acid (RNA) and its polymers in single-stranded or double-stranded forms. Unless otherwise specified, this term refers to a nucleic acid that has similar binding properties to a reference nucleic acid and is naturally occurring. It contains known analogs of natural nucleotides that are metabolized in a manner similar to that of the nucleotides being metabolized. Includes nucleic acids. Unless otherwise indicated, specific nucleic acid sequences are also conservatively modified. Variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and It implicitly includes complementary sequences and sequences that are explicitly shown. Specifically, degenerate chords Don substitution occurs when the third position of one or more selected (or all) codons is replaced. By generating sequences that are substituted with mixed bases and / or deoxyinosine residues, This can be achieved (Batzer et al., Nucleic Acid Res . 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolin i et al., Mol. Cell. Probes 8:91-98 (199 4)).

[0223] In some embodiments of the nucleic acids described herein, the nucleic acid is DNA. Yes. In some embodiments of the nucleic acids described herein, the nucleic acid is RN It is A.

[0224] In the context of this disclosure, the following abbreviations are used with respect to commonly occurring nucleic acid bases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, and "T" "U" refers to thymidine, and "U" refers to uridine.

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

[0226] The terms "peptide," "polypeptide," and "protein" are used interchangeably. Compounds used, composed of amino acid residues covalently linked by peptide bonds. This refers to the fact that a protein or peptide must contain at least two amino acids. However, there are no restrictions regarding the maximum number of amino acids that can constitute a protein or peptide sequence. There is no polypeptide. A polypeptide is two or more polypeptides joined to each other by a peptide bond. Contains any peptide or protein containing many amino acids. In addition, this term is generally used in the relevant technical field for example, peptide, oligopeptide, and There are short chains, also called oligomers, and numerous types, and in this technical field... Generally, it refers to both the long chains called proteins. "Polypeptide" is, for example, Therefore, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers Polypeptide, heterodimer, polypeptide variant, modified polypeptide, derivative, ana Logs and fusion proteins are included. Polypeptides include natural peptides and recombinant peptides. , or a combination of these.

[0227] The term "signaling pathway" refers to the signaling pathway from one part of a cell to another part of a cell. This refers to the biochemical relationships between various signal transduction molecules that play a role in signal transmission. The term "cell surface receptor" refers to a receptor that receives signals and transmits them across the cell membrane. This includes molecules and molecular complexes that can perform certain actions.

[0228] The term "active otoferrin protein" is used in wild-type mammals, in other respects. In the auditory hair cells (e.g., auditory endohair cells), full-length otoferrin protein It replaces both wild-type alleles that code for quality, and in the auditory hair cells of that mammal. When expressed, it approximates the normal hearing level of similar mammals that are completely wild-type. The DNA-encoded tan is what gives a mammal that level of hearing. It means protein. Non-limiting examples of active otoferrin proteins include full-length otoferrin proteins. Protein (for example, any of the full-length otoferrin proteins described herein) )

[0229] For example, active otoferrin proteins have 1 to approximately 240 amino acid substitutions. 1 amino acid substitution to approximately 235 amino acid substitutions, 1 amino acid substitution to approximately 230 amino acid substitutions, 1 a Mino acid substitution ~ approximately 225 amino acid substitutions, 1 amino acid substitution ~ approximately 220 amino acid substitutions, 1 amino acid Acid substitution ~ approximately 215 amino acid substitutions, 1 amino acid substitution ~ approximately 210 amino acid substitutions, 1 amino acid substitution Substitutions ~ Approximately 205 amino acid substitutions, 1 amino acid substitution ~ Approximately 200 amino acid substitutions, 1 amino acid substitution ~ Approximately 195 amino acid substitutions, 1 amino acid substitution to approximately 190 amino acid substitutions, 1 amino acid substitution to approximately 1 85 amino acid substitutions, 1 amino acid substitution to approximately 180 amino acid substitutions, 1 amino acid substitution to approximately 175 Amino acid substitution, 1 amino acid substitution to approximately 170 amino acid substitutions, 1 amino acid substitution to approximately 165 amino acids No acid substitution, 1 amino acid substitution to approximately 160 amino acid substitutions, 1 amino acid substitution to approximately 155 amino acids Substitution, 1 amino acid substitution to approximately 150 amino acid substitutions, 1 amino acid substitution to approximately 145 amino acid substitutions , 1 amino acid substitution to approximately 140 amino acid substitutions, 1 amino acid substitution to approximately 135 amino acid substitutions, 1 Amino acid substitutions ~ approximately 130 amino acid substitutions, 1 amino acid substitution ~ approximately 125 amino acid substitutions, 1 amino acid substitution Acid substitutions ~ approximately 120 amino acid substitutions, 1 amino acid substitutions ~ approximately 115 amino acid substitutions, 1 amino acid Substitutions ~ approximately 110 amino acid substitutions, 1 amino acid substitution ~ approximately 105 amino acid substitutions, 1 amino acid substitution ~Approximately 100 amino acid substitutions, 1 amino acid substitution ~Approximately 95 amino acid substitutions, 1 amino acid substitution ~Approximately 9 0 amino acid substitutions, 1 amino acid substitution to approximately 85 amino acid substitutions, 1 amino acid substitution to approximately 80 amino acids Acid substitution, 1 amino acid substitution to approximately 75 amino acid substitutions, 1 amino acid substitution to approximately 70 amino acid substitutions, 1 amino acid substitution to approximately 65 amino acid substitutions, 1 amino acid substitution to approximately 60 amino acid substitutions, 1 amino Acid substitution ~ approximately 55 amino acid substitutions, 1 amino acid substitution ~ approximately 50 amino acid substitutions, 1 amino acid substitution ~ Approximately 45 amino acid substitutions, 1 amino acid substitution to approximately 40 amino acid substitutions, 1 amino acid substitution to approximately 35 amino acid substitutions Mino acid substitution, 1 amino acid substitution to approximately 30 amino acid substitution, 1 amino acid substitution to approximately 25 amino acid substitution Substitution, 1 amino acid substitution to approximately 20 amino acid substitutions, 1 amino acid substitution to approximately 15 amino acid substitutions, 1 a Mino acid substitution ~ approximately 10 amino acid substitutions, 1 amino acid substitution ~ approximately 9 amino acid substitutions, 1 amino acid substitution ~8 amino acid substitutions, 1 amino acid substitution ~ approximately 7 amino acid substitutions, 1 amino acid substitution ~ approximately 6 amino acid substitutions Acid substitution, 1 amino acid substitution to approximately 5 amino acid substitutions, 1 amino acid substitution to approximately 4 amino acid substitutions, 1 Mino acid substitution ~ approximately 3 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 2 amino acid substitutions Acid substitution ~ approximately 235 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 2 amino acid substitutions Acid substitution ~ approximately 225 amino acid substitutions, approximately 2 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 215 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 210 amino acid substitutions, approximately 2 Mino acid substitution ~ approximately 205 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 200 amino acid substitutions, approximately 2 Mino acid substitutions ~ approximately 195 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 190 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 185 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 180 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 175 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 170 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 165 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 155 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 150 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 145 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 140 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 135 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 130 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 125 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 120 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 115 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 110 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 105 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 100 amino acid substitutions, approximately 2 amino acid substitutions Mino acid substitution ~ approximately 95 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 90 amino acid substitutions, approximately 2 amino acid substitutions Acid substitution ~ approximately 85 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 80 amino acid substitutions, approximately 2 amino acid substitutions ~ Approximately 75 amino acid substitutions, approximately 2 amino acid substitutions ~ Approximately 70 amino acid substitutions, approximately 2 amino acid substitutions ~ Approximately 65 amino acid substitutions, approximately 2 amino acid substitutions to approximately 60 amino acid substitutions, approximately 2 amino acid substitutions to approximately 5 5 amino acid substitutions, approximately 2 amino acid substitutions to approximately 50 amino acid substitutions, approximately 2 amino acid substitutions to approximately 45 amino acid substitutions Mino acid substitution, approximately 2 amino acid substitutions to approximately 40 amino acid substitutions, approximately 2 amino acid substitutions to approximately 35 amino acid substitutions Acid substitution, approximately 2 amino acid substitutions to approximately 30 amino acid substitutions, approximately 2 amino acid substitutions to approximately 25 amino acid substitutions Substitutions, approximately 2 amino acid substitutions to approximately 20 amino acid substitutions, approximately 2 amino acid substitutions to approximately 15 amino acid substitutions, Approximately 2 amino acid substitutions to approximately 10 amino acid substitutions, approximately 2 amino acid substitutions to approximately 9 amino acid substitutions, approximately 2 Mino acid substitutions ~ approximately 8 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 7 amino acid substitutions, approximately 2 amino acid substitutions Replacement ~ approximately 6 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 5 amino acid substitutions, approximately 2 amino acid substitutions ~ approximately 4 Amino acid substitutions, approximately 3 amino acid substitutions to approximately 240 amino acid substitutions, approximately 3 amino acid substitutions to approximately 235 Amino acid substitutions, approximately 3 amino acid substitutions to approximately 230 amino acid substitutions, approximately 3 amino acid substitutions to approximately 225 Amino acid substitutions, approximately 3 amino acid substitutions to approximately 220 amino acid substitutions, approximately 3 amino acid substitutions to approximately 2 15 amino acid substitutions, approximately 3 amino acid substitutions to approximately 210 amino acid substitutions, approximately 3 amino acid substitutions to approximately 2 0.5 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 200 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 95 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 190 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 85 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 180 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 75 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 170 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 65 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 55 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 150 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 45 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 140 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 35 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 130 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 25 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 120 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 15 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 110 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 1 0.5 amino acid substitutions, approximately 3 amino acid substitutions to approximately 100 amino acid substitutions, approximately 3 amino acid substitutions to approximately 9 5 amino acid substitutions, approximately 3 amino acid substitutions to approximately 90 amino acid substitutions, approximately 3 amino acid substitutions to approximately 85 amino acid substitutions Mino acid substitution, approximately 3 amino acid substitutions to approximately 80 amino acid substitutions, approximately 3 amino acid substitutions to approximately 75 amino acid substitutions Acid substitution, approximately 3 amino acid substitutions to approximately 70 amino acid substitutions, approximately 3 amino acid substitutions to approximately 65 amino acid substitutions 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 ~ approximately 40 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 35 amino acid substitutions, approximately 3 amino acid substitutions No acid substitution ~ approximately 30 amino acid substitutions, approximately 3 amino acid substitutions ~ approximately 25 amino acid substitutions, approximately 3 amino acid substitutions Substitutions: ~20 amino acid substitutions, ~3 amino acid substitutions, ~15 amino acid substitutions, ~3 amino acid substitutions ~approximately 10 amino acid substitutions, approximately 3 amino acid substitutions ~approximately 9 amino acid substitutions, approximately 3 amino acid substitutions ~approximately 8 Amino acid substitution, approximately 3 amino acid substitutions to approximately 7 amino acid substitutions, approximately 3 amino acid substitutions to approximately 6 amino acid substitutions Substitutions, approximately 3 amino acid substitutions to approximately 5 amino acid substitutions, approximately 4 amino acid substitutions to approximately 240 amino acid substitutions Approximately 4 amino acid substitutions to approximately 235 amino acid substitutions, approximately 4 amino acid substitutions to approximately 230 amino acid substitutions Approximately 4 amino acid substitutions to approximately 225 amino acid substitutions, approximately 4 amino acid substitutions to approximately 220 amino acids Substitutions, approximately 4 amino acid substitutions to approximately 215 amino acid substitutions, approximately 4 amino acid substitutions to approximately 210 amino acids Substitutions, approximately 4 amino acid substitutions to approximately 205 amino acid substitutions, approximately 4 amino acid substitutions to approximately 200 amino acid substitutions Substitutions, approximately 4 amino acid substitutions to approximately 195 amino acid substitutions, approximately 4 amino acid substitutions to approximately 190 amino acids Substitutions, approximately 4 amino acid substitutions to approximately 185 amino acid substitutions, approximately 4 amino acid substitutions to approximately 180 amino acid substitutions Substitutions, approximately 4 amino acid substitutions to approximately 175 amino acid substitutions, approximately 4 amino acid substitutions to approximately 170 amino acids Substitutions, approximately 4 amino acid substitutions to approximately 165 amino acid substitutions, approximately 4 amino acid substitutions to approximately 160 amino acid substitutions Substitutions, approximately 4 amino acid substitutions to approximately 155 amino acid substitutions, approximately 4 amino acid substitutions to approximately 150 amino acids Substitutions, approximately 4 amino acid substitutions to approximately 145 amino acid substitutions, approximately 4 amino acid substitutions to approximately 140 amino acid substitutions Substitutions, approximately 4 amino acid substitutions to approximately 135 amino acid substitutions, approximately 4 amino acid substitutions to approximately 130 amino acid substitutions Substitutions, approximately 4 amino acid substitutions to approximately 125 amino acid substitutions, approximately 4 amino acid substitutions to approximately 120 amino acid substitutions Substitutions, approximately 4 amino acid substitutions to approximately 115 amino acid substitutions, approximately 4 amino acid substitutions to approximately 110 amino acids Substitutions, approximately 4 amino acid substitutions to approximately 105 amino acid substitutions, approximately 4 amino acid substitutions to approximately 100 amino acid substitutions Substitutions, approximately 4 amino acid substitutions to approximately 95 amino acid substitutions, approximately 4 amino acid substitutions to approximately 90 amino acid substitutions Approximately 4 amino acid substitutions to approximately 85 amino acid substitutions, 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 Mino acid substitution ~ approximately 65 amino acid substitutions, approximately 4 amino acid substitutions ~ approximately 60 amino acid substitutions, approximately 4 amino acid substitutions Acid substitution ~ approximately 55 amino acid substitutions, approximately 4 amino acid substitutions ~ approximately 50 amino acid substitutions, approximately 4 amino acid substitutions ~ Approximately 45 amino acid substitutions, approximately 4 amino acid substitutions ~ Approximately 40 amino acid substitutions, approximately 4 amino acid substitutions ~ Approximately 35 amino acid substitutions, approximately 4 amino acid substitutions to approximately 30 amino acid substitutions, approximately 4 amino acid substitutions to approximately 2 5 amino acid substitutions, approximately 4 amino acid substitutions to approximately 20 amino acid substitutions, approximately 4 amino acid substitutions to approximately 15 amino acid substitutions Mino acid substitution, approximately 4 amino acid substitutions to approximately 10 amino acid substitutions, approximately 4 amino acid substitutions to approximately 9 amino acid substitutions Substitution, approximately 4 amino acid substitutions to approximately 8 amino acid substitutions, approximately 4 amino acid substitutions to approximately 7 amino acid substitutions, approximately 4 amino acid substitutions to approximately 6 amino acid substitutions, approximately 5 amino acid substitutions to approximately 240 amino acid substitutions, approximately 5 Mino acid substitution ~ approximately 235 amino acid substitutions, approximately 5 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 5 amino acid substitutions Mino acid substitutions ~ approximately 225 amino acid substitutions, approximately 5 amino acid substitutions ~ approximately 220 amino acid substitutions, approximately 5 amino acid substitutions to approximately 215 amino acid substitutions, approximately 5 amino acid substitutions to approximately 210 amino acid substitutions, approximately 5 amino acid substitutions to approximately 205 amino acid substitutions, approximately 5 amino acid substitutions to approximately 200 amino acid substitutions, approximately 5 amino acid substitutions to approximately 195 amino acid substitutions, approximately 5 amino acid substitutions to approximately 190 amino acid substitutions, approximately 5 amino acid substitutions to approximately 185 amino acid substitutions, approximately 5 amino acid substitutions to approximately 180 amino acid substitutions, approximately 5 amino acid substitutions to approximately 175 amino acid substitutions, approximately 5 amino acid substitutions to approximately 170 amino acid substitutions, approximately 5 amino acid substitutions to approximately 165 amino acid substitutions, approximately 5 amino acid substitutions to approximately 160 amino acid substitutions, approximately 5 amino acid substitutions to approximately 155 amino acid substitutions, approximately 5 amino acid substitutions to approximately 150 amino acid substitutions, approximately 5 amino acid substitutions to approximately 145 amino acid substitutions, approximately 5 amino acid substitutions to approximately 140 amino acid substitutions, approximately 5 amino acid substitutions to approximately 135 amino acid substitutions, approximately 5 amino acid substitutions to approximately 130 amino acid substitutions, approximately 5 amino acid substitutions to approximately 125 amino acid substitutions, approximately 5 amino acid substitutions to approximately 120 amino acid substitutions, approximately 5 amino acid substitutions to approximately 115 amino acid substitutions, approximately 5 amino acid substitutions to approximately 110 amino acid substitutions, approximately 5 amino acid substitutions to approximately 105 amino acid substitutions, approximately 5 amino acid substitutions to approximately 100 amino acid substitutions, approximately 5 amino acid substitutions to approximately 95 amino acid substitutions, approximately 5 amino acid substitutions to approximately 90 amino acid substitutions, approximately 5 Mino acid substitution ~ approximately 85 amino acid substitutions, approximately 5 amino acid substitutions ~ approximately 80 amino acid substitutions, approximately 5 amino acid substitutions Acid substitution ~ approximately 75 amino acid substitutions, approximately 5 amino acid substitutions ~ approximately 70 amino acid substitutions, approximately 5 amino acid substitutions ~approximately 65 amino acid substitutions, approximately 5 amino acid substitutions~ Approximately 60 amino acid substitutions, approximately 5 amino acid substitutions~ Approximately 55 amino acid substitutions, approximately 5 amino acid substitutions to approximately 50 amino acid substitutions, approximately 5 amino acid substitutions to approximately 4 5 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 Mino acid substitution, approximately 5 amino acid substitutions to approximately 30 amino acid substitutions, approximately 5 amino acid substitutions to approximately 25 amino acid substitutions Acid substitution, approximately 5 amino acid substitution to approximately 20 amino acid substitution, approximately 5 amino acid substitution to approximately 15 amino acid substitution Substitution, approximately 5 amino acid substitutions to approximately 10 amino acid substitutions, approximately 5 amino acid substitutions to approximately 9 amino acid substitutions, approximately 5 amino acid substitutions to approximately 8 amino acid substitutions, approximately 5 amino acid substitutions to approximately 7 amino acid substitutions, approximately 6 amino acid substitutions Acid substitution ~ approximately 240 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 235 amino acid substitutions, approximately 6 amino Acid substitution ~ approximately 230 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 225 amino acid substitutions, approximately 6 amino acid Substitution substitutions ~ approximately 220 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 215 amino acid substitutions, approximately 6 amino acid substitutions Acid substitutions ~ approximately 210 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 205 amino acid substitutions, approximately 6 amino acid substitutions No acid substitution ~ approximately 200 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 6 amino acid substitutions No acid substitutions ~ approximately 190 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 185 amino acid substitutions, approximately 6 amino acid substitutions No acid substitution ~ approximately 180 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 175 amino acid substitutions, approximately 6 amino acid substitutions No acid substitution ~ approximately 170 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 165 amino acid substitutions, approximately 6 amino acid substitutions No acid substitution ~ approximately 160 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 155 amino acid substitutions, approximately 6 amino acid substitutions No acid substitution ~ approximately 150 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 145 amino acid substitutions, approximately 6 amino acid substitutions No acid substitutions ~ approximately 140 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 135 amino acid substitutions, approximately 6 amino acid substitutions No acid substitution ~ approximately 130 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 125 amino acid substitutions, approximately 6 amino acid substitutions No acid substitution ~ approximately 120 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 115 amino acid substitutions, approximately 6 amino acid substitutions No acid substitutions ~ approximately 110 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 105 amino acid substitutions, approximately 6 amino acid substitutions Acid substitutions ~ approximately 100 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 95 amino acid substitutions, approximately 6 amino acid substitutions Acid substitution ~ approximately 90 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 85 amino acid substitutions, approximately 6 amino acid substitutions ~ Approximately 80 amino acid substitutions, approximately 6 amino acid substitutions ~ Approximately 75 amino acid substitutions, approximately 6 amino acid substitutions ~ Approximately 70 amino acid substitutions, approximately 6 amino acid substitutions to approximately 65 amino acid substitutions, approximately 6 amino acid substitutions to approximately 6 0 amino acid substitutions, approximately 6 amino acid substitutions to approximately 55 amino acid substitutions, approximately 6 amino acid substitutions to approximately 50 amino acid substitutions Mino acid substitution, approximately 6 amino acid substitutions to approximately 45 amino acid substitutions, approximately 6 amino acid substitutions to approximately 40 amino acid substitutions Acid substitution, approximately 6 amino acid substitutions to approximately 35 amino acid substitutions, approximately 6 amino acid substitutions to approximately 30 amino acid substitutions Substitution, approximately 6 amino acid substitutions to approximately 25 amino acid substitutions, approximately 6 amino acid substitutions to approximately 20 amino acid substitutions, Approximately 6 amino acid substitutions to approximately 15 amino acid substitutions, approximately 6 amino acid substitutions to approximately 10 amino acid substitutions, approximately 6 Amino acid substitutions ~ approximately 9 amino acid substitutions, approximately 6 amino acid substitutions ~ approximately 8 amino acid substitutions, approximately 7 amino acid substitutions Substitutions ~ approximately 240 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 235 amino acid substitutions, approximately 7 amino acids Substitutions ~ approximately 230 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 225 amino acid substitutions, approximately 7 amino acids Substitution substitutions ~ approximately 220 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 215 amino acid substitutions, approximately 7 amino acid substitutions No acid substitutions ~ approximately 210 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 205 amino acid substitutions, approximately 7 amino acid substitutions Acid substitutions ~ approximately 200 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 7 amino acid substitutions Acid substitutions ~ approximately 190 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 185 amino acid substitutions, approximately 7 amino acid substitutions No acid substitution ~ approximately 180 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 175 amino acid substitutions, approximately 7 amino acid substitutions Acid substitutions ~ approximately 170 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 165 amino acid substitutions, approximately 7 amino acid substitutions Acid substitutions ~ approximately 160 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 155 amino acid substitutions, approximately 7 amino acid substitutions Acid substitutions ~ approximately 150 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 145 amino acid substitutions, approximately 7 amino acid substitutions Acid substitutions ~ approximately 140 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 135 amino acid substitutions, approximately 7 amino acid substitutions Acid substitutions ~ approximately 130 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 125 amino acid substitutions, approximately 7 amino acid substitutions No acid substitution ~ approximately 120 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 115 amino acid substitutions, approximately 7 amino acid substitutions Acid substitutions ~ approximately 110 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 105 amino acid substitutions, approximately 7 amino acid substitutions No acid substitution ~ approximately 100 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 95 amino acid substitutions, approximately 7 amino acid substitutions Acid substitution ~ approximately 90 amino acid substitutions, approximately 7 amino acid substitutions ~ approximately 85 amino acid substitutions, approximately 7 amino acid substitutions Substitutions ~ Approximately 80 amino acid substitutions, approximately 7 amino acid substitutions ~ Approximately 75 amino acid substitutions, approximately 7 amino acid substitutions ~ Approximately 70 amino acid substitutions, approximately 7 amino acid substitutions to approximately 65 amino acid substitutions, approximately 7 amino acid substitutions to approximately 6 0 amino acid substitutions, approximately 7 amino acid substitutions to approximately 55 amino acid substitutions, approximately 7 amino acid substitutions to approximately 50 amino acid substitutions Mino acid substitution, approximately 7 amino acid substitutions to approximately 45 amino acid substitutions, approximately 7 amino acid substitutions to approximately 40 amino acid substitutions Acid substitution, approximately 7 amino acid substitutions to approximately 35 amino acid substitutions, approximately 7 amino acid substitutions to approximately 30 amino acid substitutions Substitution, approximately 7 amino acid substitutions to approximately 25 amino acid substitutions, approximately 7 amino acid substitutions to approximately 20 amino acid substitutions, Approximately 7 amino acid substitutions to approximately 15 amino acid substitutions, approximately 7 amino acid substitutions to approximately 10 amino acid substitutions, approximately 7 Amino acid substitutions ~ approximately 9 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 8 amino acid substitutions Acid substitutions ~ approximately 235 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 8 amino acid substitutions Acid substitutions ~ approximately 225 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 220 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 215 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 210 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 205 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 200 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 190 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 185 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 180 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 175 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 170 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 165 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 155 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 150 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 145 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 140 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 135 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 130 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 125 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 120 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 115 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 110 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 105 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 100 amino acid substitutions, approximately 8 Amino acid substitutions ~ approximately 95 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 90 amino acid substitutions, approximately 8 amino acid substitutions Acid substitutions ~ approximately 85 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 80 amino acid substitutions, approximately 8 amino acid substitutions Substitutions ~ approximately 75 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 70 amino acid substitutions, approximately 8 amino acid substitutions ~Approximately 65 amino acid substitutions, approximately 8 amino acid substitutions~Approximately 60 amino acid substitutions, approximately 8 amino acid substitutions~Approximately 55 amino acid substitutions, approximately 8 amino acid substitutions ~ approximately 50 amino acid substitutions, approximately 8 amino acid substitutions ~ 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 No acid substitution, approximately 8 amino acid substitutions to approximately 30 amino acid substitutions, approximately 8 amino acid substitutions to approximately 25 amino acid substitutions 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 acids Substitutions, approximately 10 amino acid substitutions to approximately 225 amino acid substitutions, approximately 10 amino acid substitutions to approximately 220 Amino acid substitutions, approximately 10 amino acid substitutions to approximately 215 amino acid substitutions, approximately 10 amino acid substitutions to approximately 2 10 amino acid substitutions, approximately 10 amino acid substitutions ~ Approximately 205 amino acid substitutions, approximately 10 amino acid substitutions ~ Approximately 200 amino acid substitutions, approximately 10 amino acid substitutions to approximately 195 amino acid substitutions, approximately 10 amino acid substitutions Amino acid substitutions: approximately 190 amino acid substitutions, approximately 10 amino acid substitutions, approximately 185 amino acid substitutions, approximately 10 amino acid substitutions Acid substitution ~ approximately 180 amino acid substitutions, approximately 10 amino acid substitutions ~ approximately 175 amino acid substitutions, approximately 10 Mino acid substitutions ~ approximately 170 amino acid substitutions, approximately 10 amino acid substitutions ~ approximately 165 amino acid substitutions, approximately 1 0 amino acid substitutions to approximately 160 amino acid substitutions, approximately 10 amino acid substitutions to approximately 155 amino acid substitutions, Approximately 10 amino acid substitutions to approximately 150 amino acid substitutions, approximately 10 amino acid substitutions to approximately 145 amino acid substitutions Substitutions: approximately 10 amino acid substitutions to approximately 140 amino acid substitutions, approximately 10 amino acid substitutions to approximately 135 amino acid substitutions. Acid substitution, approximately 10 amino acid substitutions to approximately 130 amino acid substitutions, approximately 10 amino acid substitutions to approximately 125 amino acid substitutions Mino acid substitution, approximately 10 amino acid substitutions to approximately 120 amino acid substitutions, approximately 10 amino acid substitutions to approximately 11 5 amino acid substitutions, approximately 10 amino acid substitutions ~ approximately 110 amino acid substitutions, approximately 10 amino acid substitutions ~ approximately 105 amino acid substitutions, approximately 10 amino acid substitutions to approximately 100 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 95 amino acid substitutions, approximately 10 amino acid substitutions~Approximately 90 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 85 amino acid substitutions, approximately 10 amino acid substitutions~Approximately 80 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 75 amino acid substitutions, approximately 10 amino acid substitutions~Approximately 70 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 65 amino acid substitutions, approximately 10 amino acid substitutions~Approximately 60 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 55 amino acid substitutions, approximately 10 amino acid substitutions~Approximately 50 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 45 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 40 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 35 amino acid substitutions, approximately 10 amino acid substitutions~Approximately 30 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 25 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 20 amino acid substitutions, approximately 10 amino acid substitutions ~Approximately 15 amino acid substitutions, approximately 15 amino acid substitutions ~Approximately 240 amino acid substitutions, approximately 15 amino acid substitutions Substitutions: Approximately 235 amino acid substitutions, approximately 15 amino acid substitutions, approximately 230 amino acid substitutions, approximately 15 amino acids Acid substitution ~ approximately 225 amino acid substitutions, approximately 15 amino acid substitutions, substitution ~ approximately 220 amino acid substitutions, approximately 1 5 amino acid substitutions to approximately 215 amino acid substitutions, approximately 15 amino acid substitutions to approximately 210 amino acid substitutions, Approximately 15 amino acid substitutions to approximately 205 amino acid substitutions, approximately 15 amino acid substitutions to approximately 200 amino acid substitutions Substitutions: approximately 15 amino acid substitutions to approximately 195 amino acid substitutions, approximately 15 amino acid substitutions to approximately 190 amino acid substitutions. Acid substitution, approximately 15 amino acid substitutions to approximately 185 amino acid substitutions, approximately 15 amino acid substitutions to approximately 180 amino acid substitutions Mino acid substitution, approximately 15 amino acid substitutions to approximately 175 amino acid substitutions, approximately 15 amino acid substitutions to approximately 17 0 amino acid substitutions, approximately 15 amino acid substitutions ~ approximately 165 amino acid substitutions, approximately 15 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 15 amino acid substitutions to approximately 155 amino acid substitutions, approximately 15 amino acid substitutions ~Approximately 150 amino acid substitutions, approximately 15 amino acid substitutions~Approximately 145 amino acid substitutions, approximately 15 amino acids Substitutions ~ approximately 140 amino acid substitutions, approximately 15 amino acid substitutions ~ approximately 135 amino acid substitutions, approximately 15 amino acid substitutions Acid substitutions ~ approximately 130 amino acid substitutions, approximately 15 amino acid substitutions ~ approximately 125 amino acid substitutions, approximately 15 Amino acid substitutions ~ approximately 120 amino acid substitutions, approximately 15 amino acid substitutions ~ approximately 115 amino acid substitutions, approximately 15 amino acid substitutions to approximately 110 amino acid substitutions, approximately 15 amino acid substitutions to approximately 105 amino acid substitutions Approximately 15 amino acid substitutions to approximately 100 amino acid substitutions, approximately 15 amino acid substitutions to approximately 95 amino acid substitutions Substitutions: approximately 15 amino acid substitutions to approximately 90 amino acid substitutions, approximately 15 amino acid substitutions to approximately 85 amino acid substitutions. Substitutions: approximately 15 amino acid substitutions to approximately 80 amino acid substitutions, approximately 15 amino acid substitutions to approximately 75 amino acid substitutions. Substitutions: approximately 15 amino acid substitutions to approximately 70 amino acid substitutions, approximately 15 amino acid substitutions to approximately 65 amino acid substitutions. Substitutions: approximately 15 amino acid substitutions to approximately 60 amino acid substitutions, approximately 15 amino acid substitutions to approximately 55 amino acid substitutions. Substitutions: approximately 15 amino acid substitutions to approximately 50 amino acid substitutions, approximately 15 amino acid substitutions to approximately 45 amino acid substitutions. Substitutions: approximately 15 amino acid substitutions to approximately 40 amino acid substitutions, approximately 15 amino acid substitutions to approximately 35 amino acid substitutions. Substitutions: approximately 15 amino acid substitutions to approximately 30 amino acid substitutions, approximately 15 amino acid substitutions to approximately 25 amino acid substitutions. Substitutions: approximately 15 amino acid substitutions to approximately 20 amino acid substitutions, approximately 20 amino acid substitutions to approximately 240 amino acid substitutions. Substitutions, approximately 20 amino acid substitutions to approximately 235 amino acid substitutions, approximately 20 amino acid substitutions to approximately 230 amino acid substitutions No acid substitution, approximately 20 amino acid substitutions to approximately 225 amino acid substitutions, approximately 20 amino acid substitutions to approximately 2 20 amino acid substitutions, approximately 20 amino acid substitutions ~ approximately 215 amino acid substitutions, approximately 20 amino acid substitutions ~ Approximately 210 amino acid substitutions, approximately 20 amino acid substitutions to approximately 205 amino acid substitutions, approximately 20 amino acid substitutions Amino acid substitutions: approximately 200 amino acid substitutions, approximately 20 amino acid substitutions, approximately 195 amino acid substitutions, approximately 20 amino acid substitutions. Acid substitution ~ approximately 190 amino acid substitutions, approximately 20 amino acid substitutions ~ approximately 185 amino acid substitutions, approximately 20 Mino acid substitutions ~ approximately 180 amino acid substitutions, approximately 20 amino acid substitutions ~ approximately 175 amino acid substitutions, approximately 2 0 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 Substitution, approximately 20 amino acid substitutions to approximately 150 amino acid substitutions, approximately 20 amino acid substitutions to approximately 145 amino acid substitutions Acid substitution, approximately 20 amino acid substitutions to approximately 140 amino acid substitutions, approximately 20 amino acid substitutions to approximately 135 amino acid substitutions Mino acid substitution, approximately 20 amino acid substitutions to approximately 130 amino acid substitutions, approximately 20 amino acid substitutions to approximately 12 5 Amino acid substitution, approximately 20 amino acid substitutions to approximately 120 amino acid substitutions, approximately 20 amino acid substitutions to approximately 1 15 amino acid substitutions, approximately 20 amino acid substitutions ~ approximately 110 amino acid substitutions, approximately 20 amino acid substitutions ~ Approximately 105 amino acid substitutions, approximately 20 amino acid substitutions to approximately 100 amino acid substitutions, approximately 20 amino acid substitutions Substitutions of approximately 95 amino acids, approximately 20 amino acids, approximately 90 amino acids, approximately 20 amino acids Substitutions: Approximately 85 amino acid substitutions, approximately 20 amino acid substitutions, approximately 80 amino acid substitutions, approximately 20 amino acid substitutions. Substitutions of approximately 75 amino acids, approximately 20 amino acids, approximately 70 amino acids, approximately 20 amino acids Substitutions of approximately 65 amino acids, approximately 20 amino acids, approximately 60 amino acids, approximately 20 amino acids Substitutions of approximately 55 amino acids, approximately 20 amino acids, approximately 50 amino acids, approximately 20 amino acids Replacement ~ approximately 45 amino acid substitutions, approximately 20 amino acid substitutions ~ approximately 40 amino acid substitutions, approximately 20 amino acid substitutions Substitutions of approximately 35 amino acids, approximately 20 amino acids, approximately 30 amino acids, approximately 20 amino acids Replacement ~ approximately 25 amino acid substitutions, approximately 25 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 25 amino acids Substitutions ~ approximately 235 amino acid substitutions, approximately 25 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 25 amino acid substitutions Acid substitutions ~ approximately 225 amino acid substitutions, approximately 25 amino acid substitutions ~ approximately 220 amino acid substitutions, approximately 25 amino acid substitutions to approximately 215 amino acid substitutions, approximately 25 amino acid substitutions to approximately 210 amino acid substitutions Approximately 25 amino acid substitutions to approximately 205 amino acid substitutions, approximately 25 amino acid substitutions to approximately 200 amino acids Substitutions, approximately 10 amino acid substitutions to approximately 195 amino acid substitutions, approximately 25 amino acid substitutions to approximately 190 amino acid substitutions No acid substitution, approximately 25 amino acid substitutions to approximately 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 1 70 amino acid substitutions, approximately 25 amino acid substitutions ~ approximately 165 amino acid substitutions, approximately 25 amino acid substitutions ~ Approximately 160 amino acid substitutions, approximately 25 amino acid substitutions to approximately 155 amino acid substitutions, approximately 25 amino acid substitutions Amino acid substitutions: approximately 150 amino acid substitutions, approximately 25 amino acid substitutions, approximately 145 amino acid substitutions, approximately 25 amino acid substitutions. Acid substitution ~ approximately 140 amino acid substitutions, approximately 25 amino acid substitutions ~ approximately 135 amino acid substitutions, approximately 25 Mino acid substitution ~ approximately 130 amino acid substitutions, approximately 25 amino acid substitutions ~ approximately 125 amino acid substitutions, approximately 2 5 amino acid substitutions to approximately 120 amino acid substitutions, approximately 25 amino acid substitutions to approximately 115 amino acid substitutions, Approximately 25 amino acid substitutions to approximately 110 amino acid substitutions, approximately 25 amino acid substitutions to approximately 105 amino acid substitutions Substitution, approximately 25 amino acid substitutions to approximately 100 amino acid substitutions, approximately 25 amino acid substitutions to approximately 95 amino acid substitutions Substitutions, approximately 25 amino acid substitutions to approximately 90 amino acid substitutions, approximately 25 amino acid substitutions to approximately 85 amino acids Substitutions, approximately 25 amino acid substitutions to approximately 80 amino acid substitutions, approximately 25 amino acid substitutions to approximately 75 amino acids Substitutions, approximately 25 amino acid substitutions to approximately 70 amino acid substitutions, approximately 25 amino acid substitutions to approximately 65 amino acids Substitutions, approximately 25 amino acid substitutions to approximately 60 amino acid substitutions, approximately 25 amino acid substitutions to approximately 55 amino acids Substitutions, approximately 25 amino acid substitutions to approximately 50 amino acid substitutions, approximately 25 amino acid substitutions to approximately 45 amino acid substitutions Substitutions, approximately 25 amino acid substitutions to approximately 40 amino acid substitutions, approximately 25 amino acid substitutions to approximately 35 amino acid substitutions Substitutions, approximately 25 amino acid substitutions to approximately 30 amino acid substitutions, approximately 30 amino acid substitutions to approximately 240 amino acid substitutions Acid substitution, approximately 30 amino acid substitutions to approximately 235 amino acid substitutions, approximately 30 amino acid substitutions to approximately 230 amino acid substitutions Mino acid substitution, approximately 30 amino acid substitutions ~ approximately 225 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 220 amino acid substitutions, approximately 30 amino acid substitutions to approximately 215 amino acid substitutions, approximately 30 amino acid substitutions ~Approximately 210 amino acid substitutions, approximately 30 amino acid substitutions~Approximately 205 amino acid substitutions, approximately 30 amino acids Substitutions ~ approximately 200 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 30 amino acid substitutions Acid substitutions ~ approximately 190 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 185 amino acid substitutions, approximately 30 Amino acid substitutions ~ approximately 180 amino acid substitutions, approximately 30 amino acid substitutions ~ 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 Approximately 30 amino acid substitutions to approximately 160 amino acid substitutions, approximately 30 amino acid substitutions to approximately 155 amino acids Substitutions, approximately 30 amino acid substitutions to approximately 150 amino acid substitutions, approximately 30 amino acid substitutions to approximately 145 amino acid substitutions Acid substitution, approximately 30 amino acid substitutions to approximately 140 amino acid substitutions, approximately 30 amino acid substitutions to approximately 135 Amino acid substitution, approximately 30 amino acid substitutions to approximately 130 amino acid substitutions, approximately 30 amino acid substitutions to approximately 1 25 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 120 amino acid substitutions, approximately 30 amino acid substitutions ~ Approximately 115 amino acid substitutions, approximately 30 amino acid substitutions to approximately 110 amino acid substitutions, approximately 30 amino acid substitutions Substitutions: Approximately 105 amino acid substitutions, approximately 30 amino acid substitutions, approximately 100 amino acid substitutions, approximately 30 amino acids Acid substitution ~ approximately 95 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 90 amino acid substitutions, approximately 30 amino Acid substitution ~ approximately 85 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 80 amino acid substitutions, approximately 30 amino acid substitutions Acid substitution ~ approximately 75 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 70 amino acid substitutions, approximately 30 amino acid substitutions Acid substitution ~ approximately 65 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 60 amino acid substitutions, approximately 30 amino acid substitutions Acid substitution ~ approximately 55 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 50 amino acid substitutions, approximately 30 amino acid substitutions Acid substitution ~ approximately 45 amino acid substitutions, approximately 30 amino acid substitutions ~ approximately 40 amino acid substitutions, approximately 30 amino acid substitutions Acid substitution ~ approximately 35 amino acid substitutions, approximately 35 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 35 amino acid substitutions Acid substitutions ~ approximately 235 amino acid substitutions, approximately 35 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 35 Amino acid substitutions: approximately 225 amino acid substitutions, approximately 35 amino acid substitutions, approximately 220 amino acid substitutions Approximately 35 amino acid substitutions to approximately 215 amino acid substitutions, approximately 35 amino acid substitutions to approximately 210 amino acids Substitutions, approximately 35 amino acid substitutions to approximately 205 amino acid substitutions, approximately 35 amino acid substitutions to approximately 200 amino acid substitutions No acid substitution, approximately 35 amino acid substitutions to approximately 195 amino acid substitutions, approximately 35 amino acid substitutions to approximately 190 Amino acid substitutions, approximately 35 amino acid substitutions to approximately 185 amino acid substitutions, approximately 35 amino acid substitutions to approximately 1 80 amino acid substitutions, approximately 35 amino acid substitutions ~ Approximately 175 amino acid substitutions, approximately 35 amino acid substitutions ~ Approximately 170 amino acid substitutions, approximately 35 amino acid substitutions to approximately 165 amino acid substitutions, approximately 35 amino acid substitutions Amino acid substitutions: approximately 160 amino acid substitutions, approximately 35 amino acid substitutions, approximately 155 amino acid substitutions, approximately 35 amino acid substitutions. Acid substitution ~ approximately 150 amino acid substitutions, approximately 35 amino acid substitutions ~ approximately 145 amino acid substitutions, approximately 35 Mino acid substitutions ~ approximately 140 amino acid substitutions, approximately 35 amino acid substitutions ~ approximately 135 amino acid substitutions, approximately 3 5 amino acid substitutions to approximately 130 amino acid substitutions, 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 Substitution, approximately 35 amino acid substitutions to approximately 110 amino acid substitutions, approximately 35 amino acid substitutions to approximately 105 amino acid substitutions Acid substitution, approximately 35 amino acid substitutions to approximately 100 amino acid substitutions, approximately 35 amino acid substitutions to approximately 95 amino acid substitutions No acid substitution, approximately 35 amino acid substitutions to approximately 90 amino acid substitutions, approximately 35 amino acid substitutions to approximately 85 amino acid substitutions Acid substitution, approximately 35 amino acid substitutions to approximately 80 amino acid substitutions, approximately 35 amino acid substitutions to approximately 75 amino acid substitutions No acid substitution, approximately 35 amino acid substitutions to approximately 70 amino acid substitutions, approximately 35 amino acid substitutions to approximately 65 amino acid substitutions No acid substitution, approximately 35 amino acid substitutions to approximately 60 amino acid substitutions, approximately 35 amino acid substitutions to approximately 55 amino acid substitutions No acid substitution, approximately 35 amino acid substitutions to approximately 50 amino acid substitutions, approximately 35 amino acid substitutions to approximately 45 amino acid substitutions No acid substitution, approximately 35 amino acid substitutions to approximately 40 amino acid substitutions, approximately 40 amino acid substitutions to approximately 240 amino acid substitutions Mino acid substitution, approximately 40 amino acid substitutions to approximately 235 amino acid substitutions, approximately 40 amino acid substitutions to approximately 23 0 amino acid substitutions, approximately 40 amino acid substitutions to approximately 225 amino acid substitutions, approximately 40 amino acid substitutions ~Approximately 220 amino acid substitutions, approximately 40 amino acid substitutions ~Approximately 215 amino acid substitutions, approximately 40 amino acids Substitutions ~ approximately 210 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 205 amino acid substitutions, approximately 40 amino acid substitutions Acid substitutions ~ approximately 200 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 40 Amino acid substitutions ~ approximately 190 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 185 amino acid substitutions, approximately 40 amino acid substitutions to approximately 180 amino acid substitutions, approximately 40 amino acid substitutions to approximately 175 amino acid substitutions Approximately 40 amino acid substitutions to approximately 170 amino acid substitutions, approximately 40 amino acid substitutions to approximately 165 amino acids Substitutions, approximately 40 amino acid substitutions to approximately 160 amino acid substitutions, approximately 40 amino acid substitutions to approximately 155 amino acid substitutions No acid substitution, approximately 40 amino acid substitutions to approximately 150 amino acid substitutions, approximately 40 amino acid substitutions to approximately 145 Amino acid substitution, approximately 40 amino acid substitutions to approximately 140 amino acid substitutions, approximately 40 amino acid substitutions to approximately 1 35 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 130 amino acid substitutions, approximately 40 amino acid substitutions ~ Approximately 125 amino acid substitutions, approximately 40 amino acid substitutions to approximately 120 amino acid substitutions, approximately 40 amino acid substitutions Substitutions: Approximately 115 amino acid substitutions, approximately 40 amino acid substitutions, approximately 110 amino acid substitutions, approximately 40 amino acid substitutions. Acid substitution ~ approximately 105 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 100 amino acid substitutions, approximately 40 amino acid substitutions Mino acid substitution ~ approximately 95 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 90 amino acid substitutions, approximately 40 amino acid substitutions amino acid substitution ~ approximately 85 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 80 amino acid substitutions, approximately 40 amino acid substitutions Mino acid substitution ~ approximately 75 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 70 amino acid substitutions, approximately 40 amino acid substitutions Mino acid substitution ~ approximately 65 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 60 amino acid substitutions, approximately 40 amino acid substitutions amino acid substitution ~ approximately 55 amino acid substitutions, approximately 40 amino acid substitutions ~ approximately 50 amino acid substitutions, approximately 40 amino acid substitutions Mino acid substitution ~ approximately 45 amino acid substitutions, approximately 45 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 45 Amino acid substitutions ~ approximately 235 amino acid substitutions, approximately 45 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 45 amino acid substitutions to approximately 225 amino acid substitutions, approximately 45 amino acid substitutions to approximately 220 amino acids Substitutions, approximately 45 amino acid substitutions to approximately 215 amino acid substitutions, approximately 45 amino acid substitutions to approximately 210 amino acid substitutions No acid substitution, approximately 45 amino acid substitutions to approximately 205 amino acid substitutions, approximately 45 amino acid substitutions to approximately 200 Amino acid substitutions, approximately 45 amino acid substitutions to approximately 195 amino acid substitutions, approximately 45 amino acid substitutions to approximately 1 90 amino acid substitutions, approximately 45 amino acid substitutions ~ approximately 185 amino acid substitutions, approximately 45 amino acid substitutions ~ Approximately 180 amino acid substitutions, approximately 45 amino acid substitutions to approximately 175 amino acid substitutions, approximately 45 amino acid substitutions Amino acid substitutions: approximately 170 amino acid substitutions, approximately 45 amino acid substitutions, approximately 165 amino acid substitutions, approximately 45 amino acid substitutions. Acid substitution ~ approximately 160 amino acid substitutions, approximately 45 amino acid substitutions ~ approximately 155 amino acid substitutions, approximately 45 Mino acid substitution ~ approximately 150 amino acid substitutions, approximately 45 amino acid substitutions ~ approximately 145 amino acid substitutions, approximately 4 5 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 Substitution, approximately 45 amino acid substitutions to approximately 120 amino acid substitutions, approximately 45 amino acid substitutions to approximately 115 amino acid substitutions Acid substitution, approximately 45 amino acid substitutions to approximately 110 amino acid substitutions, approximately 45 amino acid substitutions to approximately 105 amino acid substitutions Mino acid substitution, approximately 45 amino acid substitutions to approximately 100 amino acid substitutions, approximately 45 amino acid substitutions to approximately 95 Amino acid substitutions, approximately 45 amino acid substitutions to approximately 90 amino acid substitutions, approximately 45 amino acid substitutions to approximately 85 Amino acid substitutions, approximately 45 amino acid substitutions to approximately 80 amino acid substitutions, approximately 45 amino acid substitutions to approximately 75 Amino acid substitutions, approximately 45 amino acid substitutions to approximately 70 amino acid substitutions, approximately 45 amino acid substitutions to approximately 65 Amino acid substitution, approximately 45 amino acid substitutions to approximately 60 amino acid substitutions, approximately 45 amino acid substitutions to approximately 55 Amino acid substitutions, approximately 45 amino acid substitutions to approximately 50 amino acid substitutions, approximately 50 amino acid substitutions to approximately 24 0 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 235 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 50 amino acid substitutions to approximately 225 amino acid substitutions, approximately 50 amino acid substitutions Substitutions ~ approximately 220 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 215 amino acid substitutions, approximately 50 amino acid substitutions Acid substitutions ~ approximately 210 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 205 amino acid substitutions, approximately 50 Amino acid substitutions ~ approximately 200 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 50 amino acid substitutions to approximately 190 amino acid substitutions, approximately 50 amino acid substitutions to approximately 185 amino acid substitutions Approximately 50 amino acid substitutions to approximately 180 amino acid substitutions, approximately 50 amino acid substitutions to approximately 175 amino acids Substitutions, approximately 50 amino acid substitutions to approximately 170 amino acid substitutions, approximately 50 amino acid substitutions to approximately 165 amino acid substitutions Acid substitution, approximately 50 amino acid substitutions to approximately 160 amino acid substitutions, approximately 50 amino acid substitutions to approximately 155 Amino acid substitution, approximately 50 amino acid substitutions to approximately 150 amino acid substitutions, approximately 50 amino acid substitutions to approximately 1 45 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 140 amino acid substitutions, approximately 50 amino acid substitutions ~ about 135 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 130 amino acid substitutions, approximately 50 amino acid substitutions ~Approximately 125 amino acid substitutions, approximately 50 amino acid substitutions~Approximately 120 amino acid substitutions, approximately 50 amino acids Substitutions ~ approximately 115 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 110 amino acid substitutions, approximately 50 amino acid substitutions Acid substitutions ~ approximately 105 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 100 amino acid substitutions, approximately 50 Amino acid substitutions ~ approximately 95 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 90 amino acid substitutions, approximately 50 Amino acid substitutions ~ approximately 85 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 80 amino acid substitutions, approximately 50 Amino acid substitutions ~ approximately 75 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 70 amino acid substitutions, approximately 50 Amino acid substitutions ~ approximately 65 amino acid substitutions, approximately 50 amino acid substitutions ~ approximately 60 amino acid substitutions, approximately 50 Amino acid substitutions ~ approximately 55 amino acid substitutions, approximately 60 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 6 0 amino acid substitutions to approximately 235 amino acid substitutions, approximately 60 amino acid substitutions to approximately 230 amino acid substitutions, Approximately 60 amino acid substitutions to approximately 225 amino acid substitutions, approximately 60 amino acid substitutions to approximately 220 amino acid substitutions Acid substitution, approximately 60 amino acid substitutions to approximately 215 amino acid substitutions, approximately 60 amino acid substitutions to approximately 210 amino acid substitutions Mino acid substitution, approximately 60 amino acid substitutions to approximately 205 amino acid substitutions, approximately 60 amino acid substitutions to approximately 20 0 amino acid substitutions, approximately 60 amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 60 amino acid substitutions ~ approximately 190 amino acid substitutions, approximately 60 amino acid substitutions to approximately 185 amino acid substitutions, approximately 60 amino acid substitutions ~Approximately 180 amino acid substitutions, approximately 60 amino acid substitutions~Approximately 175 amino acid substitutions, approximately 60 amino acids Substitutions ~ approximately 170 amino acid substitutions, approximately 60 amino acid substitutions ~ approximately 165 amino acid substitutions, approximately 60 amino acid substitutions Acid substitutions ~ approximately 160 amino acid substitutions, approximately 60 amino acid substitutions ~ approximately 155 amino acid substitutions, approximately 60 Amino acid substitutions ~ approximately 150 amino acid substitutions, approximately 60 amino acid substitutions ~ approximately 145 amino acid substitutions, approximately 60 amino acid substitutions to approximately 140 amino acid substitutions, approximately 60 amino acid substitutions to approximately 135 amino acid substitutions Approximately 60 amino acid substitutions to approximately 130 amino acid substitutions, approximately 60 amino acid substitutions to approximately 125 amino acids Substitutions, approximately 60 amino acid substitutions to approximately 120 amino acid substitutions, approximately 60 amino acid substitutions to approximately 115 amino acid substitutions Acid substitution, approximately 60 amino acid substitutions to approximately 110 amino acid substitutions, approximately 60 amino acid substitutions to approximately 105 Amino acid substitution, approximately 60 amino acid substitutions to approximately 100 amino acid substitutions, approximately 60 amino acid substitutions to approximately 9 5 amino acid substitutions, approximately 60 amino acid substitutions to approximately 90 amino acid substitutions, approximately 60 amino acid substitutions to approximately 8 5 amino acid substitutions, approximately 60 amino acid substitutions to approximately 80 amino acid substitutions, approximately 60 amino acid substitutions to approximately 7 5 amino acid substitutions, approximately 60 amino acid substitutions ~ approximately 70 amino acid substitutions, approximately 60 amino acid substitutions ~ approximately 6 5 amino acid substitutions, approximately 70 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 70 amino acid substitutions ~ approximately 235 amino acid substitutions, approximately 70 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 70 amino acid substitutions ~Approximately 225 amino acid substitutions, approximately 70 amino acid substitutions ~Approximately 220 amino acid substitutions, approximately 70 amino acid substitutions Acid substitutions ~ approximately 215 amino acid substitutions, approximately 70 amino acid substitutions ~ approximately 210 amino acid substitutions, approximately 10 Amino acid substitutions ~ approximately 205 amino acid substitutions, approximately 70 amino acid substitutions ~ approximately 200 amino acid substitutions, approximately 70 amino acid substitutions to approximately 195 amino acid substitutions, approximately 70 amino acid substitutions to approximately 190 amino acid substitutions Approximately 70 amino acid substitutions to approximately 185 amino acid substitutions, approximately 70 amino acid substitutions to approximately 180 amino acids Substitutions, approximately 70 amino acid substitutions to approximately 175 amino acid substitutions, approximately 70 amino acid substitutions to approximately 170 amino acid substitutions Acid substitution, approximately 70 amino acid substitutions to approximately 165 amino acid substitutions, approximately 70 amino acid substitutions to approximately 160 Amino acid substitution, approximately 70 amino acid substitutions to approximately 155 amino acid substitutions, approximately 70 amino acid substitutions to approximately 1 50 amino acid substitutions, approximately 70 amino acid substitutions ~ approximately 145 amino acid substitutions, approximately 70 amino acid substitutions ~ Approximately 140 amino acid substitutions, approximately 70 amino acid substitutions to approximately 135 amino acid substitutions, approximately 70 amino acid substitutions Amino acid substitutions: approximately 130 amino acid substitutions, approximately 70 amino acid substitutions, approximately 125 amino acid substitutions, approximately 70 amino acids Acid substitution ~ approximately 120 amino acid substitutions, approximately 70 amino acid substitutions ~ approximately 115 amino acid substitutions, approximately 70 Mino acid substitutions ~ approximately 110 amino acid substitutions, approximately 70 amino acid substitutions ~ approximately 105 amino acid substitutions, approximately 7 0 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 acid substitutions, 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 acids Substitution, approximately 80 amino acid substitutions, substitutions ~ approximately 220 amino acid substitutions, approximately 80 amino acid substitutions ~ approximately 215 Amino acid substitution, approximately 80 amino acid substitutions to approximately 210 amino acid substitutions, approximately 80 amino acid substitutions to approximately 2 0.5 amino acid substitutions, approximately 80 amino acid substitutions ~ Approximately 200 amino acid substitutions, approximately 80 amino acid substitutions ~ Approximately 195 amino acid substitutions, approximately 80 amino acid substitutions to approximately 190 amino acid substitutions, approximately 80 amino acid substitutions Substitutions: Approximately 185 amino acid substitutions, approximately 80 amino acid substitutions, approximately 180 amino acid substitutions, approximately 80 amino acids Acid substitution ~ approximately 175 amino acid substitutions, approximately 80 amino acid substitutions ~ approximately 170 amino acid substitutions, approximately 80 amino acid substitutions Mino acid substitution ~ approximately 165 amino acid substitutions, approximately 80 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 8 0 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 amino acid substitutions to approximately 140 amino acid substitutions Substitution, approximately 80 amino acid substitutions to approximately 135 amino acid substitutions, approximately 80 amino acid substitutions to approximately 130 amino acid substitutions Acid substitution, approximately 80 amino acid substitutions to approximately 125 amino acid substitutions, approximately 80 amino acid substitutions to approximately 120 amino acid substitutions Mino acid substitution, approximately 80 amino acid substitutions to approximately 115 amino acid substitutions, approximately 80 amino acid substitutions to approximately 11 0 amino acid substitutions, approximately 80 amino acid substitutions ~ approximately 105 amino acid substitutions, approximately 80 amino acid substitutions ~ approximately 100 amino acid substitutions, approximately 80 amino acid substitutions ~ Approximately 95 amino acid substitutions, approximately 80 amino acid substitutions ~ Approximately 90 amino acid substitutions, approximately 80 amino acid substitutions ~ Approximately 85 amino acid substitutions, approximately 90 amino acid substitutions ~ Approximately 240 amino acid substitutions, approximately 90 amino acid substitutions to approximately 235 amino acid substitutions, approximately 90 amino acid substitutions Amino acid substitutions: approximately 230 amino acid substitutions, approximately 90 amino acid substitutions, approximately 225 amino acid substitutions, approximately 90 amino acid Acid substitutions ~ approximately 220 amino acid substitutions, approximately 90 amino acid substitutions ~ approximately 215 amino acid substitutions, approximately 9 0 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 Substitution, approximately 90 amino acid substitutions to approximately 190 amino acid substitutions, approximately 90 amino acid substitutions to approximately 185 amino acid substitutions Acid substitution, approximately 90 amino acid substitutions to approximately 180 amino acid substitutions, approximately 90 amino acid substitutions to approximately 175 amino acid substitutions Mino acid substitution, approximately 90 amino acid substitutions to approximately 170 amino acid substitutions, approximately 90 amino acid substitutions to approximately 16 5 amino acid substitutions, approximately 90 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 90 amino acid substitutions ~ approximately 155 amino acid substitutions, approximately 90 amino acid substitutions ~ approximately 150 amino acid substitutions, approximately 90 amino acid substitutions ~Approximately 145 amino acid substitutions, approximately 90 amino acid substitutions~Approximately 140 amino acid substitutions, approximately 90 amino acids Substitutions ~ approximately 135 amino acid substitutions, approximately 90 amino acid substitutions ~ approximately 130 amino acid substitutions, approximately 90 amino acid substitutions Acid substitutions ~ approximately 125 amino acid substitutions, approximately 90 amino acid substitutions ~ approximately 120 amino acid substitutions, approximately 90 Amino acid substitutions ~ approximately 115 amino acid substitutions, approximately 90 amino acid substitutions ~ approximately 110 amino acid substitutions, approximately 90 amino acid substitutions to approximately 105 amino acid substitutions, approximately 90 amino acid substitutions to approximately 100 amino acid substitutions Approximately 90 amino acid substitutions to approximately 95 amino acid substitutions, approximately 100 amino acid substitutions to approximately 240 amino acid substitutions Substitutions, approximately 100 amino acid substitutions to approximately 235 amino acid substitutions, approximately 100 amino acid substitutions to approximately 230 Amino acid substitution, approximately 100 amino acid substitutions to approximately 225 amino acid substitutions, approximately 100 amino acid substitutions Substitutions: approximately 220 amino acid substitutions, approximately 100 amino acid substitutions, approximately 215 amino acid substitutions, approximately 100 amino acid substitutions Mino acid substitutions ~ approximately 210 amino acid substitutions, approximately 100 amino acid substitutions ~ approximately 205 amino acid substitutions, approximately 100 amino acid substitutions to approximately 200 amino acid substitutions, approximately 100 amino acid substitutions to approximately 195 amino acids Substitution, approximately 100 amino acid substitutions to approximately 190 amino acid substitutions, approximately 100 amino acid substitutions to approximately 185 Amino acid substitution, approximately 100 amino acid substitutions ~ approximately 180 amino acid substitutions, approximately 100 amino acid substitutions ~ Approximately 175 amino acid substitutions, approximately 100 amino acid substitutions to approximately 170 amino acid substitutions, approximately 100 amino acids Acid substitution ~ approximately 165 amino acid substitutions, approximately 100 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 10 0 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 acids Acid substitution, approximately 100 amino acid substitutions to approximately 135 amino acid substitutions, approximately 100 amino acid substitutions to approximately 1 30 amino acid substitutions, approximately 100 amino acid substitutions to approximately 125 amino acid substitutions, approximately 100 amino acid substitutions Substitutions: approximately 120 amino acid substitutions, approximately 100 amino acid substitutions, approximately 115 amino acid substitutions, approximately 100 amino acid substitutions Mino acid substitution ~ approximately 110 amino acid substitutions, approximately 100 amino acid substitutions ~ approximately 105 amino acid substitutions, approximately 110 amino acid substitutions to approximately 240 amino acid substitutions, approximately 110 amino acid substitutions to approximately 235 amino acids Substitutions, approximately 110 amino acid substitutions to approximately 230 amino acid substitutions, approximately 110 amino acid substitutions to approximately 225 Amino acid substitution, approximately 110 amino acid substitutions to approximately 220 amino acid substitutions, approximately 110 amino acid substitutions Substitutions: approximately 215 amino acid substitutions, approximately 110 amino acid substitutions, approximately 210 amino acid substitutions, approximately 110 amino acid substitutions Mino acid substitutions ~ approximately 205 amino acid substitutions, approximately 110 amino acid substitutions ~ approximately 200 amino acid substitutions, approximately 110 amino acid substitutions to approximately 195 amino acid substitutions, approximately 110 amino acid substitutions to approximately 190 amino acids 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 ~ approximately 175 amino acid substitutions, approximately 110 amino acid substitutions ~ Approximately 170 amino acid substitutions, approximately 110 amino acid substitutions to approximately 165 amino acid substitutions, approximately 110 amino acids Acid substitution ~ approximately 160 amino acid substitutions, approximately 110 amino acid substitutions ~ approximately 155 amino acid substitutions, approximately 11 0 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 Acid substitution, approximately 110 amino acid substitutions to approximately 130 amino acid substitutions, approximately 110 amino acid substitutions to approximately 1 25 amino acid substitutions, approximately 110 amino acid substitutions to approximately 120 amino acid substitutions, approximately 110 amino acid substitutions Substitutions: approximately 115 amino acid substitutions, approximately 120 amino acid substitutions, approximately 240 amino acid substitutions, approximately 120 amino acid substitutions Mino acid substitutions ~ approximately 235 amino acid substitutions, approximately 120 amino acid substitutions ~ 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 Acid substitution, approximately 120 amino acid substitutions to approximately 215 amino acid substitutions, approximately 120 amino acid substitutions to approximately 2 10 amino acid substitutions, approximately 120 amino acid substitutions to approximately 205 amino acid substitutions, approximately 120 amino acid substitutions Substitutions: approximately 200 amino acid substitutions, approximately 120 amino acid substitutions, approximately 195 amino acid substitutions, approximately 120 amino acid substitutions Mino acid substitutions ~ approximately 190 amino acid substitutions, approximately 120 amino acid substitutions ~ 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 acids Substitutions, approximately 120 amino acid substitutions to approximately 170 amino acid substitutions, approximately 100 amino acid substitutions to approximately 165 Amino acid substitution, approximately 120 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 120 amino acid substitutions ~ Approximately 155 amino acid substitutions, approximately 120 amino acid substitutions to approximately 150 amino acid substitutions, approximately 120 amino acids Acid substitution ~ approximately 145 amino acid substitutions, approximately 120 amino acid substitutions ~ approximately 140 amino acid substitutions, approximately 12 0 amino acid substitutions to approximately 135 amino acid substitutions, approximately 120 amino acid substitutions to approximately 130 amino acid substitutions Approximately 120 amino acid substitutions to approximately 125 amino acid substitutions, approximately 130 amino acid substitutions to approximately 240 amino acid substitutions Acid substitution, approximately 130 amino acid substitutions to approximately 235 amino acid substitutions, approximately 130 amino acid substitutions to approximately 2 30 amino acid substitutions, approximately 130 amino acid substitutions to approximately 225 amino acid substitutions, approximately 130 amino acid substitutions Substitutions: approximately 220 amino acid substitutions, approximately 130 amino acid substitutions to approximately 215 amino acid substitutions, approximately 13 0 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 Acid substitution, approximately 130 amino acid substitutions to approximately 190 amino acid substitutions, approximately 130 amino acid substitutions to approximately 1 85 amino acid substitutions, approximately 130 amino acid substitutions to approximately 180 amino acid substitutions, approximately 130 amino acid substitutions exchange ~Approximately 175 amino acid substitutions, approximately 130 amino acid substitutions~Approximately 170 amino acid substitutions, approximately 130 amino acids Acid substitutions ~ approximately 165 amino acid substitutions, approximately 130 amino acid substitutions ~ approximately 160 amino acid substitutions, approximately 1 30 amino acid substitutions to approximately 155 amino acid substitutions, approximately 130 amino acid substitutions to approximately 150 amino acid substitutions Substitution, approximately 130 amino acid substitutions to approximately 145 amino acid substitutions, approximately 130 amino acid substitutions to approximately 140 amino acid substitutions Mino acid substitution, approximately 130 amino acid substitutions ~ approximately 135 amino acid substitutions, approximately 140 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 140 amino acid substitutions to approximately 235 amino acid substitutions, approximately 140 amino acids Substitutions ~ approximately 230 amino acid substitutions, approximately 140 amino acid substitutions ~ approximately 225 amino acid substitutions, approximately 140 Amino acid substitutions: approximately 220 amino acid substitutions, approximately 140 amino acid substitutions, approximately 215 amino acid substitutions. Substitution, approximately 140 amino acid substitutions to approximately 210 amino acid substitutions, approximately 140 amino acid substitutions to approximately 205 amino acid substitutions Mino acid substitution, approximately 140 amino acid substitutions ~ approximately 200 amino acid substitutions, approximately 140 amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 140 amino acid substitutions ~ approximately 190 amino acid substitutions, approximately 140 amino acids Substitutions ~ approximately 185 amino acid substitutions, approximately 140 amino acid substitutions ~ approximately 180 amino acid substitutions, approximately 140 Amino acid substitutions ~ approximately 175 amino acid substitutions, approximately 140 amino acid substitutions ~ 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 acids Acid substitution, approximately 140 amino acid substitutions to approximately 155 amino acid substitutions, approximately 140 amino acid substitutions to approximately 15 0 amino acid substitutions, approximately 140 amino acid substitutions, approximately 145 amino acid substitutions, approximately 150 amino acid substitutions ~Approximately 240 amino acid substitutions, approximately 150 amino acid substitutions ~Approximately 235 amino acid substitutions, approximately 150 amino acids Acid substitutions ~ approximately 230 amino acid substitutions, approximately 150 amino acid substitutions ~ approximately 225 amino acid substitutions, approximately 1 50 amino acid substitutions to approximately 220 amino acid substitutions, approximately 150 amino acid substitutions to approximately 215 amino acid substitutions Acid substitution, approximately 150 amino acid substitutions to approximately 210 amino acid substitutions, approximately 150 amino acid substitutions to approximately 20 5 amino acid substitutions, approximately 150 amino acid substitutions ~ approximately 200 amino acid substitutions, approximately 150 amino acid substitutions ~Approximately 195 amino acid substitutions, approximately 150 amino acid substitutions~Approximately 190 amino acid substitutions, approximately 150 amino acids Acid substitutions ~ approximately 185 amino acid substitutions, approximately 150 amino acid substitutions ~ approximately 180 amino acid substitutions, approximately 1 50 amino acid substitutions to approximately 175 amino acid substitutions, approximately 150 amino acid substitutions to approximately 170 amino acid substitutions Substitution, approximately 150 amino acid substitutions to approximately 165 amino acid substitutions, approximately 150 amino acid substitutions to approximately 160 amino acid substitutions Mino acid substitution, or substitution of approximately 150 amino acids to approximately 155 amino acids, approximately 160 amino acids Substitutions: approximately 240 amino acid substitutions, approximately 160 amino acid substitutions, approximately 235 amino acid substitutions, approximately 160 amino acid substitutions Mino acid substitutions ~ approximately 230 amino acid substitutions, approximately 160 amino acid substitutions ~ approximately 225 amino acid substitutions, approximately 160 amino acid substitutions to approximately 220 amino acid substitutions, approximately 160 amino acid substitutions to approximately 215 amino acid substitutions Acid substitution, approximately 160 amino acid substitutions to approximately 210 amino acid substitutions, approximately 160 amino acid substitutions to approximately 2 0.5 amino acid substitutions, approximately 160 amino acid substitutions to approximately 200 amino acid substitutions, approximately 160 amino acid substitutions Substitutions: approximately 195 amino acid substitutions, approximately 160 amino acid substitutions, approximately 190 amino acid substitutions, approximately 160 amino acid substitutions Mino acid substitution ~ approximately 185 amino acid substitutions, approximately 160 amino acid substitutions ~ approximately 180 amino acid substitutions, approximately 160 amino acid substitutions to approximately 175 amino acid substitutions, approximately 160 amino acid substitutions to approximately 170 amino acids 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 ~ approximately 235 amino acid substitutions, approximately 170 amino acid substitutions ~ Approximately 230 amino acid substitutions, approximately 170 amino acid substitutions to approximately 225 amino acid substitutions, approximately 170 amino acids Acid substitutions ~ approximately 220 amino acid substitutions, approximately 170 amino acid substitutions ~ 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 acids Substitutions, approximately 170 amino acid substitutions to approximately 200 amino acid substitutions, approximately 170 amino acid substitutions to approximately 195 Amino acid substitutions, approximately 170 amino acid substitutions ~ approximately 190 amino acid substitutions, approximately 170 amino acid substitutions ~ Approximately 185 amino acid substitutions, approximately 170 amino acid substitutions to approximately 180 amino acid substitutions, approximately 170 amino acids Acid substitution ~ approximately 175 amino acid substitutions, approximately 180 amino acid substitutions ~ approximately 240 amino acid substitutions, approximately 18 0 amino acid substitutions to approximately 235 amino acid substitutions, approximately 180 amino acid substitutions to approximately 230 amino acid substitutions Approximately 180 amino acid substitutions to approximately 225 amino acid substitutions, approximately 180 amino acid substitutions to approximately 220 Amino acid substitutions, approximately 180 amino acid substitutions ~ approximately 215 amino acid substitutions, approximately 180 amino acid substitutions ~ Approximately 210 amino acid substitutions, approximately 180 amino acid substitutions to approximately 205 amino acid substitutions, approximately 180 amino acids Acid substitution ~ approximately 200 amino acid substitutions, approximately 180 amino acid substitutions ~ approximately 195 amino acid substitutions, approximately 18 0 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 Acid substitution, approximately 190 amino acid substitutions to approximately 230 amino acid substitutions, approximately 190 amino acid substitutions to approximately 2 25 amino acid substitutions, approximately 190 amino acid substitutions ~ approximately 220 amino acid substitutions, approximately 190 amino acids Acid substitution ~ approximately 215 amino acid substitutions, approximately 190 amino acid substitutions ~ approximately 210 amino acid substitutions, approximately 19 0 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 Acid substitution, approximately 200 amino acid substitutions to approximately 235 amino acid substitutions, approximately 200 amino acid substitutions to approximately 2 30 amino acid substitutions, approximately 200 amino acid substitutions to approximately 225 amino acid substitutions, approximately 200 amino acid substitutions Substitutions: approximately 220 amino acid substitutions, approximately 200 amino acid substitutions to approximately 215 amino acid substitutions, approximately 20 0 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 Acid substitution, approximately 205 amino acid substitutions to approximately 230 amino acid substitutions, approximately 205 amino acid substitutions to approximately 2 25 amino acid substitutions, approximately 205 amino acid substitutions ~ approximately 220 amino acid substitutions, approximately 205 amino acid substitutions Acid substitution ~ approximately 215 amino acid substitutions, approximately 205 amino acid substitutions ~ approximately 210 amino acid substitutions, approximately 21 0 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 Acid substitution, approximately 210 amino acid substitutions ~ approximately 220 amino acid substitutions, approximately 210 amino acid substitutions ~ Approximately 215 amino acid substitutions, approximately 215 amino acid substitutions to approximately 240 amino acid substitutions, approximately 215 amino Acid substitution ~ approximately 235 amino acid substitutions, approximately 215 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 21 5 amino acid substitutions to approximately 225 amino acid substitutions, approximately 215 amino acid substitutions to approximately 220 amino acid substitutions Substitutions, approximately 220 amino acid substitutions to approximately 240 amino acid substitutions, approximately 220 amino acid substitutions to approximately 235 Amino acid substitution, approximately 220 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 220 amino acid substitutions ~ Approximately 225 amino acid substitutions, approximately 225 amino acid substitutions to approximately 240 amino acid substitutions, approximately 225 amino Acid substitution ~ approximately 235 amino acid substitutions, approximately 225 amino acid substitutions ~ approximately 230 amino acid substitutions, approximately 23 0 amino acid substitutions to approximately 240 amino acid substitutions, approximately 230 amino acid substitutions to approximately 235 amino acid substitutions wild-type, full-length otoferrin protein (e.g., wild-type, human, full-length otoferrin) It may contain a sequence of proteins. Those skilled in the art will know that wild-type otoferintan derived from different species is a wild-type otoferintan derived from different species. Amino acids that are conserved across proteins are (more so than amino acids that are not conserved across different species). It should not be mutated because it is likely to be involved in activity, but it originates from a wild species. Non-conserved amino acids between different types of otoferrin proteins are mutated without losing activity. Understand that this is possible.

[0230] Active ferrin proteins are, for example, 1 amino acid to approximately 200 amino acids, 1 amino acid to Approximately 195 amino acids, 1 amino acid to approximately 190 amino acids, 1 amino acid to approximately 185 amino acids, 1 Amino acids ~ approximately 180 amino acids, 1 amino acid ~ approximately 175 amino acids, 1 amino acid ~ approximately 170 amino acids Mino acids, 1 amino acid to approximately 165 amino acids, 1 amino acid to approximately 160 amino acids, 1 amino acid to Approximately 155 amino acids, 1 amino acid to approximately 150 amino acids, 1 amino acid to approximately 145 amino acids, 1 Amino acids ~ approximately 140 amino acids, 1 amino acid ~ approximately 135 amino acids, 1 amino acid ~ approximately 130 amino acids Mino acids, 1 amino acid to approximately 125 amino acids, 1 amino acid to approximately 120 amino acids, 1 amino acid to Approximately 115 amino acids, 1 amino acid to approximately 110 amino acids, 1 amino acid to approximately 105 amino acids, 1 Amino acids ~ approximately 100 amino acids, 1 amino acid ~ approximately 95 amino acids, 1 amino acid ~ approximately 90 amino acids Acid, 1 amino acid to approximately 85 amino acids, 1 amino acid to approximately 80 amino acids, 1 amino acid to approximately 75 amino acids Mino acids, 1 amino acid to approximately 70 amino acids, 1 amino acid to approximately 65 amino acids, 1 amino acid to approximately 6 0 amino acids, 1 amino acid to approximately 55 amino acids, 1 amino acid to approximately 50 amino acids, 1 amino acid to Approximately 45 amino acids, 1 amino acid to approximately 40 amino acids, 1 amino acid to approximately 35 amino acids, 1 amino acid Acid ~ approximately 30 amino acids, 1 amino acid ~ approximately 25 amino acids, 1 amino acid ~ approximately 20 amino acids, 1 A Mino acids ~ approximately 15 amino acids, 1 amino acid ~ approximately 10 amino acids, 1 amino acid ~ approximately 9 amino acids, 1 Amino acids ~ approximately 8 amino acids, 1 amino acid ~ approximately 7 amino acids, 1 amino acid ~ approximately 6 amino acids, 1 A Mino acids ~ approximately 5 amino acids, 1 amino acid ~ approximately 4 amino acids, 1 amino acid ~ approximately 3 amino acids, approximately 2 amino acids Mino acids ~ approximately 200 amino acids, approximately 2 amino acids ~ approximately 195 amino acids, approximately 2 amino acids ~ approximately 190 Amino acids, approximately 2 amino acids to approximately 185 amino acids, approximately 2 amino acids to approximately 180 amino acids, approximately 2 Mino acids ~ approximately 175 amino acids, approximately 2 amino acids ~ approximately 170 amino acids, approximately 2 amino acids ~ approximately 165 Amino acids, approximately 2 amino acids to approximately 160 amino acids, approximately 2 amino acids to approximately 155 amino acids, approximately 2 Mino acids ~ approximately 150 amino acids, approximately 2 amino acids ~ approximately 145 amino acids, approximately 2 amino acids ~ approximately 140 Amino acids, approximately 2 amino acids to approximately 135 amino acids, approximately 2 amino acids to approximately 130 amino acids, approximately 2 Mino acids ~ approximately 125 amino acids, approximately 2 amino acids ~ approximately 120 amino acids, approximately 2 amino acids ~ approximately 115 Amino acids, approximately 2 amino acids to approximately 110 amino acids, approximately 2 amino acids to approximately 105 amino acids, approximately 2 Mino acids ~ approximately 100 amino acids, approximately 2 amino acids ~ approximately 95 amino acids, approximately 2 amino acids ~ approximately 90 amino acids 0 amino acids, approximately 2 amino acids to approximately 85 amino acids, approximately 2 amino acids to approximately 80 amino acids, approximately 2 amino acids to approximately 2 amino acids Approximately 75 amino acids, approximately 2 to approximately 70 amino acids, approximately 2 to approximately 65 amino acids, approximately 2 Amino acids ~ approximately 60 amino acids, approximately 2 amino acids ~ approximately 55 amino acids, approximately 2 amino acids ~ approximately 50 amino acids 0 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 to approximately 30 amino acids, approximately 2 to approximately 25 amino acids, approximately 2 Amino acids ~ approximately 20 amino acids, approximately 2 amino acids ~ approximately 15 amino acids, approximately 2 amino acids ~ approximately 10 amino acids 0 amino acids, approximately 2 to 9 amino acids, approximately 2 to 8 amino acids, approximately 2 to 7 amino acids Amino acids, approximately 2 amino acids to approximately 6 amino acids, approximately 2 amino acids to approximately 5 amino acids, approximately 2 amino acids to Approximately 4 amino acids, approximately 3 to approximately 200 amino acids, approximately 3 to approximately 195 amino acids, approximately 3 amino acids to approximately 190 amino acids, approximately 3 amino acids to approximately 185 amino acids, approximately 3 amino acids to approximately 1 80 amino acids, approximately 3 amino acids to approximately 175 amino acids, approximately 3 amino acids to approximately 170 amino acids, approximately 3 amino acids to approximately 165 amino acids, approximately 3 amino acids to approximately 160 amino acids, approximately 3 amino acids to approximately 1 55 amino acids, approximately 3 amino acids to approximately 150 amino acids, approximately 3 amino acids to approximately 145 amino acids, approximately 3 amino acids to approximately 140 amino acids, approximately 3 amino acids to approximately 135 amino acids, approximately 3 amino acids to approximately 1 30 amino acids, approximately 3 amino acids to approximately 125 amino acids, approximately 3 amino acids to approximately 120 amino acids, approximately 3 amino acids to approximately 115 amino acids, approximately 3 amino acids to approximately 110 amino acids, approximately 3 amino acids to approximately 1 0.5 amino acids, approximately 3 amino acids to approximately 100 amino acids, approximately 3 amino acids to approximately 95 amino acids, approximately 3 Amino acids ~ approximately 90 amino acids, approximately 3 amino acids ~ approximately 85 amino acids, approximately 3 amino acids ~ approximately 80 amino acids 0 amino acids, approximately 3 amino acids to approximately 75 amino acids, approximately 3 amino acids to approximately 70 amino acids, approximately 3 amino acids to Approximately 65 amino acids, approximately 3 to approximately 60 amino acids, approximately 3 to approximately 55 amino acids, approximately 3 Amino acids ~ approximately 50 amino acids, approximately 3 amino acids ~ approximately 45 amino acids, approximately 3 amino acids ~ approximately 40 amino acids No acids, approximately 3 amino acids to approximately 35 amino acids, approximately 3 amino acids to approximately 30 amino acids, approximately 3 amino acids to Approximately 25 amino acids, approximately 3 to approximately 20 amino acids, approximately 3 to approximately 15 amino acids, approximately 3 Amino acids ~ approximately 10 amino acids, approximately 3 amino acids ~ approximately 9 amino acids, approximately 3 amino acids ~ approximately 8 amino acids Approximately 3 to 7 amino acids, approximately 3 to 6 amino acids, approximately 3 to 5 amino acids 0 amino acids, approximately 4 amino acids to approximately 200 amino acids, approximately 4 amino acids to approximately 195 amino acids, approximately 4 amino acids Acid ~ approximately 190 amino acids, approximately 4 amino acids ~ approximately 185 amino acids, approximately 4 amino acids ~ approximately 180 amino acids 0 amino acids, approximately 4 amino acids to approximately 175 amino acids, approximately 4 amino acids to approximately 170 amino acids, approximately 4 amino acids Acid ~ approximately 165 amino acids, approximately 4 amino acids ~ approximately 160 amino acids, approximately 4 amino acids ~ approximately 155 amino acids 0 amino acids, approximately 4 amino acids to approximately 150 amino acids, approximately 4 amino acids to approximately 145 amino acids, approximately 4 amino acids Acid ~ approximately 140 amino acids, approximately 4 amino acids ~ approximately 135 amino acids, approximately 4 amino acids ~ approximately 130 amino acids 0 amino acids, approximately 4 amino acids to approximately 125 amino acids, approximately 4 amino acids to approximately 120 amino acids, approximately 4 amino acids Acid ~ approximately 115 amino acids, approximately 4 amino acids ~ approximately 110 amino acids, approximately 4 amino acids ~ approximately 105 amino acids 0 amino acids, approximately 4 amino acids to approximately 100 amino acids, approximately 4 amino acids to approximately 95 amino acids, approximately 4 amino acids ~90 amino acids, approximately 4 amino acids ~ approximately 85 amino acids, approximately 4 amino acids ~ 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 Mino acids, approximately 4 amino acids to approximately 60 amino acids, approximately 4 amino acids to approximately 55 amino acids, approximately 4 amino acids ~50 amino acids, approximately 4 amino acids ~45 amino acids, approximately 4 amino acids ~40 amino acids, approximately 4 amino acids to approximately 35 amino acids, approximately 4 amino acids to approximately 30 amino acids, approximately 4 amino acids to approximately 25 amino acids Mino acids, approximately 4 amino acids to approximately 20 amino acids, approximately 4 amino acids to approximately 15 amino acids, approximately 4 amino acids ~approximately 10 amino acids, approximately 4 amino acids ~approximately 9 amino acids, approximately 4 amino acids ~approximately 8 amino acids, approximately 4 amino acids Mino acids ~ approximately 7 amino acids, approximately 4 amino acids ~ approximately 6 amino acids, approximately 5 amino acids ~ approximately 200 amino acids Approximately 5 amino acids to approximately 195 amino acids, approximately 5 amino acids to approximately 190 amino acids, approximately 5 amino acids to Approximately 185 amino acids, approximately 5 to approximately 180 amino acids, approximately 5 to approximately 175 amino acids Approximately 5 amino acids to approximately 170 amino acids, approximately 5 amino acids to approximately 165 amino acids, approximately 5 amino acids to Approximately 160 amino acids, approximately 5 to 155 amino acids, approximately 5 to 150 amino acids Approximately 5 amino acids to approximately 145 amino acids, approximately 5 amino acids to approximately 140 amino acids, approximately 5 amino acids to Approximately 135 amino acids, approximately 5 to approximately 130 amino acids, approximately 5 to approximately 125 amino acids Approximately 5 amino acids to approximately 120 amino acids, approximately 5 amino acids to approximately 115 amino acids, approximately 5 amino acids to Approximately 110 amino acids, approximately 5 to 105 amino acids, approximately 5 to 100 amino acids Approximately 5 amino acids to approximately 95 amino acids, approximately 5 amino acids to approximately 90 amino acids, approximately 5 amino acids to approximately 8 5 amino acids, approximately 5 amino acids to approximately 80 amino acids, approximately 5 amino acids to approximately 75 amino acids, approximately 5 amino acids 0 amino acids ~ approximately 70 amino acids, approximately 5 amino acids ~ approximately 65 amino acids, approximately 5 amino acids ~ approximately 60 amino acids Approximately 5 amino acids to approximately 55 amino acids, approximately 5 amino acids to approximately 50 amino acids, approximately 5 amino acids to approximately 4 5 amino acids, approximately 5 amino acids to approximately 40 amino acids, approximately 5 amino acids to approximately 35 amino acids, approximately 5 amino acids 0 amino acids ~ approximately 30 amino acids, approximately 5 amino acids ~ approximately 25 amino acids, approximately 5 amino acids ~ approximately 20 amino acids Approximately 5 amino acids to approximately 15 amino acids, approximately 5 amino acids to approximately 10 amino acids, approximately 5 amino acids to approximately 9 Amino acids, approximately 5 amino acids to approximately 8 amino acids, approximately 5 amino acids to approximately 7 amino acids, approximately 6 amino acids to Approximately 200 amino acids, approximately 6 to 195 amino acids, approximately 6 to 190 amino acids Approximately 6 amino acids to approximately 185 amino acids, approximately 6 amino acids to approximately 180 amino acids, approximately 6 amino acids to Approximately 175 amino acids, approximately 6 to approximately 170 amino acids, approximately 6 to approximately 165 amino acids Approximately 6 amino acids to approximately 160 amino acids, approximately 6 amino acids to approximately 155 amino acids, approximately 6 amino acids to Approximately 150 amino acids, approximately 6 to 145 amino acids, approximately 6 to 140 amino acids Approximately 6 amino acids to approximately 135 amino acids, approximately 6 amino acids to approximately 130 amino acids, approximately 6 amino acids to Approximately 125 amino acids, approximately 6 to approximately 120 amino acids, approximately 6 to approximately 115 amino acids Approximately 6 amino acids to approximately 110 amino acids, approximately 6 amino acids to approximately 105 amino acids, approximately 6 amino acids to Approximately 100 amino acids, approximately 6 to approximately 95 amino acids, approximately 6 to approximately 90 amino acids, approximately 6 amino acids to approximately 85 amino acids, approximately 6 amino acids to approximately 80 amino acids, approximately 6 amino acids to approximately 75 amino acids Mino acids, approximately 6 amino acids to approximately 70 amino acids, approximately 6 amino acids to approximately 65 amino acids, approximately 6 amino acids ~60 amino acids, approximately 6 amino acids ~ approximately 55 amino acids, approximately 6 amino acids ~ approximately 50 amino acids, approximately 6 amino acids to approximately 45 amino acids, approximately 6 amino acids to approximately 40 amino acids, approximately 6 amino acids to approximately 35 amino acids Mino acids, approximately 6 amino acids to approximately 30 amino acids, approximately 6 amino acids to approximately 25 amino acids, approximately 6 amino acids ~approximately 20 amino acids, approximately 6 amino acids ~ approximately 15 amino acids, approximately 6 amino acids ~ approximately 10 amino acids, approximately 6 amino acids to approximately 9 amino acids, approximately 6 amino acids to approximately 8 amino acids, approximately 7 amino acids to approximately 200 amino acids 0 amino acids, approximately 7 amino acids to approximately 195 amino acids, approximately 7 amino acids to approximately 190 amino acids, approximately 7 amino acids Acid ~ approximately 185 amino acids, approximately 7 amino acids ~ approximately 180 amino acids, approximately 7 amino acids ~ approximately 175 amino acids 0 amino acids, approximately 7 amino acids to approximately 170 amino acids, approximately 7 amino acids to approximately 165 amino acids, approximately 7 amino acids Acid ~ approximately 160 amino acids, approximately 7 amino acids ~ approximately 155 amino acids, approximately 7 amino acids ~ approximately 150 amino acids 0 amino acids, approximately 7 amino acids to approximately 145 amino acids, approximately 7 amino acids to approximately 140 amino acids, approximately 7 amino acids Acid ~ approximately 135 amino acids, approximately 7 amino acids ~ approximately 130 amino acids, approximately 7 amino acids ~ approximately 125 amino acids 0 acids, approximately 7 amino acids to approximately 120 amino acids, approximately 7 amino acids to approximately 115 amino acids, approximately 7 amino acids Acid ~ approximately 110 amino acids, approximately 7 amino acids ~ approximately 105 amino acids, approximately 7 amino acids ~ approximately 100 amino acids 0 amino acids, approximately 7 amino acids to approximately 95 amino acids, approximately 7 amino acids to approximately 90 amino acids, approximately 7 amino acids to approximately 7 amino acids Approximately 85 amino acids, approximately 7 amino acids to approximately 80 amino acids, approximately 7 amino acids to approximately 75 amino acids, approximately 7 Amino acids ~ approximately 70 amino acids, approximately 7 amino acids ~ approximately 65 amino acids, approximately 7 amino acids ~ approximately 60 amino acids 0 amino acids, approximately 7 amino acids to approximately 55 amino acids, approximately 7 amino acids to approximately 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 ~ approximately 30 amino acids, approximately 7 amino acids ~ approximately 25 amino acids, approximately 7 amino acids ~ approximately 20 amino acids No 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 7 amino acids 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 1 80 amino acids, approximately 8 amino acids to approximately 175 amino acids, approximately 8 amino acids to approximately 170 amino acids, approximately 8 amino acids to approximately 165 amino acids, approximately 8 amino acids to approximately 160 amino acids, approximately 8 amino acids to approximately 1 55 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 1 30 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 1 0.5 amino acids, approximately 8 amino acids to approximately 100 amino acids, approximately 8 amino acids to approximately 95 amino acids, approximately 8 Amino acids ~ approximately 90 amino acids, approximately 8 amino acids ~ approximately 85 amino acids, approximately 8 amino acids ~ approximately 80 amino acids 0 amino acids, approximately 8 amino acids to approximately 75 amino acids, approximately 8 amino acids to approximately 70 amino acids, approximately 8 amino acids to Approximately 65 amino acids, approximately 8 amino acids to approximately 60 amino acids, approximately 8 amino acids to approximately 55 amino acids, approximately 8 Amino acids ~ approximately 50 amino acids, approximately 8 amino acids ~ approximately 45 amino acids, approximately 8 amino acids ~ approximately 40 amino acids of Acid, approximately 8 amino acids to approximately 35 amino acids, approximately 8 amino acids to approximately 30 amino acids, approximately 8 amino acids to approximately 25 amino acids, approximately 8 amino acids to approximately 20 amino acids, approximately 8 amino acids to approximately 15 amino acids, approximately 8 Mino acids ~ approximately 10 amino acids, approximately 10 amino acids ~ approximately 200 amino acids, approximately 10 amino acids ~ approximately 19 5 amino acids, approximately 10 to 190 amino acids, approximately 10 to 185 amino acids, Approximately 10 amino acids to approximately 180 amino acids, approximately 10 amino acids to approximately 175 amino acids, approximately 10 amino acids Acid ~ approximately 170 amino acids, approximately 10 amino acids ~ approximately 165 amino acids, approximately 10 amino acids ~ approximately 160 Amino acids, approximately 10 amino acids to approximately 155 amino acids, approximately 10 amino acids to approximately 150 amino acids, approximately 10 amino acids to approximately 145 amino acids, approximately 10 amino acids to approximately 140 amino acids, approximately 10 amino acids ~Approximately 135 amino acids, approximately 10 amino acids~Approximately 130 amino acids, approximately 10 amino acids~Approximately 125 amino acids Mino acids, approximately 10 amino acids to approximately 120 amino acids, approximately 10 amino acids to approximately 115 amino acids, approximately 1 0 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 to 95 amino acids, approximately 10 to 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 ~75 amino acids, ~10 amino acids~70 amino acids, ~10 amino acids~65 amino acids Approximately 10 amino acids to approximately 60 amino acids, approximately 10 amino acids to approximately 55 amino acids, approximately 10 amino acids ~50 amino acids, ~10 amino acids~45 amino acids, ~10 amino acids~40 amino acids Approximately 10 amino acids to approximately 35 amino acids, approximately 10 amino acids to approximately 30 amino acids, approximately 10 amino acids ~25 amino acids, ~10 amino acids~20 amino acids, ~10 amino acids~15 amino acids Approximately 15 amino acids to approximately 200 amino acids, approximately 15 amino acids to approximately 195 amino acids, approximately 15 amino acids 0.5 amino acids ~ approximately 190 amino acids, approximately 15 amino acids ~ approximately 185 amino acids, approximately 15 amino acids ~ approximately 18 0 amino acids, approximately 15 to 175 amino acids, approximately 15 to 170 amino acids, Approximately 15 amino acids to approximately 165 amino acids, approximately 15 amino acids to approximately 160 amino acids, approximately 15 amino acids Acid ~ approximately 155 amino acids, approximately 15 amino acids ~ approximately 150 amino acids, approximately 15 amino acids ~ approximately 145 Amino acids, approximately 15 amino acids to approximately 140 amino acids, approximately 15 amino acids to approximately 135 amino acids, approximately 15 amino acids to approximately 130 amino acids, approximately 15 amino acids to approximately 125 amino acids, approximately 15 amino acids ~Approximately 120 amino acids, approximately 15 amino acids~Approximately 115 amino acids, approximately 15 amino acids~Approximately 110 amino acids Mino acids, approximately 15 amino acids to approximately 105 amino acids, approximately 15 amino acids to approximately 100 amino acids, approximately 1 5 amino acids to approximately 95 amino acids, approximately 15 amino acids to approximately 90 amino acids, approximately 15 amino acids to approximately 8 5 amino acids, approximately 15 amino acids to approximately 80 amino acids, approximately 15 amino acids to approximately 75 amino acids, approximately 1 5 amino acids to approximately 70 amino acids, approximately 15 amino acids to approximately 65 amino acids, approximately 15 amino acids to approximately 6 0 amino acids, approximately 15 amino acids to approximately 55 amino acids, approximately 15 amino acids to approximately 50 amino acids, approximately 1 5 amino acids to approximately 45 amino acids, approximately 15 amino acids to approximately 40 amino acids, approximately 15 amino acids to approximately 3 5 amino acids, approximately 15 amino acids to approximately 30 amino acids, approximately 15 amino acids to approximately 25 amino acids, approximately 1 5 amino acids to approximately 20 amino acids, approximately 20 amino acids to approximately 200 amino acids, approximately 20 amino acids to approximately 195 amino acids, approximately 20 amino acids to approximately 190 amino acids, approximately 20 amino acids to approximately 185 amino acids Acid, approximately 20 amino acids to approximately 180 amino acids, approximately 20 amino acids to approximately 175 amino acids, approximately 20 Mino acids ~ approximately 170 amino acids, approximately 20 amino acids ~ approximately 165 amino acids, approximately 20 amino acids ~ approximately 1 60 amino acids, approximately 20 to 155 amino acids, approximately 20 to 150 amino acids Approximately 20 amino acids to approximately 145 amino acids, approximately 20 amino acids to approximately 140 amino acids, approximately 20 amino acids No acids ~ approximately 135 amino acids, approximately 20 amino acids ~ approximately 130 amino acids, approximately 20 amino acids ~ approximately 12 5 amino acids, approximately 20 to 120 amino acids, approximately 20 to 115 amino acids, Approximately 20 amino acids to approximately 110 amino acids, approximately 20 amino acids to approximately 105 amino acids, approximately 20 amino acids Acid ~ approximately 100 amino acids, approximately 20 amino acids ~ approximately 95 amino acids, approximately 20 amino acids ~ approximately 90 amino acids No acids, approximately 20 amino acids to approximately 85 amino acids, approximately 20 amino acids to approximately 80 amino acids, approximately 20 amino acids 0.5 amino acids, No acids, approximately 20 to 60 amino acids, approximately 20 to 55 amino acids, approximately 20 amino acids 0.50 amino acids, 20 amino acids to 45 amino acids, 20 amino acids to 40 amino acids No acids, approximately 20 to 35 amino acids, approximately 20 to 30 amino acids, approximately 20 amino acids 0.5 amino acids ~ approximately 25 amino acids, approximately 25 amino acids ~ approximately 200 amino acids, approximately 25 amino acids ~ approximately 195 amino acids 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 ~Approximately 170 amino acids, approximately 25 amino acids~Approximately 165 amino acids, approximately 25 amino acids~Approximately 160 amino acids Mino acids, approximately 25 amino acids to approximately 155 amino acids, approximately 25 amino acids to approximately 150 amino acids, approximately 2 5 amino acids to approximately 145 amino acids, approximately 25 amino acids to approximately 140 amino acids, approximately 25 amino acids to Approximately 135 amino acids, approximately 25 amino acids to approximately 130 amino acids, approximately 25 amino acids to approximately 125 amino acids No acids, approximately 25 amino acids to approximately 120 amino acids, approximately 25 amino acids to approximately 115 amino acids, approximately 25 Amino acids ~ approximately 110 amino acids, approximately 25 amino acids ~ approximately 105 amino acids, approximately 25 amino acids ~ approximately 100 amino acids, approximately 25 to 95 amino acids, approximately 25 to 90 amino acids, Approximately 25 amino acids to approximately 85 amino acids, approximately 25 amino acids to approximately 80 amino acids, approximately 25 amino acids to Approximately 75 amino acids, approximately 25 to approximately 70 amino acids, approximately 25 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 to 45 amino acids, approximately 25 to 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 to approximately 195 amino acids, approximately 30 to approximately 190 amino acids No acids, approximately 30 amino acids to approximately 185 amino acids, approximately 30 amino acids to approximately 180 amino acids, approximately 30 Amino acids ~ approximately 175 amino acids, approximately 30 amino acids ~ approximately 170 amino acids, approximately 30 amino acids ~ approximately 165 amino acids, approximately 30 amino acids to approximately 160 amino acids, approximately 30 amino acids to approximately 155 amino acids Acid, approximately 30 amino acids to approximately 150 amino acids, approximately 30 amino acids to approximately 145 amino acids, approximately 30 Mino acids ~ approximately 140 amino acids, approximately 30 amino acids ~ approximately 135 amino acids, approximately 30 amino acids ~ approximately 1 30 amino acids, approximately 30 to approximately 125 amino acids, approximately 30 to approximately 120 amino acids Approximately 30 amino acids to approximately 115 amino acids, approximately 30 amino acids to approximately 110 amino acids, approximately 30 amino acids 0.5 amino acids ~ approximately 105 amino acids, approximately 30 amino acids ~ approximately 100 amino acids, approximately 30 amino acids ~ approximately 95 amino acids Amino acids, approximately 30 amino acids to approximately 90 amino acids, approximately 30 amino acids to approximately 85 amino acids, approximately 30 Amino acids ~ approximately 80 amino acids, approximately 30 amino acids ~ approximately 75 amino acids, approximately 30 amino acids ~ 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 ~ approximately 55 amino acids, approximately 30 amino acids ~ approximately 50 amino acids, approximately 30 amino acids ~ 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 acids ~ approximately 200 amino acids, approximately 35 amino acids ~ approximately 195 amino acids, approximately 35 amino acids ~ approximately 190 amino acids, approximately 35 amino acids to approximately 185 amino acids, approximately 35 amino acids to approximately 180 amino acids Acid, approximately 35 amino acids to approximately 175 amino acids, approximately 35 amino acids to approximately 170 amino acids, approximately 35 amino acids Mino acids ~ approximately 165 amino acids, approximately 35 amino acids ~ approximately 160 amino acids, approximately 35 amino acids ~ approximately 1 55 amino acids, approximately 35 amino acids to approximately 150 amino acids, approximately 35 amino acids to approximately 145 amino acids Approximately 35 amino acids to approximately 140 amino acids, approximately 35 amino acids to approximately 135 amino acids, approximately 35 amino acids 0.5 amino acids ~ approximately 130 amino acids, approximately 35 amino acids ~ approximately 125 amino acids, approximately 35 amino acids ~ approximately 12 0 amino acids, approximately 35 to 115 amino acids, approximately 35 to 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 Acids ~ approximately 95 amino acids, approximately 35 amino acids ~ approximately 90 amino acids, approximately 35 amino acids ~ approximately 85 amino acids Acid, approximately 35 amino acids to approximately 80 amino acids, approximately 35 amino acids to approximately 75 amino acids, approximately 35 amino acids Acids ~ approximately 70 amino acids, approximately 35 amino acids ~ approximately 65 amino acids, approximately 35 amino acids ~ approximately 60 amino acids Acid, approximately 35 amino acids to approximately 55 amino acids, approximately 35 amino acids to approximately 50 amino acids, approximately 35 amino acids Acid ~ approximately 45 amino acids, approximately 35 amino acids ~ approximately 40 amino acids, approximately 40 amino acids ~ approximately 200 amino acids No acids, approximately 40 amino acids to approximately 195 amino acids, approximately 40 amino acids to approximately 190 amino acids, approximately 40 Amino acids ~ approximately 185 amino acids, approximately 40 amino acids ~ approximately 180 amino acids, approximately 40 amino acids ~ approximately 175 amino acids, approximately 40 amino acids to approximately 170 amino acids, approximately 40 amino acids to approximately 165 amino acids Acid, approximately 40 amino acids to approximately 160 amino acids, approximately 40 amino acids to approximately 155 amino acids, approximately 40 Mino acids ~ approximately 150 amino acids, approximately 40 amino acids ~ approximately 145 amino acids, approximately 40 amino acids ~ approximately 1 40 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 No acids ~ approximately 115 amino acids, approximately 40 amino acids ~ approximately 110 amino acids, approximately 40 amino acids ~ approximately 10 5 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 ~Approximately 190 amino acids, approximately 45 amino acids~Approximately 185 amino acids, approximately 45 amino acids~Approximately 180 amino acids Mino acids, approximately 45 amino acids to approximately 175 amino acids, approximately 45 amino acids to approximately 170 amino acids, approximately 4 5 amino acids ~ approximately 165 amino acids, approximately 45 amino acids ~ approximately 160 amino acids, approximately 45 amino acids ~ Approximately 155 amino acids, approximately 45 amino acids to approximately 150 amino acids, approximately 45 amino acids to approximately 145 amino acids No acids, approximately 45 amino acids to approximately 140 amino acids, approximately 45 amino acids to approximately 135 amino acids, approximately 45 Amino acids ~ approximately 130 amino acids, approximately 45 amino acids ~ approximately 125 amino acids, approximately 45 amino acids ~ approximately 120 amino acids, approximately 45 amino acids to approximately 115 amino acids, approximately 45 amino acids to approximately 110 amino acids Acid, approximately 45 amino acids to approximately 105 amino acids, approximately 45 amino acids to approximately 100 amino acids, approximately 45 amino acids Mino acids ~ approximately 95 amino acids, approximately 45 amino acids ~ approximately 90 amino acids, approximately 45 amino acids ~ approximately 85 amino acids Mino acids, approximately 45 amino acids to approximately 80 amino acids, approximately 45 amino acids to approximately 75 amino acids, approximately 45 amino acids Mino acids ~ approximately 70 amino acids, approximately 45 amino acids ~ approximately 65 amino acids, approximately 45 amino acids ~ approximately 60 amino acids Mino acids, approximately 45 amino acids to approximately 55 amino acids, approximately 45 amino acids to approximately 50 amino acids, approximately 50 amino acids Mino acids ~ approximately 200 amino acids, approximately 50 amino acids ~ approximately 195 amino acids, approximately 50 amino acids ~ approximately 1 90 amino acids, approximately 50 to 185 amino acids, approximately 50 to 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 No acids ~ approximately 165 amino acids, approximately 50 amino acids ~ approximately 160 amino acids, approximately 50 amino acids ~ approximately 15 5 amino acids, approximately 50 to 150 amino acids, approximately 50 to 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 Acid ~ approximately 130 amino acids, approximately 50 amino acids ~ approximately 125 amino acids, approximately 50 amino acids ~ 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 ~Approximately 95 amino acids, approximately 50 amino acids~approximately 90 amino acids, approximately 50 amino acids~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 ~ Approximately 70 amino acids, approximately 50 to 65 amino acids, approximately 50 to 60 amino acids, Approximately 50 amino acids to approximately 55 amino acids, approximately 55 amino acids to approximately 200 amino acids, approximately 55 amino acids ~Approximately 195 amino acids, approximately 55 amino acids~Approximately 190 amino acids, approximately 55 amino acids~Approximately 185 amino acids Mino acids, approximately 55 amino acids to approximately 180 amino acids, approximately 55 amino acids to approximately 175 amino acids, approximately 5 5 amino acids to approximately 170 amino acids, approximately 55 amino acids to approximately 165 amino acids, approximately 55 amino acids to Approximately 160 amino acids, approximately 55 to 155 amino acids, approximately 55 to 150 amino acids No acids, approximately 55 amino acids to approximately 145 amino acids, approximately 55 amino acids to approximately 140 amino acids, approximately 55 Amino acids ~ approximately 135 amino acids, approximately 55 amino acids ~ approximately 130 amino acids, approximately 55 amino acids ~ approximately 125 amino acids, approximately 55 amino acids to approximately 120 amino acids, approximately 55 amino acids to approximately 115 amino acids Acid, approximately 55 amino acids to approximately 110 amino acids, approximately 55 amino acids to approximately 105 amino acids, approximately 55 amino acids Mino acids ~ approximately 100 amino acids, approximately 55 amino acids ~ approximately 95 amino acids, approximately 55 amino acids ~ approximately 90 Amino acids, approximately 55 amino acids to approximately 85 amino acids, approximately 55 amino acids to approximately 80 amino acids, approximately 55 Amino acids ~ approximately 75 amino acids, approximately 55 amino acids ~ approximately 70 amino acids, approximately 55 amino acids ~ approximately 65 amino acids Amino acids, approximately 55 amino acids to approximately 60 amino acids, approximately 60 amino acids to approximately 200 amino acids, approximately 6 0 amino acids to approximately 195 amino acids, approximately 60 amino acids to approximately 190 amino acids, approximately 60 amino acids to approximately 60 amino acids Approximately 185 amino acids, approximately 60 to 180 amino acids, approximately 60 to 175 amino acids No acids, approximately 60 amino acids to approximately 170 amino acids, approximately 60 amino acids to approximately 165 amino acids, approximately 60 Amino acids ~ approximately 160 amino acids, approximately 60 amino acids ~ approximately 155 amino acids, approximately 60 amino acids ~ approximately 150 amino acids, approximately 60 amino acids to approximately 145 amino acids, approximately 60 amino acids to approximately 140 amino acids Acid, approximately 60 amino acids to approximately 135 amino acids, approximately 60 amino acids to approximately 130 amino acids, approximately 60 amino acids Mino acids ~ approximately 125 amino acids, approximately 60 amino acids ~ approximately 120 amino acids, approximately 60 amino acids ~ approximately 1 15 amino acids, approximately 60 to 110 amino acids, approximately 60 to 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 Acid ~ approximately 90 amino acids, approximately 60 amino acids ~ approximately 85 amino acids, approximately 60 amino acids ~ approximately 80 amino acids Acid, approximately 60 amino acids to approximately 75 amino acids, approximately 60 amino acids to approximately 70 amino acids, approximately 60 amino acids Acid ~ approximately 65 amino acids, approximately 65 amino acids ~ approximately 200 amino acids, approximately 65 amino acids ~ approximately 195 amino acids Mino acids, approximately 65 amino acids to approximately 190 amino acids, approximately 65 amino acids to approximately 185 amino acids, approximately 6 5 amino acids to approximately 180 amino acids, approximately 65 amino acids to approximately 175 amino acids, approximately 65 amino acids to Approximately 170 amino acids, approximately 65 to 165 amino acids, approximately 65 to 160 amino acids No acids, approximately 65 amino acids to approximately 155 amino acids, approximately 65 amino acids to approximately 150 amino acids, approximately 65 Amino acids ~ approximately 145 amino acids, approximately 65 amino acids ~ approximately 140 amino acids, approximately 65 amino acids ~ approximately 135 amino acids, approximately 65 amino acids to approximately 130 amino acids, approximately 65 amino acids to approximately 125 amino acids Acid, approximately 65 amino acids to approximately 120 amino acids, approximately 65 amino acids to approximately 115 amino acids, approximately 65 amino acids Mino acids ~ approximately 110 amino acids, approximately 65 amino acids ~ approximately 105 amino acids, approximately 65 amino acids ~ approximately 1 00 amino acids, approximately 65 amino acids to approximately 95 amino acids, approximately 65 amino acids to approximately 90 amino acids, approximately 65 amino acids to approximately 85 amino acids, approximately 65 amino acids to approximately 80 amino acids, approximately 65 amino acids to approximately 75 amino acids, approximately 65 to 70 amino acids, approximately 70 to 200 amino acids, Approximately 70 amino acids to approximately 195 amino acids, approximately 70 amino acids to approximately 190 amino acids, approximately 70 amino acids Acid ~ approximately 185 amino acids, approximately 70 amino acids ~ approximately 180 amino acids, approximately 70 amino acids ~ approximately 175 Amino acids, approximately 70 amino acids to approximately 170 amino acids, approximately 70 amino acids to approximately 165 amino acids, approximately 70 amino acids to approximately 160 amino acids, approximately 70 amino acids to approximately 155 amino acids, approximately 70 amino acids ~Approximately 150 amino acids, approximately 70 amino acids~Approximately 145 amino acids, approximately 70 amino acids~Approximately 140 amino acids Mino acids, approximately 70 amino acids to approximately 135 amino acids, approximately 70 amino acids to approximately 130 amino acids, approximately 7 0 amino acids to approximately 125 amino acids, approximately 70 amino acids to approximately 120 amino acids, approximately 70 amino acids to approximately 70 amino acids Approximately 115 amino acids, approximately 70 to 110 amino acids, approximately 70 to 105 amino acids No acids, approximately 70 amino acids to approximately 100 amino acids, approximately 70 amino acids to approximately 95 amino acids, approximately 70 amino acids Mino acids ~ approximately 90 amino acids, approximately 70 amino acids ~ approximately 85 amino acids, approximately 70 amino acids ~ approximately 80 amino acids Mino acids, approximately 70 amino acids to approximately 75 amino acids, approximately 75 amino acids to approximately 200 amino acids, approximately 75 Amino acids ~ approximately 195 amino acids, approximately 75 amino acids ~ approximately 190 amino acids, approximately 75 amino acids ~ approximately 185 amino acids, approximately 75 amino acids to approximately 180 amino acids, approximately 75 amino acids to approximately 175 amino acids Acid, approximately 75 amino acids to approximately 170 amino acids, approximately 75 amino acids to approximately 165 amino acids, approximately 75 amino acids Mino acids ~ approximately 160 amino acids, approximately 75 amino acids ~ approximately 155 amino acids, approximately 75 amino acids ~ approximately 1 50 amino acids, approximately 75 to 145 amino acids, approximately 75 to 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 0.5 amino acids ~ approximately 125 amino acids, approximately 75 amino acids ~ approximately 120 amino acids, approximately 75 amino acids ~ approximately 11 5 amino acids, approximately 75 amino acids to approximately 110 amino acids, approximately 75 amino acids to approximately 105 amino acids, Approximately 75 amino acids to approximately 100 amino acids, approximately 75 amino acids to approximately 95 amino acids, approximately 75 amino acids ~90 amino acids, ~75 amino acids~85 amino acids, ~75 amino acids~80 amino acids Approximately 80 amino acids to approximately 200 amino acids, approximately 80 amino acids to approximately 195 amino acids, approximately 80 amino acids 0.0 amino acids ~ approximately 190 amino acids, approximately 80 amino acids ~ approximately 185 amino acids, approximately 80 amino acids ~ approximately 18 0 amino acids, approximately 80 to 175 amino acids, approximately 80 to 170 amino acids, Approximately 80 amino acids to approximately 165 amino acids, approximately 80 amino acids to approximately 160 amino acids, approximately 80 amino acids Acid ~ approximately 155 amino acids, approximately 80 amino acids ~ approximately 150 amino acids, approximately 80 amino acids ~ approximately 145 amino acids Amino acids, approximately 80 amino acids to approximately 140 amino acids, approximately 80 amino acids to approximately 135 amino acids, approximately 80 amino acids to approximately 130 amino acids, approximately 80 amino acids to approximately 125 amino acids, approximately 80 amino acids ~Approximately 120 amino acids, approximately 80 amino acids~Approximately 115 amino acids, approximately 80 amino acids~Approximately 110 amino acids Mino acids, approximately 80 amino acids to approximately 105 amino acids, approximately 80 amino acids to approximately 100 amino acids, approximately 8 0 amino acids to approximately 95 amino acids, approximately 80 amino acids to approximately 90 amino acids, approximately 80 amino acids to approximately 8 5 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 Acid ~ approximately 180 amino acids, approximately 85 amino acids ~ approximately 175 amino acids, approximately 85 amino acids ~ 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 ~Approximately 145 amino acids, approximately 85 amino acids~Approximately 140 amino acids, approximately 85 amino acids~Approximately 135 amino acids Mino acids, approximately 85 amino acids to approximately 130 amino acids, approximately 85 amino acids to approximately 125 amino acids, approximately 8 5 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 to 105 amino acids, approximately 85 to 100 amino acids No acids, approximately 85 amino acids to approximately 95 amino acids, approximately 85 amino acids to approximately 90 amino acids, approximately 90 amino acids 0.0 amino acids ~ approximately 200 amino acids, approximately 90 amino acids ~ approximately 195 amino acids, approximately 90 amino acids ~ approximately 19 0 amino acids, approximately 90 to 185 amino acids, approximately 90 to 180 amino acids, Approximately 90 amino acids to approximately 175 amino acids, approximately 90 amino acids to approximately 170 amino acids, approximately 90 amino acids Acid ~ approximately 165 amino acids, approximately 90 amino acids ~ approximately 160 amino acids, approximately 90 amino acids ~ approximately 155 amino acids Amino acids, approximately 90 amino acids to approximately 150 amino acids, approximately 90 amino acids to approximately 145 amino acids, approximately 90 amino acids to approximately 140 amino acids, approximately 90 amino acids to approximately 135 amino acids, approximately 90 amino acids ~Approximately 130 amino acids, approximately 90 amino acids~Approximately 125 amino acids, approximately 90 amino acids~Approximately 120 amino acids Mino acids, approximately 90 amino acids to approximately 115 amino acids, approximately 90 amino acids to approximately 110 amino acids, approximately 9 0 amino acids to approximately 105 amino acids, approximately 90 amino acids to approximately 100 amino acids, approximately 90 amino acids to approximately 90 amino acids Approximately 95 amino acids, approximately 95 to approximately 200 amino acids, approximately 95 to approximately 195 amino acids Acid, approximately 95 amino acids to approximately 190 amino acids, approximately 95 amino acids to approximately 185 amino acids, approximately 95 amino acids Mino acids ~ approximately 180 amino acids, approximately 95 amino acids ~ approximately 175 amino acids, approximately 95 amino acids ~ approximately 1 70 amino acids, approximately 95 to 165 amino acids, approximately 95 to 160 amino acids Approximately 95 amino acids to approximately 155 amino acids, approximately 95 amino acids to approximately 150 amino acids, approximately 95 amino acids 0.5 amino acids ~ approximately 145 amino acids, approximately 95 amino acids ~ approximately 140 amino acids, approximately 95 amino acids ~ approximately 13 5 amino acids, approximately 95 to 130 amino acids, approximately 95 to 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 Acid ~ approximately 110 amino acids, approximately 95 amino acids ~ approximately 105 amino acids, approximately 95 amino acids ~ 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 10 0 amino acids to approximately 180 amino acids, approximately 100 amino acids to approximately 175 amino acids, approximately 100 amino acids Acid ~ approximately 170 amino acids, approximately 100 amino acids ~ approximately 165 amino acids, approximately 100 amino acids ~ approximately 1 60 amino acids, approximately 100 amino acids to approximately 155 amino acids, approximately 100 amino acids to approximately 150 amino acids No acids, approximately 100 amino acids to approximately 145 amino acids, approximately 100 amino acids to approximately 140 amino acids, approximately 100 amino acids to approximately 135 amino acids, approximately 100 amino acids to approximately 130 amino acids, approximately 100 amino acids Mino acids ~ approximately 125 amino acids, approximately 100 amino acids ~ approximately 120 amino acids, approximately 100 amino acids ~ Approximately 115 amino acids, approximately 100 to 110 amino acids, approximately 100 to 105 amino acids Amino acids, approximately 105 amino acids to approximately 200 amino acids, approximately 105 amino acids to approximately 195 amino acids Approximately 105 amino acids to approximately 190 amino acids, approximately 105 amino acids to approximately 185 amino acids, approximately 10 5 amino acids to approximately 180 amino acids, approximately 105 amino acids to approximately 175 amino acids, approximately 105 amino acids Acid ~ approximately 170 amino acids, approximately 105 amino acids ~ approximately 165 amino acids, approximately 105 amino acids ~ approximately 1 60 amino acids, approximately 105 amino acids to approximately 155 amino acids, approximately 105 amino acids to approximately 150 amino acids No acids, approximately 105 amino acids to approximately 145 amino acids, approximately 105 amino acids to approximately 140 amino acids, approximately 105 amino acids to approximately 135 amino acids, approximately 105 amino acids to approximately 130 amino acids, approximately 105 amino acids Mino acids ~ approximately 125 amino acids, approximately 105 amino acids ~ approximately 120 amino acids, approximately 105 amino acids ~ Approximately 115 amino acids, approximately 105 to 110 amino acids, approximately 110 to 200 amino acids Amino acids, approximately 110 amino acids to approximately 195 amino acids, approximately 110 amino acids to approximately 190 amino acids Approximately 110 amino acids to approximately 185 amino acids, approximately 110 amino acids to approximately 180 amino acids, approximately 11 0 amino acids to approximately 175 amino acids, approximately 110 amino acids to approximately 170 amino acids, approximately 110 amino acids Acid ~ approximately 165 amino acids, approximately 110 amino acids ~ approximately 160 amino acids, approximately 110 amino acids ~ approximately 1 55 amino acids, approximately 110 amino acids to approximately 150 amino acids, approximately 110 amino acids to approximately 145 amino acids No 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 Mino acids ~ approximately 120 amino acids, approximately 110 amino acids ~ approximately 115 amino acids, approximately 115 amino acids ~ Approximately 200 amino acids, approximately 115 to approximately 195 amino acids, approximately 115 to approximately 190 amino acids Amino acids, approximately 115 amino acids to approximately 185 amino acids, approximately 115 amino acids to approximately 180 amino acids Approximately 115 amino acids to approximately 175 amino acids, approximately 115 amino acids to approximately 170 amino acids, approximately 11 5 amino acids to approximately 165 amino acids, approximately 115 amino acids to approximately 160 amino acids, approximately 115 amino acids Acid ~ approximately 155 amino acids, approximately 115 amino acids ~ approximately 150 amino acids, approximately 115 amino acids ~ approximately 1 45 amino acids, approximately 115 amino acids to approximately 140 amino acids, approximately 115 amino acids to approximately 135 amino acids of Acid, approximately 115 amino acids to approximately 130 amino acids, approximately 115 amino acids to approximately 125 amino acids, approximately 1 15 amino acids to approximately 120 amino acids, approximately 120 amino acids to approximately 200 amino acids, approximately 120 amino acids 0 amino acids ~ approximately 195 amino acids, approximately 120 amino acids ~ approximately 190 amino acids, approximately 120 amino acids ~ approximately 185 amino acids, approximately 120 amino acids to approximately 180 amino acids, approximately 120 amino acids to approximately 175 amino acids Mino acids, approximately 120 to 170 amino acids, approximately 120 to 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 ~ approximately 150 amino acids, approximately 120 amino acids ~ approximately 145 amino acids, approximately 120 amino acids ~140 amino acids, ~120 amino acids~135 amino acids, ~120 amino acids~13 0 amino acids, approximately 120 amino acids to approximately 125 amino acids, approximately 125 amino acids to approximately 200 amino acids Acid, approximately 125 amino acids to approximately 195 amino acids, approximately 125 amino acids to approximately 190 amino acids, approximately 1 25 amino acids to approximately 185 amino acids, approximately 125 amino acids to approximately 180 amino acids, approximately 125 amino acids No acids ~ approximately 175 amino acids, approximately 125 amino acids ~ approximately 170 amino acids, approximately 125 amino acids ~ approximately 165 amino acids, approximately 125 amino acids to approximately 160 amino acids, approximately 125 amino acids to approximately 155 amino acids Mino acids, approximately 125 amino acids to approximately 150 amino acids, 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 ~ approximately 130 amino acids, approximately 130 amino acids ~ approximately 200 amino acids, approximately 130 amino acids ~Approximately 195 amino acids, approximately 130 amino acids~Approximately 190 amino acids, approximately 130 amino acids~Approximately 18 5 amino acids, approximately 130 amino acids to approximately 180 amino acids, approximately 130 amino acids to approximately 175 amino acids Acid, approximately 130 amino acids to approximately 170 amino acids, approximately 130 amino acids to approximately 165 amino acids, approximately 1 30 amino acids to approximately 160 amino acids, approximately 130 amino acids to approximately 155 amino acids, approximately 130 amino acids 0.0 amino acids ~ approximately 150 amino acids, approximately 130 amino acids ~ approximately 145 amino acids, approximately 130 amino acids ~ approximately 140 amino acids, approximately 130 amino acids to approximately 135 amino acids, approximately 135 amino acids to approximately 200 amino acids Mino acids, approximately 135 amino acids to approximately 195 amino acids, approximately 135 amino acids to approximately 190 amino acids, Approximately 135 amino acids to approximately 185 amino acids, approximately 135 amino acids to approximately 180 amino acids, approximately 135 Amino acids ~ approximately 175 amino acids, approximately 135 amino acids ~ approximately 170 amino acids, approximately 135 amino acids ~Approximately 165 amino acids, approximately 135 amino acids~Approximately 160 amino acids, approximately 135 amino acids~Approximately 15 5 amino acids, approximately 135 amino acids to approximately 150 amino acids, approximately 135 amino acids to approximately 145 amino acids Acid, approximately 135 amino acids to approximately 140 amino acids, approximately 140 amino acids to approximately 200 amino acids, approximately 1 40 amino acids to approximately 195 amino acids, approximately 140 amino acids to approximately 190 amino acids, approximately 140 amino acids 0.5 amino acids ~ approximately 185 amino acids, approximately 140 amino acids ~ approximately 180 amino acids, approximately 140 amino acids ~ approximately 175 amino acids, approximately 140 amino acids to approximately 170 amino acids, approximately 140 amino acids to approximately 165 amino acids Mino acids, approximately 140 amino acids to approximately 160 amino acids, approximately 140 amino acids to approximately 155 amino acids, Approximately 140 amino acids to approximately 150 amino acids, approximately 40 amino acids to approximately 145 amino acids, approximately 145 amino acids Mino acids ~ approximately 200 amino acids, approximately 145 amino acids ~ approximately 195 amino acids, approximately 145 amino acids ~ Approximately 190 amino acids, approximately 145 to 185 amino acids, approximately 145 to 180 amino acids Amino acids, approximately 145 amino acids to approximately 175 amino acids, approximately 145 amino acids to approximately 170 amino acids Approximately 145 amino acids to approximately 165 amino acids, approximately 145 amino acids to approximately 160 amino acids, approximately 14 5 amino acids to approximately 155 amino acids, approximately 145 amino acids to approximately 150 amino acids, approximately 150 amino acids Acid ~ approximately 200 amino acids, approximately 150 amino acids ~ approximately 195 amino acids, approximately 150 amino acids ~ approximately 1 90 amino acids, approximately 150 amino acids to approximately 185 amino acids, approximately 150 amino acids to approximately 180 amino acids No 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 Mino acids ~ approximately 155 amino acids, approximately 155 amino acids ~ approximately 200 amino acids, approximately 155 amino acids ~ Approximately 195 amino acids, approximately 155 to 190 amino acids, approximately 155 to 185 amino acids Amino acids, approximately 155 amino acids to approximately 180 amino acids, approximately 155 amino acids to approximately 175 amino acids Approximately 155 amino acids to approximately 170 amino acids, approximately 155 amino acids to approximately 165 amino acids, approximately 15 5 amino acids to approximately 160 amino acids, approximately 160 amino acids to approximately 200 amino acids, approximately 160 amino acids Acid ~ approximately 195 amino acids, approximately 160 amino acids ~ approximately 190 amino acids, approximately 160 amino acids ~ approximately 1 85 amino acids, approximately 160 amino acids to approximately 180 amino acids, approximately 160 amino acids to approximately 175 amino acids No acids, approximately 160 amino acids to approximately 170 amino acids, approximately 160 amino acids to approximately 165 amino acids, approximately 165 amino acids to approximately 200 amino acids, approximately 165 amino acids to approximately 195 amino acids, approximately 165 amino acids Mino acids ~ approximately 190 amino acids, approximately 165 amino acids ~ approximately 185 amino acids, approximately 165 amino acids ~ Approximately 180 amino acids, approximately 165 to 175 amino acids, approximately 165 to 170 amino acids 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 17 0 amino acids to approximately 180 amino acids, approximately 170 amino acids to approximately 175 amino acids, approximately 175 amino acids Acid ~ approximately 200 amino acids, approximately 175 amino acids ~ approximately 195 amino acids, approximately 175 amino acids ~ approximately 1 90 amino acids, approximately 175 amino acids to approximately 185 amino acids, approximately 175 amino acids to approximately 180 amino acids No acids, approximately 180 amino acids to approximately 200 amino acids, approximately 180 amino acids to approximately 195 amino acids, approximately 180 amino acids to approximately 190 amino acids, approximately 180 amino acids to approximately 185 amino acids, approximately 185 amino acids Mino acids ~ approximately 200 amino acids, approximately 185 amino acids ~ approximately 195 amino acids, approximately 185 amino acids ~ Approximately 190 amino acids, approximately 190 amino acids to approximately 200 amino acids, approximately 190 amino acids to approximately 195 Wild-type Otoferii with approximately 195 to 200 amino acid deletions, full-length Otoferii It may contain sequences of proteins (e.g., wild-type, human, full-length otoferrin protein). Two or more amino acids are missing from the wild-type, full-length otoferrin protein sequence. In some embodiments where the amino acids are missing, two or more are missing from the full-length protein. The sequence can be continuous. Two or more amino acids are wild-type, full-length oval. In other embodiments, deletions from the toferin protein sequence include two or more deletions. The missing amino acids are not contiguous in the sequence of the wild-type, full-length protein. Those skilled in the art will know that different... The amino acids conserved between wild-type and full-length otoferrin proteins derived from the species are, Because it is more likely to be involved in activity than amino acids that are not conserved between different species, deletions are It should not be conserved, but conserved between wild-type and full-length otoferrin proteins from different species. Understand that unprocessed amino acids can be deleted without losing their activity. In some cases, active otoferrin proteins are, for example, 1 amino acid to about 100 amino acids. No acids, 1 amino acid to approximately 95 amino acids, 1 amino acid to approximately 90 amino acids, 1 amino acid to approximately 85 Amino acids, 1 amino acid to approximately 80 amino acids, 1 amino acid to approximately 75 amino acids, 1 amino acid to approximately 70 amino acids, 1 amino acid to approximately 65 amino acids, 1 amino acid to approximately 60 amino acids, 1 amino acid ~Approximately 55 amino acids, 1 amino acid ~Approximately 50 amino acids, 1 amino acid ~Approximately 45 amino acids, 1 amino acid 1 amino acid ~ approximately 40 amino acids, 1 amino acid ~ approximately 35 amino acids, 1 amino acid ~ approximately 30 amino acids, 1 Amino acids ~ approximately 25 amino acids, 1 amino acid ~ approximately 20 amino acids, 1 amino acid ~ approximately 15 amino acids , 1 amino acid to approximately 10 amino acids, 1 amino acid to approximately 9 amino acids, 1 amino acid to approximately 8 amino acids , 1 amino acid to approximately 7 amino acids, 1 amino acid to approximately 6 amino acids, 1 amino acid to approximately 5 amino acids, 1 amino acid to approximately 4 amino acids, 1 amino acid to approximately 3 amino acids, approximately 2 amino acids to approximately 100 amino acids Acid, approximately 2 amino acids to approximately 95 amino acids, approximately 2 amino acids to approximately 90 amino acids, approximately 2 amino acids to approximately 85 amino acids, approximately 2 amino acids ~ approximately 80 amino acids, approximately 2 amino acids ~ approximately 75 amino acids, approximately 2 amino acids Mino acids ~ approximately 70 amino acids, approximately 2 amino acids ~ approximately 65 amino acids, approximately 2 amino acids ~ approximately 60 amino acids Acid, 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 Mino acids ~ approximately 30 amino acids, approximately 2 amino acids ~ approximately 25 amino acids, approximately 2 amino acids ~ approximately 20 amino acids Acid, 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 ~6 amino acids, approximately 2 amino acids ~ approximately 5 amino acids, approximately 2 amino acids ~ approximately 4 amino acids, approximately 3 amino acids 0 amino acids ~ approximately 100 amino acids, approximately 3 amino acids ~ approximately 95 amino acids, approximately 3 amino acids ~ approximately 90 amino acids Acid, approximately 3 amino acids to approximately 85 amino acids, approximately 3 amino acids to approximately 80 amino acids, approximately 3 amino acids to approximately 75 amino acids, approximately 3 amino acids ~ approximately 70 amino acids, approximately 3 amino acids ~ approximately 65 amino acids, approximately 3 amino acids Mino acids ~ approximately 60 amino acids, approximately 3 amino acids ~ approximately 55 amino acids, approximately 3 amino acids ~ approximately 50 amino acids Acid, approximately 3 amino acids to approximately 45 amino acids, approximately 3 amino acids to approximately 40 amino acids, approximately 3 amino acids to approximately 35 amino acids, approximately 3 amino acids to approximately 30 amino acids, approximately 3 amino acids to approximately 25 amino acids, approximately 3 amino acids Mino acids ~ approximately 20 amino acids, approximately 3 amino acids ~ approximately 15 amino acids, approximately 3 amino acids ~ approximately 10 amino acids Acid, approximately 3 amino acids to approximately 9 amino acids, approximately 3 amino acids to approximately 8 amino acids, approximately 3 amino acids to approximately 7 amino acids Mino acids, approximately 3 to 6 amino acids, approximately 3 to 5 amino acids, approximately 4 to 6 amino acids 100 amino acids, approximately 4 amino acids ~ approximately 95 amino acids, approximately 4 amino acids ~ approximately 90 amino acids, approximately 4 Amino acids ~ approximately 85 amino acids, approximately 4 amino acids ~ approximately 80 amino acids, approximately 4 amino acids ~ approximately 75 amino acids 0 amino acids, approximately 4 amino acids to approximately 70 amino acids, approximately 4 amino acids to approximately 65 amino acids, approximately 4 amino acids to Approximately 60 amino acids, approximately 4 to approximately 55 amino acids, approximately 4 to approximately 50 amino acids, approximately 4 Amino acids ~ approximately 45 amino acids, approximately 4 amino acids ~ approximately 40 amino acids, approximately 4 amino acids ~ approximately 35 amino acids No acids, approximately 4 amino acids to approximately 30 amino acids, approximately 4 amino acids to approximately 25 amino acids, approximately 4 amino acids to Approximately 20 amino acids, approximately 4 to approximately 15 amino acids, approximately 4 to approximately 10 amino acids, approximately 4 Amino acids ~ approximately 9 amino acids, approximately 4 amino acids ~ approximately 8 amino acids, approximately 4 amino acids ~ approximately 7 amino acids, Approximately 4 to 6 amino acids, approximately 5 to 100 amino acids, approximately 5 to 95 amino acids Amino acids, approximately 5 amino acids to approximately 90 amino acids, approximately 5 amino acids to approximately 85 amino acids, approximately 5 amino acids Acid ~ approximately 80 amino acids, approximately 5 amino acids ~ approximately 75 amino acids, approximately 5 amino acids ~ approximately 70 amino acids, Approximately 5 amino acids to approximately 65 amino acids, approximately 5 amino acids to approximately 60 amino acids, approximately 5 amino acids to approximately 55 amino acids Amino acids, approximately 5 amino acids to approximately 50 amino acids, approximately 5 amino acids to approximately 45 amino acids, approximately 5 amino acids Acid ~ approximately 40 amino acids, approximately 5 amino acids ~ approximately 35 amino acids, approximately 5 amino acids ~ approximately 30 amino acids, Approximately 5 amino acids to approximately 25 amino acids, approximately 5 amino acids to approximately 20 amino acids, approximately 5 amino acids to approximately 15 Amino acids, approximately 5 amino acids to approximately 10 amino acids, approximately 5 amino acids to approximately 9 amino acids, approximately 5 amino acids ~approximately 8 amino acids, approximately 5 amino acids ~approximately 7 amino acids, approximately 6 amino acids ~approximately 100 amino acids, approximately 6 Amino acids ~ approximately 95 amino acids, approximately 6 amino acids ~ approximately 90 amino acids, approximately 6 amino acids ~ approximately 85 amino acids No acids, approximately 6 amino acids to approximately 80 amino acids, approximately 6 amino acids to approximately 75 amino acids, approximately 6 amino acids to Approximately 70 amino acids, approximately 6 to approximately 65 amino acids, approximately 6 to approximately 60 amino acids, approximately 6 Amino acids ~ approximately 55 amino acids, approximately 6 amino acids ~ approximately 50 amino acids, approximately 6 amino acids ~ approximately 45 amino acids 0 amino acids, approximately 6 amino acids to approximately 40 amino acids, approximately 6 amino acids to approximately 35 amino acids, approximately 6 amino acids to Approximately 30 amino acids, approximately 6 amino acids to approximately 25 amino acids, approximately 6 amino acids to approximately 20 amino acids, approximately 6 Amino acids ~ approximately 15 amino acids, approximately 6 amino acids ~ approximately 10 amino acids, approximately 6 amino acids ~ approximately 9 amino acids Acid, approximately 6 amino acids to approximately 8 amino acids, approximately 7 amino acids to approximately 100 amino acids, approximately 7 amino acids to approximately 95 amino acids, approximately 7 amino acids ~ approximately 90 amino acids, approximately 7 amino acids ~ approximately 85 amino acids, approximately 7 amino acids Mino acids ~ approximately 80 amino acids, approximately 7 amino acids ~ approximately 75 amino acids, approximately 7 amino acids ~ approximately 70 amino acids Acid, approximately 7 amino acids to approximately 65 amino acids, approximately 7 amino acids to approximately 60 amino acids, approximately 7 amino acids to approximately 55 amino acids, approximately 7 amino acids ~ approximately 50 amino acids, approximately 7 amino acids ~ approximately 45 amino acids, approximately 7 amino acids Mino acids ~ approximately 40 amino acids, approximately 7 amino acids ~ approximately 35 amino acids, approximately 7 amino acids ~ approximately 30 amino acids Acid, 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 0 amino acids ~ approximately 100 amino acids, approximately 8 amino acids ~ approximately 95 amino acids, approximately 8 amino acids ~ approximately 90 amino acids Acid, approximately 8 amino acids to approximately 85 amino acids, approximately 8 amino acids to approximately 80 amino acids, approximately 8 amino acids to approximately 75 amino acids, approximately 8 amino acids ~ approximately 70 amino acids, approximately 8 amino acids ~ approximately 65 amino acids, approximately 8 amino acids Mino acids ~ approximately 60 amino acids, approximately 8 amino acids ~ approximately 55 amino acids, approximately 8 amino acids ~ approximately 50 amino acids Acid, approximately 8 amino acids to approximately 45 amino acids, approximately 8 amino acids to approximately 40 amino acids, approximately 8 amino acids to approximately 35 amino acids, approximately 8 amino acids ~ approximately 30 amino acids, approximately 8 amino acids ~ approximately 25 amino acids, approximately 8 Mino acids ~ approximately 20 amino acids, approximately 8 amino acids ~ approximately 15 amino acids, approximately 8 amino acids ~ approximately 10 amino acids Acid, approximately 10 amino acids to approximately 100 amino acids, approximately 10 amino acids to approximately 95 amino acids, approximately 10 amino acids 0 amino acids ~ approximately 90 amino acids, approximately 10 amino acids ~ approximately 85 amino acids, approximately 10 amino acids ~ approximately 80 amino acids No acids, approximately 10 amino acids to approximately 75 amino acids, approximately 10 amino acids to approximately 70 amino acids, approximately 10 amino acids 0.5 amino acids, 10 amino acids to 60 amino acids, 10 amino acids to 55 amino acids No acids, approximately 10 to 50 amino acids, approximately 10 to 45 amino acids, approximately 10 amino acids 0 amino acids ~ approximately 40 amino acids, approximately 10 amino acids ~ approximately 35 amino acids, approximately 10 amino acids ~ approximately 30 amino acids No acids, approximately 10 to 25 amino acids, approximately 10 to 20 amino acids, approximately 10 amino acids No acids ~ approximately 15 amino acids, approximately 20 amino acids ~ approximately 100 amino acids, approximately 20 amino acids ~ approximately 95 amino acids Mino acids, approximately 20 amino acids to approximately 90 amino acids, approximately 20 amino acids to approximately 85 amino acids, approximately 20 amino acids Mino acids ~ approximately 80 amino acids, approximately 20 amino acids ~ approximately 75 amino acids, approximately 20 amino acids ~ approximately 70 amino acids Mino acids, approximately 20 amino acids to approximately 65 amino acids, approximately 20 amino acids to approximately 60 amino acids, approximately 20 amino acids Mino acids ~ approximately 55 amino acids, approximately 20 amino acids ~ approximately 50 amino acids, approximately 20 amino acids ~ approximately 45 amino acids Mino acids, approximately 20 to 40 amino acids, approximately 20 to 35 amino acids, approximately 20 amino acids Mino acids ~ approximately 30 amino acids, approximately 20 amino acids ~ approximately 25 amino acids, approximately 30 amino acids ~ approximately 100 amino acids Amino acids, approximately 30 amino acids to approximately 95 amino acids, approximately 30 amino acids to approximately 90 amino acids, approximately 30 Amino acids ~ approximately 85 amino acids, approximately 30 amino acids ~ approximately 80 amino acids, approximately 30 amino acids ~ approximately 75 amino acids Amino acids, approximately 30 amino acids to approximately 70 amino acids, approximately 30 amino acids to approximately 65 amino acids, approximately 30 Amino acids ~ approximately 60 amino acids, approximately 30 amino acids ~ approximately 55 amino acids, approximately 30 amino acids ~ 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 ~ approximately 35 amino acids, approximately 40 amino acids ~ approximately 100 amino acids, approximately 40 amino acids ~ approximately 9 5 amino acids, approximately 40 amino acids to approximately 90 amino acids, approximately 40 amino acids to approximately 85 amino acids, approximately 4 0 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 75 amino acids, approximately 40 amino acids to approximately 7 0 amino acids, approximately 40 amino acids to approximately 65 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 4 0 amino acids to approximately 55 amino acids, approximately 40 amino acids to approximately 50 amino acids, approximately 40 amino acids to approximately 4 5 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 to 100 amino acids, approximately 60 to 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 to 75 amino acids, approximately 60 to 70 amino acids, Approximately 60 to 65 amino acids, approximately 70 to 100 amino acids, approximately 70 amino acids ~Approximately 95 amino acids, approximately 70 amino acids~approximately 90 amino acids, approximately 70 amino acids~approximately 85 amino acids Approximately 70 to 80 amino acids, approximately 70 to 75 amino acids, approximately 80 amino acids ~Approximately 100 amino acids, approximately 80 amino acids~Approximately 95 amino acids, approximately 80 amino acids~Approximately 90 amino acids Acid, approximately 80 amino acids to approximately 85 amino acids, approximately 90 amino acids to approximately 100 amino acids, approximately 90 amino acids 0.5 amino acids, or approximately 95 to 100 amino acids, from their N-terminus The sequence of wild-type, full-length otoferrin protein with / or removal from its C-terminus It may include. In some embodiments, the active otoferrin protein is, for example, 1 to 50 amino acids. Mino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 35 amino acids No acids, 1 to 30 amino acids, 1 to 25 amino acids, 1 to 20 amino acids Acid, 1 amino acid to 15 amino acids, 1 amino acid to 10 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 6 amino acids, 1 amino acid Acids up to 5 amino acids, 1 amino acid to 4 amino acids, 1 amino acid to 3 amino acids, approximately 2 amino acids to 5 amino acids 0 amino acids, approximately 2 amino acids to 45 amino acids, approximately 2 amino acids to 40 amino acids, approximately 2 amino acids ~35 amino acids, approximately 2 amino acids ~30 amino acids, approximately 2 amino acids ~25 amino acids, approximately 2 amino acids 20 amino acids, approximately 2 amino acids to 15 amino acids, approximately 2 amino acids to 10 amino acids, approximately 2 Amino acids ~9 amino acids, approximately 2 amino acids ~8 amino acids, approximately 2 amino acids ~7 amino acids, approximately 2 amino acids Mino acids ~6 amino acids, approximately 2 amino acids ~5 amino acids, approximately 2 amino acids ~4 amino acids, approximately 3 amino acids 0.50 amino acids, approximately 3 amino acids to 45 amino acids, approximately 3 amino acids to 40 amino acids, approximately 3 Amino acids ~ 35 amino acids, approximately 3 amino acids ~ 30 amino acids, approximately 3 amino acids ~ 25 amino acids, Approximately 3 to 20 amino acids, approximately 3 to 15 amino acids, approximately 3 to 10 amino acids Acid, approximately 3-9 amino acids, approximately 3-8 amino acids, approximately 3-7 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 No acids, approximately 4 to 30 amino acids, approximately 4 to 25 amino acids, approximately 4 to 20 amino acids Amino acids, approximately 4 amino acids to 15 amino acids, approximately 4 amino acids to 10 amino acids, approximately 4 amino acids to 9 amino acids, approximately 4-8 amino acids, approximately 4-7 amino acids, approximately 4-6 amino acids Amino acids, approximately 5 amino acids to 50 amino acids, approximately 5 amino acids to 45 amino acids, approximately 5 amino acids to 40 amino acids, approximately 5 amino acids to 35 amino acids, approximately 5 amino acids to 30 amino acids, approximately 5 amino acids Acid ~25 amino acids, approximately 5 amino acids ~20 amino acids, approximately 5 amino acids ~15 amino acids, approximately 5 amino acids Mino acids ~ 10 amino acids, approximately 5 amino acids ~ 9 amino acids, approximately 5 amino acids ~ 8 amino acids, approximately 5 amino acids Mino acids ~7 amino acids, approximately 6 amino acids ~50 amino acids, approximately 6 amino acids ~45 amino acids, approximately 6 Amino acids ~40 amino acids, approximately 6 amino acids ~35 amino acids, approximately 6 amino acids ~30 amino acids, Approximately 6 to 25 amino acids, approximately 6 to 20 amino acids, approximately 6 to 15 amino acids Acid, approximately 6-10 amino acids, approximately 6-9 amino acids, approximately 6-8 amino acids Acid, approximately 7 amino acids to 50 amino acids, approximately 7 amino acids to 45 amino acids, approximately 7 amino acids to 40 amino acids Mino acids, approximately 7 to 35 amino acids, approximately 7 to 30 amino acids, approximately 7 to 2 amino acids 5 amino acids, approximately 7 amino acids to 20 amino acids, approximately 7 amino acids to 15 amino acids, approximately 7 amino acids ~10 amino acids, approximately 7 amino acids ~9 amino acids, approximately 8 amino acids ~50 amino acids, approximately 8 amino acids Acid ~45 amino acids, approximately 8 amino acids ~40 amino acids, approximately 8 amino acids ~35 amino acids, approximately 8 amino acids Mino acids ~ 30 amino acids, approximately 8 amino acids ~ 25 amino acids, approximately 8 amino acids ~ 20 amino acids, approximately 8 amino acids to 15 amino acids, approximately 8 amino acids to 10 amino acids, approximately 10 amino acids to 50 amino acids Acid, approximately 10 amino acids to 45 amino acids, approximately 10 amino acids to 40 amino acids, approximately 10 amino acids to 35 amino acids, approximately 10 amino acids to 30 amino acids, approximately 10 amino acids to 25 amino acids, approximately 10 Amino acids ~20 amino acids, approximately 10 amino acids ~15 amino acids, approximately 15 amino acids ~50 amino acids Acid, approximately 15 amino acids to 45 amino acids, approximately 15 amino acids to 40 amino acids, approximately 15 amino acids to 35 amino acids, approximately 15 amino acids to 30 amino acids, approximately 15 amino acids to 25 amino acids, approximately 15 Amino acids ~20 amino acids, approximately 20 amino acids ~50 amino acids, approximately 20 amino acids ~45 amino acids Acid, approximately 20-40 amino acids, approximately 20-35 amino acids, approximately 20- 30 amino acids, approximately 20-25 amino acids, approximately 25-50 amino acids, approximately 25 Amino acids ~45 amino acids, approximately 25 amino acids ~40 amino acids, approximately 25 amino acids ~35 amino acids Acid, approximately 25-30 amino acids, approximately 30-50 amino acids, approximately 30- 45 amino acids, approximately 30-40 amino acids, approximately 30-35 amino acids, approximately 35 Amino acids ~50 amino acids, approximately 35 amino acids ~45 amino acids, approximately 35 amino acids ~40 amino acids Acid, approximately 40-50 amino acids, approximately 40-45 amino acids, or approximately 45 amino acids It may contain the sequence of wild-type, full-length otoferrin protein with approximately 50 amino acids inserted. In some cases, the inserted amino acids are in a sequence that is continuous with the wild-type, full-length protein sequence. It can be inserted. In some cases, the inserted amino acid is consecutive in the sequence of the wild-type, full-length protein. It is not inserted as a sequence. As can be understood in the art, amino acids are interspecies It can be inserted into the less conserved wild-type, full-length protein sequence.

[0231] Scope: Throughout this disclosure, various aspects of this disclosure may be presented in scope form. The formulas used are for convenience and brevity only and do not necessarily reflect the scope of this disclosure. It should be understood that this should not be considered a rigid limitation. Therefore, the scope description is: A disclosure of all possible sub-ranges and the specific n...

Claims

1. Recombinant AAV vector of sequence number 96.

2. Recombinant AAV vector of sequence number 105.

3. From 5' to 3', The 5' ITR sequence of sequence number 97, Sequence ID 98 CMV initial enhancer element, Sequence ID 99 chicken beta element CAG promoter containing the cutin gene sequence and the chimeric intron of SEQ ID NO: 100 —and, OTOF cDNA containing exons 1 through 21 (up to), 5' OTOF coding. Domain and, The SD intron sequence of sequence number 102, The AK recombinant inducible sequence of sequence number 103, The 3' ITR sequence of sequence number 104 and Recombinant AAV vectors containing [specific vectors].

4. The recombination according to claim 3, wherein the 5'OTOF coding region is sequence number 101. AAV vector.

5. The 5'OTOF coding region is at least 70% of the sequence number 101. At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99%, at least 99.5%, and They are at least 99.9% identical and are the same as the one coded by sequence number 101. A recombinant AAV vector according to claim 3 or 4, encoding a mino acid sequence.

6. From 5' to 3', The 5' ITR sequence of sequence number 97, The AK recombinant inducible sequence of sequence number 103, The SA intron sequence of sequence number 106, OTOF cDNA containing exons 22 to 48 (up to), 3'OTOF c The loading area and, The bgH polyA sequence of sequence number 108, The 3' ITR sequence of sequence number 104 and Recombinant AAV vectors containing [specific vectors].

7. The recombination according to claim 6, wherein the 3'OTOF coding region is sequence number 107. AAV vector.

8. The 3'OTOF coding region is at least 70% of the sequence number 107. At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99%, at least 99.5%, and They are at least 99.9% identical and are the same as the one coded by sequence number 107. A recombinant AAV vector according to claim 6 or 7, encoding a mino acid sequence.

9. Capsid encapsulation by Anc80 capsid as described in any one of claims 1 to 8 Recombinant AAV particles (rAAV particles) containing the described recombinant AAV vector.

10. The claim 9 states that the Anc80 capsid comprises the polypeptide of Sequence ID No.

109. The rAAV particle.

11. A first rAAV particle comprising the recombinant AAV vector described in claim 1, and the description described in claim 2. A composition comprising a second rAAV particle containing a recombinant AAV vector.

12. First rAAV particle comprising the recombinant AAV vector according to any one of claims 3 to 5 A child and a second rAA comprising the recombinant AAV vector according to any one of claims 6 to 8 A composition containing V particles.

13. The first and second rAAV particles, each independently, are formed by the Anc80 capsid. The composition according to claim 11 or 12, which is encapsulated in a capsid.

14. The Anc80 capsid comprises the polypeptide of Sequence ID No. 109, as described in claim 13. The composition of the material.

15. When the composition is introduced into human cells, the recombinant AAV vector according to claims 1 and 2 is formed. - However, concatemer formation or homologous recombination occurs between them, thereby within the cell Forming a recombinant nucleic acid encoding a full-length otoferrin protein, as described in claim 11. The composition of the material.

16. When the composition is introduced into human cells, the recombinant cells described in any one of claims 3 to 5 AAV vector and recombinant AAV vector according to any one of claims 6 to 8, Concatemer formation or homologous recombination occurs, thereby allowing full-length cells to be formed within the cell. The set according to claim 12, which forms a recombinant nucleic acid encoding toferrin protein. Finished product.

17. Claim 15 or 16 further comprises at least one pharmaceutically acceptable excipient. The composition of the material.

18. A therapeutically effective amount of the composition according to any one of claims 11 to 17 is administered to the cochlea of ​​a mammal. A method, including the steps to be implemented.

19. The method according to claim 18, wherein the mammal is a human.

20. The aforementioned mammal has been previously identified as having a missing otoferrin gene. The method according to claim 18 or 19.

21. A method for increasing the expression of full-length otoferrin protein in mammalian cells, Introducing the composition according to any one of claims 11 to 17 into the mammalian cells Methods, including the top.

22. The method according to claim 21, wherein the mammalian cell is an inner hair cell.

23. The method according to claim 21 or 22, wherein the mammalian cell is a human cell.

24. The aforementioned mammalian cells have been previously determined to have a deficient otoferrin gene. The method according to any one of claims 21 to 23.

25. Increases the expression of full-length otoferrin protein in the inner hair cells of the mammalian cochlea. A method comprising, the therapeutically effective amount of the composition according to any one of claims 11 to 17, A method comprising the step of introducing the substance into the cochlea of ​​a mammal.

26. The aforementioned mammal has been previously identified as having a missing otoferrin gene. The method according to claim 25.

27. The method according to claim 25 or 26, wherein the mammal is a human.

28. In subjects identified as having a deficiency in the otoferrin gene, asymptomatic sensorineural A method for treating hearing loss, wherein the therapeutically effective amount is described in any one of claims 11 to 17. A method comprising the step of administering the composition described above to the cochlea of ​​the subject.

29. The method according to claim 28, wherein the subject is a human being.

30. Before the administration step, it is confirmed that the subject has a deficient otoferrin gene. The method according to claim 28 or 29, further comprising the step of determining.

31. Claim 28, wherein the composition is administered to the cochlea using a microcatheter. The method described in any one of items 30.

32. The microcatheter enters the middle ear cavity via the external auditory canal, and the microcatheter The method according to claim 31, wherein the end of the part is shaped to be in contact with the RWM.

33. The distal end of the microcatheter has a diameter of at least 10 to 1,000 microns. The method according to claim 31 or 32, wherein both consist of a single microneedle.

34. Claim 2, the composition is administered to the cochlea using a device including a needle. The method described in any one of items 8 to 30.

35. The method according to claim 34, wherein the needle includes a curved portion and an angled tip. 。

36. A kit comprising the composition according to any one of claims 11 to 17.

37. The kit according to claim 36, wherein the composition is pre-filled into the device.

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

39. The microcatheter enters the middle ear cavity via the external auditory canal, and the microcatheter The kit according to claim 38, wherein the end of the kit has a shape that allows it to come into contact with the RWM.

40. The distal end of the microcatheter has a diameter of at least 10 to 1,000 microns. The kit according to claim 38 or 39, each consisting of a single microneedle.

41. The kit according to claim 36, further comprising a device.

42. The claim states that the device is the device shown in any one of Figures 82 to 87. The kit described in item 41.

43. Claim 4, the device includes a needle with a curved portion and an angled tip. The kit described in item 2.

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