Materials and methods for delivering nucleic acids to cochlear and vestibular cells

Abstract

Provided are materials and methods for the efficient delivery of nucleic acids to cochlear and vestibular cells, as well as methods for treating sensory transduction disorders associated with genetic abnormalities. [Solution] Provided are an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector encoding a capsid having at least about 85% sequence identity to Anc80 and comprising a promoter that induces expression of harmonin a, harmonin b, or harmonin c polypeptides selected from the group consisting of Espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter, and a method for treating Usher syndrome in a subject using the vector.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 455,197, filed February 6, 2017, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Hereditary hearing loss is a serious disorder with few treatment options other than cochlear implants. Hereditary hearing impairment is often due to a single gene defect. Prelingual hearing loss is diagnosed in 1 / 500 infants, with approximately 50% of cases having a genetic etiology. Usher syndrome, associated with numerous distinct clinical subtypes, each of which can result from mutations in any of a number of different genes, is responsible for 3–6% of childhood hearing loss. One of the more common genetic defects, estimated to account for 1–2% of all hereditary hearing loss, occurs in the TMC1 gene. USH1, the most severe form of Usher syndrome, is associated with abnormalities in six genes: USH1, MYO7A (myosin 7a), USH1C (harmonin), CDH23 (cadherin 23), PCDH15 (protocadherin 15), SANS (also known as USH1G), and CIB2 (calcium and integrin-binding protein 2).

[0003] The inner ear, e.g., the cochlea, and particularly the inner and outer hair cells (IHCs and OHCs) in the cochlea, are attractive targets for polynucleotide therapy to intervene in hearing loss and deafness of various etiologies, particularly monogenic forms of inherited deafness. However, efficiently targeting and transducing IHCs and OHCs, as well as other inner ear cells that may be relevant for gene therapy, has been challenging. Summary of the Invention

[0004] overview The present invention provides compositions and methods for targeting cells (e.g., inner or outer hair cells) of a subject's inner ear to express a transgene encoding a polypeptide of interest (e.g., TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 USH1C (e.g., harmonin a, b, or c)). In one embodiment, an inner ear hair cell-targeting AAV is administered to the inner ear of a subject with a genetic abnormality in hearing and / or vestibular mechanosensation.

[0005] As shown herein, adeno-associated viruses (AAVs) containing an ancestral scaffold capsid protein called Anc80 or specific Anc80 capsid proteins (e.g., Anc80-0065) efficiently target various cells in the inner ear, including IHCs and OHCs in vivo.

[0006] Gene transfer to inner ear cells in animal models has limited efficacy due to limited transduction of one or more cell types, such as outer hair cells. However, the novel gene delivery modalities described herein, including new compositions and methods based on AAV containing the Anc80 capsid protein, provide highly efficient gene transfer to inner ear cells, including both IHCs and OHCs.

[0007] In one aspect, the present invention provides an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector containing a polynucleotide encoding a human USH1 polypeptide, wherein the USH1 polypeptide is myosin 7a, harmonin (e.g., harmonin a, harmonin b, or harmonin c), cadherin 23, protocadherin 15, SANS, and calcium and integrin binding protein 2, or any other polypeptide described herein.

[0008] In another aspect, the present invention provides an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector encoding a capsid having at least about 85% sequence identity to Anc80L65 and containing a promoter that drives expression of a human TMC1 or TMC2 polynucleotide.

[0009] In another aspect, the present invention provides an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector containing a promoter that drives expression of a downstream polynucleotide, the promoter being an Espin promoter, a PCDH15 promoter, a PTPRQ promoter, or a TMHS (LHFPL5) promoter.

[0010] In another aspect, the present invention provides a cell containing the inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector of the above aspect.

[0011] In another aspect, the invention provides a method for expressing a polypeptide in the inner ear of a subject, comprising contacting cells of the inner ear with an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector encoding a polypeptide of interest, wherein the AAV vector transfects at least about 70% of the cells of the inner ear and contains ancestral AAV sequences.

[0012] In another aspect, the invention provides a method for expressing a polypeptide in the inner ear of a subject, comprising contacting cells of the inner ear with a synthetic adeno-associated virus (AAV) vector encoding a human polypeptide of interest, wherein the AAV vector encodes a capsid having at least about 85% sequence identity to Anc80L65.

[0013] In another aspect, the present invention provides a method for treating a sensory transduction disorder in a subject, comprising contacting cells of the subject with an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector, wherein the vector contains a polynucleotide encoding a human USH1 polypeptide, and the USH1 polypeptide is any one or more of myosin 7a, harmonin, cadherin 23, protocadherin 15, SANS, and calcium and integrin binding protein 2.

[0014] In another aspect, the present invention provides a method for treating a sensory transduction disorder in a subject, comprising contacting cells of the subject with an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector, wherein the vector contains a promoter that is any one of the Espin promoter, the PCDH15 promoter, the PTPRQ promoter, and the TMHS (LHFPL5) promoter.

[0015] In another aspect, the present invention provides a method of treating a sensory transduction disorder in a subject, comprising contacting a cell of the subject with an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector, wherein the vector encodes a capsid having at least about 85% sequence identity to Anc80L65 and contains a promoter operably linked to a polynucleotide encoding myosin 7a, harmonin, cadherin 23, protocadherin 15, SANS, and a calcium or integrin binding protein 2, USH1 polypeptide.

[0016] In various embodiments of the above or any other aspect of the invention described herein, the sensory transduction disorder is a genetic disease associated with a genetic alteration of a polypeptide expressed in the inner ear. In other embodiments of the above aspects, the promoter is any one or more of the Espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter. In other embodiments of the above aspects, the vector transduces inner hair cells and outer hair cells with an efficiency of at least about 70% or greater. In other embodiments of the above aspects, the harmonin polypeptide is harmonin a, harmonin b, or harmonin c. In other embodiments of the above aspects, the cell is an outer hair cell or an inner hair cell. In other embodiments of the above aspects, the vector contains a promoter that drives expression of a downstream polynucleotide, and the promoter is the Espin promoter, PCDH15 promoter, PTPRQ promoter, or TMHS (LHFPL5) promoter. In other embodiments of the above aspects, the downstream polynucleotide is TMC1, TMC2, or a USH1 polypeptide that is myosin 7a, harmonin, cadherin 23, protocadherin 15, SANS, or calcium or integrin binding protein 2. In certain embodiments of the above aspects, the harmonin polypeptide is harmonin a, harmonin b, or harmonin c. In other embodiments of the above aspects, the inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector targets inner and outer hair cells with at least about 70%, 80%, 90%, 95% or more efficiency, or even 100% efficiency. In other embodiments of the above aspects, the human polypeptide is TMC1, TMC2, harmonin a, harmonin b, or harmonin c. In other embodiments of the above aspects, the sensory transduction disorder is hearing impairment or vestibular disorder. In other embodiments of the above aspects, the sensory transduction disorder is Usher syndrome.

[0017] definition Unless otherwise specified, all scientific and technical terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Vcrlag (1991) and Hale & Marham, The HarperCollins Dictionary of Biology (1991). The following terms used herein have the meanings given below unless otherwise specified.

[0018] "Ancestral AAV sequence" refers to a designed sequence that is the product of genetic engineering resulting from analysis of naturally occurring AAVs and prediction of evolutionary ancestry.

[0019] "Anc80 polypeptide" refers to a capsid polypeptide having at least about 85% amino acid identity to the following polypeptide sequence: TIFF0007601916000001.tif55133

[0020] "Anc80 polynucleotide" refers to a nucleic acid molecule that encodes an Anc80 polypeptide.

[0021] "Seropositivity" refers to the number of people in a population who test positive for a particular disease based on serological (serum) samples. In one embodiment, seropositivity is characterized as a percentage of all samples tested or as a rate per 100,000 people tested.

[0022] " Inner ear hair cell targeting AAV " refers to an adeno-associated virus that transfects at least 70% of inner hair cells and 70% of outer hair cells after administration to the inner ear of a subject or contacting with cells derived from the inner ear in vitro.Preferably, inner ear hair cell targeting AAV is an AAV that transfects at least 90% of inner hair cells and 90% of outer hair cells after in vivo injection into the cochlea.Transfection efficiency can be evaluated using a gene encoding GFP in a mouse model.

[0023] "Mechanosensation" refers to a response to a mechanical stimulus. Touch, hearing, and balance are examples of the conversion of mechanical stimuli into neural signals. Mechanosensory input is converted into a response to the mechanical stimulus through a process called "mechanotransduction."

[0024] "TMC1 polypeptide" refers to a polypeptide or fragment thereof that has mechanotransduction channel activity and has at least about 85% amino acid sequence identity to NCBI Reference Sequence: NP_619636.2. An exemplary amino acid sequence of TMC1 is provided below. TIFF0007601916000002.tif59130

[0025] "TMC1 polynucleotide" refers to a polynucleotide that encodes a TMC1 polypeptide. The sequence of an exemplary TMC1 polynucleotide is provided in NCBI Reference Sequence: NM_138691.2, reproduced below. TIFF0007601916000003.tif160132TIFF0007601916000004.tif92133

[0026] "TMC2 polypeptide" refers to a polypeptide or fragment thereof that functions in mechanosensation and has at least about 85% or more amino acid sequence identity to NCBI Reference Sequence: NP_542789. An exemplary amino acid sequence of TMC2 is provided below. TIFF0007601916000005.tif74131

[0027] "Harmonin polypeptide" refers to a polypeptide or fragment thereof having at least about 85% amino acid sequence identity to Q9Y6N9-1 (isoform 1), Q9Y6N9-2, Q9Y6N9-3, Q9Y6N9-4, Q9Y6N9-5, which functions in mechanosensation or interacts with any one or more of USH1C, USH1G, CDH23, and MYO7A. The sequence of an exemplary harmonin a polypeptide (isoform 1) is provided below. >sp|Q9Y6N9|USH1C_HumanHarmonin OS=Homosapiens GN=USH1C PE=1 SV=3 TIFF0007601916000006.tif34128

[0028] "Ush1C polynucleotide" refers to a nucleic acid molecule that encodes a harmonin polypeptide. The sequence of an exemplary Ush1C polynucleotide, NM_005709, is provided below. TIFF0007601916000007.tif178131

[0029] Other exemplary harmonin sequences are provided below. Harmonin B >XM 011519832.2 Predicted: Homo sapiens USH1 protein network component harmonin (USH1C), transcript variant X3, mRNA TIFF0007601916000008.tif106132

[0030] Harmonin B Polypeptide TIFF0007601916000009.tif49128

[0031] Harmonin C >NM 001297764.1 Homo sapiens USH1 protein network component harmonin (USH1C), transcript variant 3, mRNA TIFF0007601916000010.tif97132

[0032] Harmonin C Polypeptide TIFF0007601916000011.tif30128

[0033] "Espin promoter" refers to a regulatory polynucleotide sequence derived from NCBI Reference Sequence: NG_015866.1 that is sufficient to drive expression of a downstream polynucleotide in a cochlear cell. In one embodiment, the Espin promoter comprises at least about 350, 500, 1000, 2000, 3000, 4000, 5000, or more base pairs upstream of the Espin coding sequence.

[0034] "Protocadherin 15 (PCDH15) promoter" refers to a regulatory polynucleotide sequence derived from NCBI Reference Sequence: NG_009191 that is sufficient to drive expression of a downstream polynucleotide in a cochlear cell. In one embodiment, the PCDH15 promoter comprises at least about 350, 500, 1000, 2000, 3000, 4000, 5000, or more base pairs upstream of the PCDH15 coding sequence.

[0035] "Protein tyrosine phosphatase receptor type Q (PTPRQ) promoter" refers to a regulatory polynucleotide sequence derived from Genel D: 374462 that is sufficient to drive expression of a downstream polynucleotide in a cochlear cell. In one embodiment, the PTPRQ promoter comprises at least about 350, 500, 1000, 2000, 3000, 4000, 5000, or more base pairs upstream of the PTPRQ coding sequence.

[0036] "Lipoma HMGIC Fusion Partner-Like 5 (LHFPL5) promoter" (also referred to as "TMHS promoter") refers to a regulatory polynucleotide sequence derived from NCBI Reference Sequence: GeneID: 222662 that is sufficient to direct expression of a downstream polynucleotide in a cochlear cell. In one embodiment, the TMHS promoter comprises at least about 350, 500, 1000, 2000, 3000, 4000, 5000 or more base pairs upstream of the PCDH15 coding sequence.

[0037] "Agent" refers to a polypeptide, polynucleotide or small chemical compound.

[0038] "Ameliorate" refers to slowing, inhibiting, attenuating, reducing, arresting, or stabilizing the onset or progression of a disease or disorder.

[0039] "Alteration" refers to a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by standard known methods, such as those described herein. As used herein, alteration includes a 10% change in expression level, preferably a 25% change in expression level, more preferably a 40% change, and most preferably a 50% or greater change.

[0040] In this disclosure, "comprises," "including," "containing," and "having," etc., can have the meaning given to them in U.S. patent law and can mean "comprises," "including," etc.; "consisting essentially of" or "consisting essentially of," etc., have the meaning given to them in U.S. patent law, and the terms are open-ended, allowing for the presence of more than what is recited, but excluding prior art aspects, so long as the presence of more than what is recited does not alter the basic or novel characteristics of what is recited.

[0041] "Detecting" refers to identifying the presence, absence, or amount of the analyte to be detected.

[0042] "Disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include genetic disorders characterized by a loss of function in proteins that function in mechanosensory transduction, e.g., expressed in the inner ear of a subject. In another embodiment, the disease is Usher syndrome (e.g., USH1) or age-related hearing loss. In one embodiment, the disease is hearing impairment associated with a genetic abnormality, e.g., an abnormality in TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, USH1C (e.g., harmonin a, b, or c).

[0043] An "effective amount" refers to the amount of an active substance required to improve the symptoms of a disease in an untreated patient. The effective amount of an active substance used to practice the present invention for the therapeutic treatment of a disease will vary depending on the method of administration, the age, weight, and health of the subject. Ultimately, the appropriate amount and dosage regimen will be determined by the attending physician or veterinarian. Such an amount is referred to as an "effective amount."

[0044] "Fragment" refers to a portion of a polypeptide or nucleic acid molecule. This portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. Fragments can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0045] "Hybridization" refers to hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0046] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components that normally accompany it when found in the natural state. "Isolated" refers to some degree of separation from a source or surroundings.

[0047] "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other substances so that any impurities do not substantially affect the biological properties of the protein or produce other adverse effects. That is, a nucleic acid or peptide of the invention is purified if, when produced by recombinant DNA techniques, it is substantially free of cellular material, viral material, or culture medium, or, when chemically synthesized, it is free of chemical precursors or other reagents. Purity and homogeneity are usually measured using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can refer to the nucleic acid or protein giving rise to essentially one band in an electrophoretic gel. In the case of proteins that can be modified, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be separately purified.

[0048] An "isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes that flank the gene in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector; recombinant DNA that is incorporated into an autonomously replicating plasmid or virus; recombinant DNA that is incorporated into the genomic DNA of a prokaryote or eukaryote; and recombinant DNA that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). In addition, the term includes an RNA molecule transcribed from the DNA molecule and recombinant DNA that is part of a hybrid gene that encodes an additional polypeptide sequence.

[0049] An "isolated polypeptide" refers to a polypeptide of the invention that has been separated from components that naturally accompany it. Generally, a polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% a polypeptide of the invention. Isolated polypeptides of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemical protein synthesis. Purity can be measured by any appropriate method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0050] "Marker" refers to any protein or polynucleotide that exhibits altered expression levels or activity associated with a disease or disorder.

[0051] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise obtaining the agent.

[0052] A "promoter" refers to a polynucleotide sufficient to direct transcription of a downstream polynucleotide.

[0053] By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0054] "Reference" refers to a standard or control condition.

[0055] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer therebetween or thereabout.

[0056] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence will generally be able to hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence will generally be able to hybridize with at least one strand of a double-stranded nucleic acid molecule.

[0057] "Hybridize" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringency conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0058] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, the concentration of detergents, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is carried out in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In a more preferred embodiment, hybridization is carried out in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization is carried out in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be readily apparent to those of skill in the art.

[0059] In most applications, washing steps following hybridization also vary in stringency. Wash stringency conditions can be determined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for washing steps are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps typically include at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, washing steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washing steps are performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is carried out in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68°C. Further modifications to these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0060] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95%, or 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0061] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine, and phenylalanine, tyrosine. In an exemplary method for measuring the degree of identity, the BLAST program can be used, e.g., -3 ~e -100 A probability score between indicates a closely related sequence.

[0062] "Subject" refers to a mammal, including a human or non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0063] "Transgene" refers to any piece of DNA that is deliberately inserted into a cell and becomes part of the genome of the organism that develops from that cell, or, in the case of nematode transgenes, becomes part of an inherited extrachromosomal array. Such transgenes may contain genes that are partially or completely heterologous (i.e., exogenous) to the transgenic organism, or may represent genes that are homologous to endogenous genes of the organism.

[0064] Ranges provided herein will be understood to be shorthand for all of the values ​​within the range. For example, a range of 1 to 50 will be understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0065] As used herein, the terms "treat," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms. It will be understood that treating a disorder or condition does not require (although does not exclude) the complete elimination of the disorder, condition, or its associated symptoms.

[0066] As used herein, the term "or" will be understood to be inclusive unless specifically stated otherwise or clear from context. Unless specifically stated otherwise or clear from context, "a," "an," and "the" will be understood to be singular or plural.

[0067] Unless specifically stated or clear from the context, the term "about" as used herein will be understood to mean within a normal range of tolerance in the art, for example, within two standard deviations of the mean value. "About" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0068] The recitation of a list of chemical groups in any definition of a variable herein includes definition of that variable as any one group or combination of listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any one embodiment or in combination with any other embodiment or portion thereof.

[0069] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0070] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the method and composition of matter belong. Although methods and materials similar or equivalent to those described herein can be used to carry out or test the method and composition of matter, suitable methods and materials are described herein. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety. [The present invention 1001] encoding a capsid having at least about 85% sequence identity to Anc80; and a promoter that induces expression of a harmonin a, harmonin b, or harmonin c polypeptide selected from the group consisting of an Espin promoter, a PCDH15 promoter, a PTPRQ promoter, and a TMHS (LHFPL5) promoter; Inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vectors. [The present invention 1002] 1001. An inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector of the present invention that transduces inner and outer hair cells with at least about 70% or higher efficiency. [The present invention 1003] A cell comprising an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector of the present invention. [The present invention 1004] The cell of the present invention 1003, which is an outer hair cell or an inner hair cell. [The present invention 1005] A method of treating Usher syndrome in a subject, comprising contacting cells of the subject with an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector of the present invention. [The present invention 1006] A method of introducing a wild-type form of a defective gene into a subject with Usher syndrome, comprising contacting the cells of the subject with an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector of the present invention. [The present invention 1007] A method for treating Usher syndrome in a subject, comprising contacting the subject's cells with an inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector, wherein the vector comprises a promoter selected from the group consisting of Espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter, and the promoter induces expression of a polynucleotide encoding a human USH1 polypeptide selected from the group consisting of myosin 7a, harmonin, cadherin 23, protocadherin 15, SANS, and calcium and integrin binding protein 2. [The present invention 1008] 1007. The method of claim 10, wherein the human polypeptide is TMC1, TMC2, harmonin a, harmonin b, or harmonin c. [The present invention 1009] The method of claim 1007, wherein the administration reverses hearing loss. [The present invention 1010] The method of claim 1009, wherein the hearing loss is partial hearing loss or complete hearing loss. [The present invention 1011] The method of claim 1009, wherein restoration of hearing function is associated with preservation of hair bundle morphology and / or restoration of mechanotransduction. [Brief explanation of the drawings]

[0071] Part 1: Highly efficient cochlear gene transfer [Figure 1-1]Figures 1A-1G provide a series of photomicrographs (A) and six graphs (B-G). In vivo cochlear transduction with native AAV serotypes and Anc80. (A) Confocal images of mouse organs of Corti counterstained with Alexa-546 phalloidin (red) and imaged for eGFP (green—shown throughout as bright staining in the middle and bottom panels). Mice were injected with 1 μL of AAV stock solution at the titers indicated above each panel. Scale bar = 50 μm. (B) Quantification of eGFP-positive IHCs in the basal and apical regions of AAV-eGFP-injected cochleae. (C) Quantification of eGFP-positive OHCs in the basal and apical regions of AAV-eGFP-injected cochleae. Injections were performed in one ear of C57BL / 6 mice: five (AAV1), four (AAV2), two (AAV8), one (AAV6), and three (Anc80). (D) Groups of sensory transduction currents recorded at P7 (left) from eGFP-negative OHCs (black) and eGFP-positive OHCs (green). Hair bundles were deflected between -0.1 and 1 μm in 0.1 μm increments. The vertical scale bar indicates 200 pA; the horizontal scale bar indicates 20 msec. Currents from eGFP-negative (black) and eGFP-positive (green) P35 IHCs are shown on the right. The vertical scale bar indicates 100 pA; the horizontal scale bar indicates 20 msec. (E) Sensory transduction current amplitudes plotted for 103 IHCs and OHCs at the ages indicated below. Data from eGFP-negative (black) and eGFP-positive (green) IHCs are shown. The number of cells in each group is indicated on the graph. Injections were performed at P1 for all mice. (F) Mean ± standard deviation (SD). ABR thresholds plotted for four Anc80-injected ears (green) and four non-injected ears (black), as well as data from one injected ear (red) that showed no eGFP fluorescence due to injection-related damage. (G) Mean ± SD. DPOAE thresholds are plotted for four Anc80-injected ears (green) and four non-injected ears (black), as well as one negative control ear with injection damage (red) that showed no eGFP fluorescence. Injection titers for data points in B–G are the same as in A. [Figure 1-2] See description of Figure 1-1. [Figure 2]Figures 2A-2D are images showing Anc80-eGFP transduction in the vestibular sensory epithelium. (A) Mouse utricle from a P1 mouse injected with 1 μL of Anc80-eGFP (1.7 × 10 GC / mL). Tissue was harvested at P10, fixed, stained with Alexa546 phalloidin (red), and imaged for eGFP (green). Morphological evaluation of multiple focal planes of eGFP-positive cells demonstrated transduction of the stereotypical flask morphology of type I cells and the columnar morphology of type II cells in all samples examined (not shown). Scale bar = 100 μm. (B) Posterior ridge of the semicircular canal from the same mouse described for panel A. Scale bar = 50 μm. (C) Sensory epithelium of the human utricle. Tissues were exposed to 10 GC Anc80.CMV.eGFP.WPRE for 24 hours, cultured for 10 days, fixed, stained with Alexa546 phalloidin (red), and imaged for eGFP fluorescence (green). Scale bar = 100 μm. (D) Higher magnification of human epithelium in the utricle stained with Alexa546 phalloidin (red) and Myo7A (blue) and imaged for eGFP (green) transduced under the same conditions as in C. White arrows in the overlay panel indicate selected eGFP-positive / Myo7A-positive cells. Scale bar = 20 μm. [Figure 3]Figures 3A-3E show images demonstrating widespread inner and outer hair cell transduction in mouse cochleae with Anc80. (A) Low-magnification image of the entire apical section of a mouse cochlea injected with 1 μL of Anc80-eGFP (1.7 × 10 GC / mL) at P1. The cochlea was harvested at P10, stained with Alexa546 phalloidin (red), and imaged for eGFP (green). Scale bar = 100 μm. (B) High-magnification image of the basal section from a different mouse cochlea injected with 1 μL of Anc80-493 eGFP (1.7 × 10 GC / mL) at P1. The cochlea was harvested at P10, stained with Alexa546 phalloidin (red), and imaged for eGFP (green). Scale bar = 20 μm. (C, D) Quantitative comparison of inner and outer hair cell transduction efficiency at equal doses for all serotypes after round window injection in P1-2 C57BL / 6 mice. Mouse cochleae in (C, D) were injected with 1.36 × 10 12 AAV1, AAV2, AAV8, and Anc80 and harvested at 7–9 days for live cell imaging and quantification by epifluorescence microscopy (n = 8 per group). (E) Dose-dependence of Anc80 hair cell transduction. Cochleae exposed to two different Anc80-eGFP titers (1.8 × 10 12 vs. 1.36 × 10 12 GC) were fixed, stained with Alexa546 phalloidin (red), and imaged for eGFP (green). Scale bar = 20 μm. [Figure 4] Figures 4A-4B are images and graphs, respectively, showing vestibular function after Anc80 cochlear transduction. Mice were injected with Anc80.CMV.eGFP through the RWM at P1 and assessed for expression and balance function on the rotarod apparatus. Expression of eGFP (green) in the vestibular tissue was demonstrated by confocal microscopy using immunofluorescent staining for Myo7A (red) (A). The rotarod data revealed no difference between injected and uninjected controls. The mean time + / - SEM for mice to fall off the apparatus is plotted. N = 3 mice, 5 trials each (injected) and 2 mice, 5 trials each (control) (B). Scale bar = 50 µm.

[0072] Part 2: Gene therapy restores hearing and vestibular function in a mouse model of Usher syndrome type 1C [Figure 5] Figures 5A–5L show images of scanning electron microscopy of the organ of Corti in P8 Ush1c c.216G>A mutant mice. (A–F) The basal, intermediate, and apical regions of the organ of Corti were imaged in P8 c.216GA (n = 3 mice) and c.216AA (n = 4 mice) mutant mice. OHC and IHC hair bundles were preserved in heterozygous mice, but some hair bundles appeared disintegrated along the organ of Corti in homozygous 216AA mice. (G–L) High-magnification images revealed fragmented, disintegrated bundles and disorganized staircase arrangements in many, but not all, OHCs (G–H) and IHCs (I–J). Examples of OHC hair bundles imaged in the central region of the organ at P8 show preserved (K) and disintegrated (L) hair bundles present in the same specimen. Asterisks indicate preserved hair bundles; arrowheads indicate disintegrated hair bundles; and arrows indicate wavy IHC bundles. Scale bars low magnification: 5 µm (A-F); high magnification: 2 µm (G), 3 µm (H), 2 µm (I-863 J) and 1 µm (K, L). [Figure 6-1]Figures 6A-6H show images demonstrating mechanotransduction in hair cells of Ush1c c.216G>A neonatal mutant mice. (A-D) The permeable styryl dye FM1-43 was used to assess the presence of open transduction channels in hair cells of c.216GA and c.216AA mice. In the organ of Corti, at P4, FM uptake in sensory hair cells of c.216AA mice was reduced (A-B, intermediate basal). Note that the IHC FM1-43 fluorescence appears dimmer because the IHC is located in a different focal plane. Left: DIC, Right: FM1-43; Scale bar 10 μm. In the utricle, FM1-43 uptake was restricted to the extrastriola region in c.216AA mutants at P6 (C; scale bar 50 μm), and utricle hair cells retained largely normal bundle morphology as assessed by DIC (D; scale bar 10 μm). The white line in panel D demarcates the border between the striola region (no uptake) and the extrastriola region (with uptake). Experiments were repeated three times. (E-H) Mechanotransduction was assessed in OHCs, IHCs, and VHCs in neonatal c.216GA and c.216AA mice (number of recorded mice: n = 7 and 6 for OHCs, n = 2 and 4 for IHCs, and n = 2 and 6 for VHCs, respectively; number of cells is indicated above the bar graph). Representative transduction currents (E), their associated current / displacement plots fitted with second-order Boltzmann functions (F), and mean peak transduction currents (G-H) are plotted. Recordings were obtained in the cochlea from P3 to P6 in the mid-organ and mid-apical turns. Transduction currents were recorded from the extrastriola and VHCs in the utricle between P5 and P7 (E-F). Although hair bundles appeared well preserved under DIC, smaller mean transduction currents were elicited in the c.216AA mutant (H). Mean peak transduction was significantly different between the two genotypes in OHCs, IHCs, and VHCs (***P<0.01, one-way ANOVA). [Figure 6-2] See description of Figure 6-1. [Figure 7]Figures 7A-7E show the expression and localization of fluorescently labeled harmonin in tissues exposed to adeno-associated viral vectors in vitro and in vivo. (A-C) Rapidly dissected P0-P1 inner ear tissues were exposed to AAV2 / 1 vectors for 24 hours and maintained in culture for 7-8 days before being fixed, counterstained (Alexa Fluor phalloidin, Invitrogen), and imaged using a Zeiss LSM confocal microscope. Numerous sensory hair cells were infected in wild-type utricles. Expression of harmonin b1 fused to EGFP was evident in most hair cells, particularly at the apical ends of sensory hair bundles (A, Scale bar: 10 μm - upper panel; 5 μm - lower panel). Similarly, EGFP::harmonin-b1 expression was evident at the tips of stereocilia in OHCs and IHCs of c.216AA and wild-type mice (B, scale bar: 10 μm; C, scale bar: 3 μm). Following injection of the AAV2 / 1.CMV.EGFP::harmonin-b1 vector at P1, EGFP signals were detected in several IHCs and OHCs in the left injected ear at P60 (D, scale bar: 30 μm). In P7 organotypic cultures exposed to AAV2 / 1.CMV.tdTomato::harmonin-a1 for 24 hours at P0, exogenous tdTomato::harmonin-a1 was detected in the cell bodies of IHCs and OHCs (E, scale bar: 5 μm). Some harmonin-a1 puncta colocalized with CTBP2 (blue; mouse anti-CTBP2 1 / 200, BD Biosciences), particularly at the base of sensory cells, likely near ribbon synapses. No expression was observed in the stereocilia bundles. [Figure 8]Figures 8A-8C show images demonstrating the recovery of mechanotransduction in hair cells of mice injected with the Anc80 harmonin vector. (A-C) Mechanotransduction currents were recorded during IHC in c.216AA non-injected control mice (n = 8 cells, 1 mouse), c.216AA mice injected with AAV2 / Anc80.CMV.harmonin-b1 at P1 (0.8 µL, n = 15 cells, 1 mouse), and c.216AA mice injected with a combination of AAV2 / Anc80.CMV.harmonin-b1 and AAV2 / Anc80.CMV.harmonin-a1 at P1 (0.5 µL + 0.5 µL, n = 7 cells, 1 mouse). Organotypic cultures were prepared at P6, and recordings were performed between P15 and P16 (9-10 days after injection). Although small mechanotransduction currents could be induced by hair bundle stimulation in c.216AA mice, larger currents were evoked in c.216AA mice injected with vectors driving harmonin b1 expression or dual expression of harmonin a1 and b1 (A). Corresponding I / X curves and double Boltzmann fit functions for each data set. Respective maximum mechanotransduction currents, Imax, were 102.1 pA (c.216AA); 424.3 pA (c.216AA + harmonin b1); and 341.1 pA (c.216AA + harmonin a1 & b1) (B). Average responses (mean ± SD) show significant restoration of transduction (***P < 0.001) in harmonin b1-injected and harmonin a1 + b1-injected mice compared to uninjected mice. Mean transduction currents were not significantly different between harmonin b1-injected and c.216GA control mice (NS P>0.5). Combining harmonin a and harmonin b did not significantly improve mechanotransduction recovery. (C) One-way ANOVA. [Figure 9-1]Figures 9A-9E show images showing ABR and DPOAE threshold recovery in mice injected with AAV2 / Anc80.CMV.harmonin-b1 at P1. (A) Representative ABR responses to a 16 kHz tone in 6-week-old c.216AA control mice and c.216AA mice injected with vectors encoding harmonin a1, harmonin b1, or a combination of the two via the RWM at P1. ABR threshold recovery approaching 30 dB SPL was measured in mice injected with harmonin b1 alone or with harmonin a1 and b1 together. (B) Average ABR responses obtained for c.216AA (n = 13); c.216GA (n = 12); c.216AA + harmonin a1 (n = 12); c.216AA + harmonin b1 (n = 19 rescues / 25 trials); and c.216AA + harmonin a1 & b1 (n = 6 rescues / 11 trials). Mean ± SE, continuous line. Dotted line: ABR threshold across the entire frequency range in mice with 16 kHz recordings shown in panel A. (C) Average DPOAE responses obtained for c.216AA (n = 13); c.216GA (n = 12); c.216AA + harmonin a1 (n = 12); c.216AA + harmonin b1 (n = 15 rescues, DPOAE < 70 dB SPL / trial 25); and c.216AA + harmonin a1 & b1 (n = 4 rescues, DPOAE < 70 dB SPL / trial 11). Mean ± SE, solid line. Dotted line: DPOAE threshold for the four mice whose recordings are shown in panel A. Arrows indicate thresholds higher than the maximal stimulus level tested. (D-E) ABR and DPOAE responses obtained at 6 weeks and 3 months in eight mice that showed initial ABR thresholds below 45 dB. Six of the eight mice were housed for 6 months and had ABR and DPOAE assessments (dotted lines). Mean ± SE. ABR and DPOAE threshold shifts were evident during the first 3 months, but at 6 months of age, auditory rescue in the lower low frequency range was still prominent. [Figure 9-2] See description of Figure 9-1. [Figure 9-3] See description of Figure 9-1. [Figure 10-1]Figures 10A-10E show images showing startle response, rotarod performance, and open-field behavioral recovery in mice injected with AAV2 / Anc80.CMV.harmonin-a1 and AAV2 / Anc80.CMV.harmonin-b1 at P1. (A) Startle responses to white noise stimuli were recorded in 6-week-old control c.216GA, c.216AA, and c.216AA-injected mice. Partial startle rescue was evident in mice injected with harmonin b1, but not harmonin a1 (data overlap with control c.216AA mice). Means are shown ± SE. (B) Rotarod performance was recorded at 4-6 weeks in control c.216GA, c.216AA, and c.216AA-injected mice. Complete recovery was observed in mice injected with harmonin b1 and harmonin a1 / b1. Harmonin a1 alone did not result in recovery. Means are shown ± SE. (C-E) Open-field observations were performed for 5 min in 6-week-old control c.216GA, c.216AA, and c.216AA and c.216GA-injected mice. Representative tracks from 2.5 min are shown (B). While c.216AA mutant mice explore the entire field and repeatedly rotate their entire bodies, c.216AA mice injected with harmonin a1, harmonin b1, or a combination of the two vectors at P1 exhibit normal behavior, similar to their heterozygous c.216GA counterparts or c.216GA mice injected with truncated vectors (C). Graphs show mean ± SD for number of rotations and distance traveled per minute. Significant recovery was observed between uninjected and injected mice. ***P<0.001. Statistical analysis by one-way ANOVA. [Figure 10-2] See description of Figure 10-1. [Figure 11]Scanning electron microscopy images of the organ of Corti in mice injected with AAV2 / Anc80.CMV.harmonin-b1. The basal, middle, and apical regions of the organ of Corti were imaged at 6 weeks in c.216GA, c.216AA, and c.216AA mice injected at P1 (RMW injection of 0.8 μl AAV2 / Anc80.CMV.harmonin-b1). OHC and IHC hair bundles were preserved in c.216GA mice but appeared disorganized along the organ of Corti in c.216AA mice. Significant hair cell loss (asterisks) and hair bundle disorganization were observed in c.216AA mice, with more pronounced degeneration at the basal end of the organ. Hair bundles in c.216AA mice lacked normal stereocilia rows. Lower rows appeared shrunken, while the highest rows were preserved in c.216AA mice (arrows). Hair cell loss and bundle collapse were also evident in rescued c.216AA mice, but hair cell survival was significantly higher in the basal and intermediate regions of the organ. Hair cell counts are summarized in the bar graph. A total of 1,824 cells were counted in c.216AA mice (four ears) and 792 in rescued c.216AA mice (two ears). Mean ± SE. Higher magnification images reveal rescue of staircase arrangements in many, but not all, cells (arrowheads) in injected c.216AA mice (arrows). Scale bar: low magnification: 5 μm; higher magnification: 1 μm. [Figure 12] Figures 12A-12L show SEM images of hair bundle morphology in P18 Ush1c c.216G>A mice. (A-C) Heterozygous c.216GA mice showed normal hair bundle morphology at P18. (D-I) Disintegrated hair bundles were observed along the sac of homozygous c.216AA mutant mice at P18. (J-L) IHC hair bundles were mildly disrupted in c.216AA mice. Distances measured from the apex: basal 3.5-4 mm; medial 1.8-2.2 mm; apical 0.6-0.8 mm. Scale bar: low magnification: 5 μm; high magnification: 1 μm. [Figure 13]Figures 13A-13J are images showing mechanotransduction properties in c.216AA mutant mice. (A-E) Analysis of mechanotransduction in neonatal OHCs from the middle and mid-apical turn of the cochlea, P3-P6. Representative current traces from ~Po = 0.5 were fitted to a double exponential decay function to assess adaptation in c.216GA and c.216AA mutants (A). The fit was used to generate fast (C) and slow (D) time constants and the extent of adaptation (E). The 10-90% operative range did not change significantly (B). The extent of adaptation in c.216AA mice was significantly lower than in heterozygous OHCs shown in this scatter plot (E). (F-J) Analysis of mechanotransduction in neonatal IHCs. The 10-90% working range values ​​were smaller in c.216GA IHC compared to c.216AA IHC (G). Adaptation was always present, albeit at a slightly slower rate and to a significantly lesser extent in c.216AA IHC (H-J). Statistical analysis is shown for each plot: *P<0.05, **P<0.01, and ***P<0.001, one-way ANOVA. [Figure 14] Figures 14A-14C are images showing the expression of fluorescently labeled harmonin a and harmonin b Anc80 vectors in the c.216AA organ of Corti 6 weeks after dual vector injection at P1. (A-C) Confocal images of the basal turn in a 6-week-old c.216AA mouse after P1 co-injection of AAV2 / Anc80.CMV.tdTomato::harmonin-a1 (0.5 μl; 4.11E^12 gc / ml) and AAV2 / Anc80.CMV.eGFP::harmonin-b1 (0.5 μl; 2.99E^12 gc / ml). 69% and 74% of the total cell population expressed eGFP (A) and tdTomato (C), respectively, and 65% expressed both markers, demonstrating successful co-transduction. Scale bar: 20 μm. [Figure 15]Figures 15A-15F show data showing analysis of ABR responses in 6-week-old control c.216GA mice and injected, rescued c.216AA mice. (A, D) Example ABR responses at 8 and 16 kHz for control c.216GA and rescued c.216AA mice. (B-C, E-F) Mean peak 1 amplitude (B-D) and latency (C-D) at 8-11.3 and 16 kHz in 6-week-old mice with comparable thresholds (n=8 c.216GA, n=5 c.216AA + Harmonin b1 RWM P1). Mean ± SE. One-way ANOVA. [Figure 16A-1] Figures 16A-16D show that the mutant form of harmonin expressed in Ush1c c.216G>A mice does not alter hair cell or auditory function. (A) Sequence alignment between wild-type harmonin b1 protein and the truncated harmonin secreted as a result of cryptic splicing and frameshifting associated with the Acadian G>A mutation in exon 3 of the Ush1c gene. (B) Semiquantitative RT-PCR from the auditory organs of P2-P3 wild-type, c.216GA, and c.216AA mutant mice confirms the expression of wild-type (450 bp) and truncated (-35 bp) harmonin in c.216GA and c.216AA mice. (C-D) Auditory brainstem responses (ABRs, C) and distortion product otoacoustic emissions (DPOAEs, D) were measured in 6-week-old c.216GA-injected mice and control c.216GA and c.216AA mice. Plots are shown as mean ± SE. No threshold shift was observed in the injected mice relative to the control 216GA mice. [Figure 16A-2] See legend to Figure 16A-1. [Figure 16B] See legend to Figure 16A-1. [Figure 16C] See legend to Figure 16A-1. [Figure 16D] See legend to Figure 16A-1. [Figure 17-1]Figures 17A-17C are images showing the restoration of correct Ush1c splicing in the inner ear of 6-week-old mice injected with AAV2 / Anc80.CMV.harmonin-b1. (A) Semiquantitative RT-PCR quantification of correctly spliced ​​(450 bp) and aberrant (415 bp) mRNA from the Ush1c.216A allele demonstrates the restoration of correct Ush1c splicing in the injected ear (I) and contralateral ear (C) of c.216AA rescue mice #1 and #2 (35 dB SPL response at 11.3 kHz from the injected ear). Mouse #3, with a poor ABR response (90 dB SPL at 11.3 kHz), shows a moderate restoration of correct mRNA expression, while mouse #4 (100 dB SPL at 11.3 kHz) shows none. While the correct splice form is not detected in uninjected c.216AA mice (mice #5 and #6), both the correct and truncated splice forms are detected in c.216GA mice (mice #7, #8, and #9). The corresponding mouse Gapdh shown in the lower panel was amplified to confirm the relative amounts of material. (B) Semiquantitative radiolabeled PCR analysis confirms the presence of AAV-mUsh1c in the injected and contralateral ears of Ush1c.216AA mice. Relative levels of AAV-mUsh1c DNA were present but reduced in mice #3 and #4. (C) Relative amounts of AAV-mUsh1c correlate with ABR thresholds. Analysis at 11.3 and 16 kHz is shown. Linear regression indicates a high correlation between the two. [Figure 17-2] See description of Figure 17-1. [Figure 18]Long-term ABR threshold recovery correlates with OHC survival in the intermediate-apical region of the auditory organ. Postmortem whole-organ-of-Corti hair cell counts were performed on the left ears of three 6-month-old non-injected c.216AA mice and five injected c.216AA mice (P1 RWM injection, 0.8 μl AAV2 / Anc80.CMV.harmonin-b1). Insert: To 5.6-16 kHz tone stimuli, two mice (#1 and #2) showed low ABR response thresholds across the entire test range (≥95 dB SPL), while three mice (#3-5) responded in the 35-55 dB SPL range. The total number of IHC and OHC hair cells was increased in injected mice. Comparison of rescued injected mice with those showing low rescue rates shows that, although the number of IHCs was not different, a significant number of OHCs was observed in rescued mice. Analysis along the entire length of the organ showed that this difference could be explained by an increase in hair cell survival from the mid to apical regions of the organ.

[0073] Part 3: Gene therapy for additional mutations involved in hearing loss [Figure 19] Figures 19A-19D show representative confocal images of the cochlea from a Ush1c mutant mouse injected with 1 ml (6 x 10 gc / ml) of Anc80-harmonin::GFP (i.e., GFP fused to the harmonin polypeptide) through the RWM at P2, harvested at P9, and stained for actin (red; 19A), Myo7a (blue; 19B), and imaged for GFP (green; 19C). A merged image of (19A), (19B), and (19C) is shown in (19D). [Figure 20] Graph showing ABR thresholds plotted as a function of sound frequency for Ush1c mutant mice (squares) and Ush1c mutant mice injected with the Anc80-Harmonin::GFP vector (circles). [Figure 21]Figures 21A-21C are representative confocal images at low (21A) and high (21B) magnification of a KCNQ4- / - cochlea injected with 1 μl (6 × 1012 gc / ml) of Anc80-KCNQ4 by RWM at P2, harvested at P9, and stained with Alexa 546 phalloidin (red) and antibody against KCNQ4 (green), relative to a high-magnification uninjected cochlea (21C). [Figure 22] 22A-22C are a series of graphs showing KCNQ4 currents in P10 wild-type mice (22A), P10 KCNQ4- / - mice (22B), and P10 KCNQ4- / - mice infused with Anc80-KCNQ4 (2.4 x 1013 gc / ml) via the RWM (22C). Cochleae were harvested 8 days after infusion. [Figure 23] A series of three images shows FM1-43 uptake (FM1-43 permeates only functional Tmcl channels) in Tmc1- / - tissue injected with the Anc80-CMV-Tmc1 vector. P2 Tmc1- / - mice were injected with the Anc80-CMV-Tmc1 vector (2.4 x 1013 gc / ml) through the RWM, and cochleae were harvested 6-7 days after injection. [Figure 24] Figure 24A shows representative panels of sensory transduction currents recorded from IHCs from a P10 wild-type mouse (left), a P10 Tmcl- / - mouse (middle), and a P10 Tmcl- / - mouse injected with Anc80-CMV-Tmcl (2.4 x 10 gc / ml) via the RWM at P2 (right). Cochleae were harvested 8 days after injection. Figure 24B is a graphical representation of the recovery rate of the mice shown in Figure 24A. The graphs in Figure 24B show the percentage of functional cells in wild-type mice (left), Tmcl- / - mice (middle), and Tmcl- / - mice injected with Anc80-CMV-Tmcl (right). [Figure 25] 1 is a graph showing distortion product otoacoustic emission (DPOAE) thresholds as a function of stimulation frequency for wild-type, Tmc1 − / − mice, and Tmc1 − / − mice injected with Anc80-CMV-Tmc1. [Figure 26]Figures 26A-26C show the effect of various promoters on expression. Figure 26A shows Anc80-Pcdh15-GFP expression in hair cells and supporting cells, with GFP and myosin 7a shown as indicated, and hair cells stained red for myosin 7a (a hair cell marker). Figure 26B shows Anc80-Myo6-GFP with inner and outer hair cells showing GFP expression. For this experiment, there was no counterstaining. Figure 26C shows Anc80-KCNQ4-GFP with outer hair cells showing GFP expression. The tissue was counterstained with phalloidin to highlight the hair cells. DETAILED DESCRIPTION OF THE INVENTION

[0074] Detailed Description The present invention provides compositions and methods for delivering and expressing proteins required for mechanosensation, including hearing and / or vestibular function (e.g., TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 USH1C (e.g., harmonin a, b, or c)) in cells of the inner ear, e.g., cochlear cells (e.g., inner hair cells or outer hair cells), of a subject having a loss or reduction in the level or activity of the protein.

[0075] The present invention is based, at least in part, on the discovery that a rationally designed synthetic vector encoding the Anc80 capsid ("Anc80 vector") is useful for efficient transgene delivery to the cochlea; this vector can also be used to deliver wild-type Ush1c to the inner ear of a mouse model of Usher syndrome. Round window membrane injection resulted in highly efficient transduction of mouse inner and outer hair cells (a substantial improvement over conventional adeno-associated virus (AAV) vectors). Anc80 round window injection was well tolerated, as indicated by sensory cell function, auditory and vestibular function, and immunological parameters. The ability of Anc80 to target a high proportion of outer hair cells, a requirement for the restoration of complex hearing function, may enable future gene therapy for hearing and balance disorders. Mice injected with the Anc80 vector encoding Ush1c demonstrated restoration of gene and protein expression, restoration of sensory cell function, rescue of complex hearing function, and restoration of hearing and balance behavior to near-wild-type levels. The data represent an unprecedented restoration of inner ear function and indicate that biologic therapies for treating hearing loss may be suitable for application to individuals with inherited inner ear disorders.

[0076] Usher Syndrome Human Usher syndrome (USH) is a rare genetic disorder that causes deafness and blindness. It is inherited as an autosomal recessive trait, affects 16,000–20,000 individuals in the United States, and accounts for 3–6% of childhood hearing loss. Usher syndrome is classified into three clinical subtypes (USH-1, 2, and 3) according to the severity of symptoms. USH-1 is the most severe subtype. Patients affected with USH-1 suffer from congenital bilateral profound sensorineural hearing loss, vestibular apathy, and prepubertal retinitis pigmentosa (progressive bilateral symmetric degeneration of retinal rod and cone function). Individuals typically do not develop speech unless they receive a cochlear implant. Currently, there is no biological treatment for Usher syndrome patients; however, early reintroduction of the wild-type form of the defective gene can allow for disease reversal.

[0077] Six usher genes are associated with USH1: MYO7A (myosin 7a), USH1C (harmonin), CDH23 (cadherin 23), PCDH15 (protocadherin 15), SANS (sans), and CIB2 (calcium and integrin-binding protein 2). These genes encode proteins involved in hair bundle morphogenesis in the inner ear and are part of the interactome (see, for example, Mathur & Yang, 2015, Biochim. Biophys. Acta, 1852:406-20). Harmonin is central to the USH1 interactome, where it binds to other usher1 proteins. Due to its PDZ (PSD-59 95 / Dlg / ZO-1) interacting domain, harmonin has been proposed to function as a scaffolding protein. In vitro binding studies showed that all other known USH1 proteins, as well as two USH2 proteins, Usherin and VLGR1, bind to the PDZ domain of harmonin. The USH1C gene consists of 28 exons that encode 10 alternatively spliced ​​forms of harmonin, classified into three distinct subclasses (a, b, and c) depending on the domain composition of the protein. The three isoforms differ in the number of PDZ protein-protein interaction domains, coiled-coil (CC) domains, and proline-serine-threonine (PST)-rich domains.

[0078] The USH1 protein is localized at the apical end of hair cells in the mechanosensory hair bundle, which is composed of hundreds of stereocilia interconnected by numerous extracellular links. Cadherin 23 and protocadherin 15 are products of the Usher gene (USH1D and USH1E, respectively) and form the sensory filaments located at the distal end of the stereocilia. Harmonin b binds to CDH23, PCDH15, F-actin, and itself. It is found at the tip of the stereocilia near the sensory filament insertion point of the hair cell, where it is thought to play a functional role in transduction and adaptation in hair cells. Harmonin b is expressed early postnatally, but its expression decreases around postnatal day 30 (P30) in both the cochlea and vestibule. Harmonin a also binds to cadherin 23 and is found in the stereocilia. A recent report revealed an additional role for harmonin a at synapses: it associates with Cav1.3 Ca 2+ channels and restricts channel availability via a ubiquitin-dependent pathway.

[0079] Several mouse models of Usher syndrome have been identified or engineered over the past decade, seven of which affect harmonin. Of these, only one model, the Ush1c c.216G>A model, recapitulates both the auditory and retinal abnormalities that characterize human Usher syndrome. Ush1c c.216G>A is a knock-in mouse model that affects the expression of all conventional harmonin isoforms due to a point mutation similar to that found in a cohort of French-Acadian USH1C patients. The mutation introduces a cryptic splice site at the end of exon 3 of the Ush1c gene. Use of this cryptic splice site generates a frameshifted transcript with a 35-bp deletion, resulting in the translation of a severely truncated protein lacking the PDZ, PST, and CC domains. Homozygous c.216AA knock-in mice suffer from severe hearing loss at 1 month of age, whereas heterozygous c.216GA mice do not exhibit any abnormal phenotypes. Cochlear histology in c.216AA mice shows disrupted hair bundles, abnormal cell columns and loss of both inner and outer hair cells in the middle and basal turns at P30.

[0080] It is demonstrated herein that AAV containing ancestral AAV capsid proteins successfully transduce hair cells and drive the expression and precise localization of harmonin splice forms.Furthermore, it is demonstrated herein that early postnatal round window membrane injection of AAV containing ancestral AAV capsid proteins described herein successfully restores hearing and vestibular function in homozygous c.216AA mice.The restoration of hearing function in injected mice is associated with the restoration of mRNA expression encoding wild-type harmonin and the preservation of hair bundle morphology and mechanotransduction.The results provided herein demonstrate that early reintroduction of wild-type harmonin using AAV containing ancestral AAV capsid proteins described herein can be useful for the treatment of USH1C.

[0081] TMC1 / TMC2 More than 40 different mutations in TMC1 that result in hearing loss have been identified. These are subdivided into 35 recessive mutations and 5 dominant mutations. The majority of recessive mutations result in severe congenital hearing loss (e.g., DFNB7 / 11), while a minority result in late-onset, moderate-to-severe hearing loss. All dominant mutations result in mid-teenage onset, progressive hearing loss (e.g., DFNA36). In particular, AAV vectors containing the Anc80 capsid protein described herein can be used to deliver non-mutant (e.g., wild-type) TMC1 or TMC2 sequences, thereby preventing hearing loss (e.g., further hearing loss) and / or restoring hearing function.

[0082] Therapeutic strategies for the treatment of hearing loss Because sensory cells in the adult mammalian cochlea lack the ability to self-repair, current treatment strategies (depending on the level and precise location of the damage) rely on amplification (hearing aids), better sound transmission (middle ear prostheses / active implants), or direct nerve stimulation (cochlear implants) to compensate for permanent damage to the primary sensory hair cells or spiral ganglion neurons that form the auditory nerve and relay acoustic information to the brain. While these approaches have been transformative, they are still far from optimal in restoring complex human hearing functions critical to modern life. Notably, key problems include limited frequency sensitivity, unnatural sound perception, and limited speech discrimination in noisy environments.

[0083] Therapeutic gene transfer into the cochlea has been proposed as an improvement over current standard treatments, ranging from age-related and environmentally induced hearing loss to genetic forms of hearing loss. More than 300 genetic loci have been associated with hereditary hearing loss, with more than 70 causative genes described (Parker & Bitner-Glindzicz, 2015, Arch. Dis. Childhood, 100:271-8). Therapeutic success in these approaches depends significantly on the safe and efficient delivery of exogenous gene constructs to the relevant therapeutic target cells in the organ of Corti (OC) in the cochlea.

[0084] The OC contains two classes of sensory hair cells: IHCs, which transduce mechanical information carried by sound into electrical signals transmitted to neuronal structures; and OHCs, which act to amplify and modulate cochlear responses, a process necessary for complex hearing function. Other potential targets in the inner ear include spiral ganglion neurons, columnar cells at the edge of the spiral lamina, which are important for maintaining the adjacent tectorial membrane or supporting cells, which have a protective function and can be induced to transdifferentiate into hair cells by early neonatal life.

[0085] Injection into the cochlear duct, which is filled with potassium-rich endolymph, can provide direct access to hair cells. However, alterations to this delicate fluid environment may disrupt the endocochlear potential and increase the risk of injection-related toxicity. The perilymph-filled spaces surrounding the cochlear duct, scala tympani, and scala vestibuli can be accessed from the middle ear through either the vestibulocochlear window membrane or the round window membrane (RWM). The RWM, the only non-bony opening to the inner ear, is relatively easily accessible in many animal models, and administration of viral vectors using this route is well tolerated. In humans, cochlear implant placement traditionally relies on surgical electrode insertion through the RWM.

[0086] Previous studies evaluating AAV serotypes in organotypic cochlear explants and in vivo cochlear injections have only resulted in partial hearing rescue in mouse models of hereditary hearing loss. Unexpectedly, adeno-associated viruses (AVVs) containing ancestral AAV capsid proteins transduce OHCs with high efficiency. This finding overcomes the low transduction rates that have limited the successful development of cochlear gene therapy using conventional AAV serotypes. AAVs containing the ancestral AAV capsid proteins described herein provide a valuable platform for inner ear gene delivery to IHCs and OHCs, as well as various other inner ear cell types affected by hereditary hearing and balance disorders. In addition to providing high transduction rates, AAVs containing the ancestral AAV capsid proteins described herein have been shown to have a similar safety profile in mice and non-human primates upon systemic injection and are antigenically distinct from circulating AAVs, offering potential benefits regarding pre-existing immunity that limits the efficacy of conventional AAV vectors.

[0087] However, the expression of TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 into cells, particularly cells in the inner ear, for example in the cochlea (or cells of the cochlea or cochlear cells). Described herein are compositions and methods that allow for highly efficient delivery of nucleic acids (e.g., AAV vectors, e.g., ANC80 vectors comprising a promoter (e.g., CMV, Espin, PCDH15, PTPRQ, TMHS (LHFPL5)) that direct the expression of a polynucleotide encoding one or more USH1Cs (e.g., harmonin a, b, or c). As used herein, inner ear cells refer, but are not limited to, inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, vestibular ganglion neurons, and supporting cells. Supporting cells refer to cells in the ear that are not excitable, e.g., cells that are not hair cells or neurons. One example of a supporting cell is a Schwann cell.

[0088] Delivery of one or more of the nucleic acids described herein to cells of the inner ear can be used to treat any number of inherited or acquired hearing disorders, generally defined by partial or complete hearing loss. The methods described herein can be used to treat hearing disorders, including, but not limited to, recessive hearing loss, dominant hearing loss, Usher syndrome and other syndromic hearing loss, and hearing loss due to trauma or aging.

[0089] Methods for producing viruses carrying specific transgenes As described herein, adeno-associated viruses (AAV) containing ancestral AAV capsid proteins are particularly efficient at delivering nucleic acids (e.g., transgenes, including polynucleotides encoding one or more of TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 USH1C (e.g., harmonin a, b, or c)) to inner ear cells; a particularly effective class of ancestral AAV capsid proteins is designated by the ancestral scaffold capsid protein designated Anc80, as shown in SEQ ID NO: 1. Anc80 vectors are an example of an inner ear hair cell-targeting AAV that advantageously transduces approximately 60%, 70%, 80%, 90%, greater than 95%, or 100% of inner or outer hair cells. One particular ancestral capsid protein that falls within the class of Anc80 ancestral capsid proteins is Anc80-0065 (SEQ ID NO: 2), although WO2015 / 054653, incorporated herein by reference in its entirety, describes several additional ancestral capsid proteins that fall within the class of Anc80 ancestral capsid proteins.

[0090] In certain embodiments, the adeno-associated virus (AAV) contains ancestral AAV capsid proteins with natural or engineered tropism for hair cells. In some embodiments, the virus is an inner ear hair cell-targeting AAV that delivers a transgene (e.g., a polynucleotide encoding one or more of TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 USH1C (e.g., harmonin a, b, or c)) to the inner ear of a subject. In some embodiments, the virus is an AAV comprising a purified capsid polypeptide. In some embodiments, the virus is an engineered virus. In some embodiments, the virus contains ancestral AAV sequences. In some embodiments, the virus is an AAV with a lower seropositivity rate than AAV2. In some embodiments, the virus is an exome-associated AAV. In some embodiments, the virus is an exome-associated AAV1. In some embodiments, the virus comprises a capsid protein having at least 95% amino acid sequence identity or homology to an Anc80 capsid protein.

[0091] The viruses described herein containing the Anc80 capsid protein can be used to deliver various nucleic acids to inner ear cells. In one embodiment, an inner ear hair cell-targeting AAV (e.g., ANC80 vector) includes a promoter (e.g., CMV, Espin, PCDH15, PTPRQ, TMHS (LHFPL5)) that induces the expression of a polynucleotide encoding one or more of TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 USH1C (e.g., harmonin a, b, or c). The nucleic acid sequence delivered to a cell for expression is often referred to as a transgene. Exemplary transgenes that can be delivered to and expressed in inner ear cells include, but are not limited to, transgenes encoding polypeptides that function in hearing and / or vestibular mechanosensation (e.g., TMC1, TMC2, MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7 (e.g., harmonin a, b, or c)), transgenes encoding neurotrophic factors (e.g., GDNV, BDNF, or HSP70), immunomodulatory proteins, or anti-oncogenic transcripts. In addition, exemplary transgenes that can be delivered to and expressed in inner ear cells also include, but are not limited to, transgenes encoding antibodies or fragments thereof, antisense, silencing, or long non-coding RNA species, or genome editing systems (e.g., genetically engineered zinc finger nucleases, transcription activator-like effector nucleases (TALENs), or CRISPRs (clustered regularly interspaced short palindromic repeats)).Additionally, representative transgenes that can be delivered to and expressed in inner ear cells include ACTG1, ADCY1, ATOHI, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, G PR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LR TOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, N T3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS , SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN, and XIA.In certain embodiments, the transgene is one or more of MY07A, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7, USH1C (e.g., harmonin a, b, or c).

[0092] Expression of a transgene can be driven by the transgene's native promoter (i.e., the promoter naturally found with the transgenic coding sequence) or by a heterologous promoter (e.g., the CMV promoter, Espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter). For example, any of the transgenes described herein can be used with its native promoter. Alternatively, any of the transgenes described herein can be used with a heterologous promoter. As used herein, a heterologous promoter refers to a promoter that does not naturally drive expression of the sequence (i.e., is not found with the sequence in nature). Exemplary heterologous promoters that can be used to drive expression of any of the transgenes described herein include, for example, the CMV promoter, the CBA promoter, the CASI promoter, the P promoter, and the EF-1 promoter, the alpha9 nicotinic receptor promoter, the prestin promoter, the Gfi1 promoter, and the Vglut3 promoter. In addition, a promoter that naturally drives expression of one of the above transgenes (e.g., the KCNQ4 promoter, the Myo7a promoter, the Myo6 promoter, or the Atoh1 promoter) can be used as a heterologous promoter to drive expression of the transgene. In other embodiments, the promoter is the Espin promoter, the PCDH15 promoter, the PTPRQ promoter, or the TMHS (LHFPL5) promoter.

[0093] Methods for generating transgenes (e.g., TMC1, TMC2, USH1C (e.g., harmonin a, b, or c), MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7) for packaging into viruses containing the Anc80 capsid protein are known in the art and utilize conventional molecular biology and recombinant nucleic acid techniques. In one embodiment, a construct is provided that includes a nucleic acid sequence encoding the Anc80 capsid protein and carries the transgene flanked by appropriate inverted terminal repeats (ITRs), which allow the transgene to be packaged within the Anc80 capsid protein.

[0094] The transgene can be packaged into an AAV containing the Anc80 capsid protein, for example, using a packaging host cell. One or more constructs described herein can be used to transiently or stably introduce components of the viral particle (e.g., rep sequence, cap sequence, inverted terminal repeat (ITR) sequence) into a packaging host cell. The viruses described herein contain at least the Anc80 capsid protein; other components of the viral particle (e.g., rep sequence, ITR sequence) can be based on an ancestral sequence or a modern sequence. In some instances, for example, the entire viral particle can be based on an ancestral sequence. Such viruses can be purified using conventional methods.

[0095] Generally, "nucleic acid" as used herein can include DNA and RNA, and can also include nucleic acids containing one or more nucleotide analogs or backbone modifications. Nucleic acids can be single-stranded or double-stranded, usually depending on their intended use. Nucleic acids that can be used in the methods described herein can be identical to known nucleic acid sequences or can differ in sequence from such known sequences. Simply by way of example, a nucleic acid (or encoded polypeptide) can have at least 75% sequence identity to a known sequence (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0096] When calculating the percent sequence identity value, two sequences are aligned and the number of perfect matches of nucleotides or amino acid residues between the two sequences is determined. The number of perfect matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to determine the percent sequence identity value. It is understood that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It is also understood that a sequence can be aligned to more than one other sequence and therefore can have different percent sequence identity values ​​between each aligned region.

[0097] The alignment of two or more sequences to determine sequence identity percentage values ​​is carried out using the computer program ClustalW and default parameters, which allows nucleic acid or polypeptide sequences to be aligned over their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW calculates the best match between a query and one or more target sequences, and aligns them to determine identity, similarity, and difference. To maximize sequence alignment, gaps of one or more residues can be inserted into the query sequence, the target sequence, or both. For pairwise alignment of nucleic acid sequences, default parameters are used (i.e., word size: 2; window size: 4; scoring method: % value; number of top diagonals: 4; and gap penalty: 5); for alignment of multiple nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weighted transitions: yes. For pairwise alignment of polypeptide sequences, the following parameters are used: word size: 1; window size: 5; scoring method: % value; number of top diagonals: 5 and gap penalty: 3. For multiple alignment of polypeptide sequences, the following parameters are used: weight matrix: BLOSUM( blo cks su bstitution m atrix); gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophobic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gap penalties: on. ClustalW can be run on the World Wide Web, for example, at the Baylor College of Medicine Search Launcher website or the European Bioinformatics Institute website.

[0098] Changes can be introduced into a nucleic acid sequence, which, when the nucleic acid sequence is a coding sequence, can result in changes in the amino acid sequence of the encoded polypeptide. For example, changes can be introduced into a nucleic acid coding sequence by using mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis) or by chemically synthesizing a nucleic acid molecule containing such changes. Such nucleic acid changes can result in conservative and / or non-conservative amino acid substitutions at one or more amino acid residues. A "conservative amino acid substitution" is one in which one amino acid residue is replaced with a different amino acid residue having a similar side chain (see, e.g., Dayhoff et al. (1978, Atlas of Protein Sequence and Structure, 5(Suppl. 3):345-352), which provides a frequency table of amino acid substitutions), whereas a non-conservative substitution is one in which an amino acid residue is replaced with an amino acid residue that does not have a similar side chain.

[0099] The nucleic acid can be contained within a construct, which may be referred to as a vector or plasmid. Constructs are commercially available or can be produced by recombinant techniques routine in the art. Constructs containing nucleic acids can have expression elements that induce and / or regulate expression of such nucleic acids and can also include sequences for maintaining the construct (e.g., origins of replication, selectable markers), and the like. Expression elements are known in the art and include, for example, promoters, introns, enhancer sequences, response elements, or inducible elements.

[0100] Pharmaceutical Composition An inner ear hair cell-targeting AAV (e.g., Anc805 vector) containing a promoter (e.g., CMV, Espin, PCDH15, PTPRQ, TMHS (LHFPL5)) and one or more polynucleotides of USH1, MYO7A, USH1C (harmonin a, b, c), CDH23, PCDH15, SANS, and CIB2, typically suspended in a physiologically compatible excipient, can be administered to a subject (e.g., a human or non-human mammal) by injection into the subject's inner ear through the round window. Suitable carriers include saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, and water. The inner ear hair cell-targeting AAV is administered in an amount sufficient to transduce or infect cells and provide sufficient levels of gene transfer and expression to provide therapeutic benefit without undue adverse effects.

[0101] The dose of an inner ear hair cell-targeting AAV administered to a subject depends primarily on factors such as the condition being treated and the age, weight, and health of the subject. For example, a therapeutically effective dose of an inner ear hair cell-targeting AAV administered to a human subject is generally about 1 x 10 genome copies (GC) of AAV. 1 ~1×10 12 (e.g., GC approximately 1 × 10 3 ~1×10 9 The concentration of the solution containing the compound ranges from about 0.1 ml to about 10 ml.

[0102] Methods for delivering nucleic acids to inner ear cells Methods for delivering nucleic acids to cells are generally known in the art, and methods for delivering transgene-containing viruses (which may also be referred to as viral particles) to inner ear cells in vivo are described herein. 8 ~about 10 12The virus particles can be administered to a subject, and the virus can be suspended in an appropriate volume (e.g., 10 μL, 50 μL, 100 μL, 500 μL, or 1000 μL), for example, in artificial perilymph.

[0103] Viruses containing a promoter (e.g., CMV, Espin, PCDH15, PTPRQ, TMHS (LHFPL5)) and a transgene (e.g., TMC1, TMC2, USH1C (e.g., harmonin a, b, or c), MYO7A, USCH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHRN, CLRN1, PDZD7) described herein can be delivered to inner ear cells (e.g., cells in the cochlea) using any number of means. For example, a therapeutically effective amount of a composition comprising viral particles containing one or more different types of transgenes described herein can be injected through the round window or oval window in a generally relatively simple (e.g., outpatient) procedure. In some embodiments, a composition comprising a therapeutically effective number of viral particles containing a transgene or containing one or more sets of different viral particles (each particle in a set can contain the same type of transgene, but each set of particles contains a different type of transgene than the other sets) as described herein can be delivered to an appropriate location in the ear during a surgical procedure (e.g., a cochleostomy or canalostomy).

[0104] In one embodiment, an inner ear hair cell-targeting AAV (e.g., Anc80 vector) comprising a promoter (e.g., CMV, Espin, PCDH15, PTPRQ, TMHS (LHFPL5)) and a polynucleotide that is one or more of USH1, MYO7A, USH1C (harmonin a, b, c), CDH23, PCDH15, SANS, and CIB2 is injected through the round window of a subject in need thereof.

[0105] In addition, delivery vehicles (e.g., polymers) are available that facilitate transfer of agents across the tympanic membrane and / or through the round window, and any such delivery vehicle can be used to deliver the viruses described herein. See, e.g., Arnold et al., 2005, Audiol. Neurootol., 10:53-63.

[0106] The compositions and methods described herein allow for highly efficient delivery of nucleic acids to inner ear cells, e.g., cochlear cells. For example, the compositions and methods described herein allow for delivery of and expression of a transgene to at least 80% (e.g., at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the inner hair cells or delivery of and expression of a transgene to at least 80% (e.g., at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the outer hair cells.

[0107] As demonstrated herein, expression of a transgene delivered using an AAV containing the Anc80 capsid protein can result in regeneration of inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons, stria vascularis, vestibular hair cells, and / or vestibular ganglion neurons (e.g., Atoh1, NF2), resulting in long-term (e.g., months, years, decades, lifelong) restoration of hearing or vestibular function.

[0108] As described in WO2015 / 054653, AAV containing the Anc80 capsid protein can be characterized by its seropositivity rate and / or the degree to which it is neutralized compared to conventional AAV (i.e., AAV that does not contain the Anc80 capsid protein). Seropositivity rate is understood in the art to refer to the proportion of subjects in a population who are seropositive (i.e., have been exposed to a particular pathogen or immunogen), and is calculated as the number of subjects in a population who produce antibodies to a particular pathogen or immunogen divided by the total number of individuals in the tested population. Determining the seropositivity rate of a virus is routinely performed in the art and generally involves using an immunoassay to determine the positivity rate of one or more antibodies in samples (e.g., blood samples) from a particular population of individuals. In addition, several methods are available for determining the degree of neutralizing antibodies in serum samples. For example, a neutralizing antibody assay measures the titer at which an experimental sample contains an antibody concentration that neutralizes infection by 50% or more compared to a control sample that does not contain the antibody. See also Fisher et al. (1997, Nature Med., 3:306-12) and Manning et al. (1998, Human Gene Ther., 9:477-85). Representative conventional AAVs include, but are not limited to, AAV8 (or viruses containing AAV8 capsid proteins) and / or AAV2 (or viruses containing AAV2 capsid proteins).

[0109] kit The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention (e.g., an inner ear hair cell-targeting AAV (e.g., an Anc80 vector) comprising a promoter (e.g., CMV, Espin, PCDH15, PTPRQ, TMHS (LHFPL5)) and a polynucleotide that is one or more of USH1, MYO7A, USH1C (harmonin a, b, c), CDH23, PCDH15, SANS, and CIB2). Such containers may be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products (reflecting approval by that agency of the manufacture, use, or sale for human administration).

[0110] The present invention also provides kits for treating or preventing a disease or disorder (or a symptom thereof) associated with abnormalities in hearing and / or vestibular mechanosensation. In one embodiment, the kit comprises an effective amount of an inner ear hair cell-targeting AAV (e.g., an Anc80 vector) in a unit dosage form, the AAV comprising a promoter (e.g., CMV, Espin, PCDH15, PTPRQ, TMHS (LHFPL5)) and a polynucleotide selected from one or more of USH1, MYO7A, USH1C (harmonin a, b, c), CDH23, PCDH15, SANS, and CIB2, together with instructions for administering an anti-angiogenic compound to a subject suffering from or susceptible to a disease or disorder associated with angiogenesis or a symptom thereof, wherein the effective amount of the anti-angiogenic compound is less than 500 mg. In a preferred embodiment, the kit includes a sterile container housing the angiogenesis inhibitory compound; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. The instructions generally include information regarding the use of the angiogenesis inhibitory compound for treating angiogenesis-associated diseases or disorders or symptoms thereof; in a preferred embodiment, the instructions include at least one of the following: a detailed description of the angiogenesis inhibitory compound; a dosing schedule and administration for treating angiogenesis-associated diseases or disorders or symptoms thereof; precautions; warnings; indications; counter labeling; overdose information; side effects; animal pharmacology; clinical trials and / or references. The instructions can be printed directly on the container (if present), provided as a label affixed to the container, or provided as a separate sheet, pamphlet, card, or folder provided in or along with the container.

[0111] Conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques within the skill of those in the art can be employed in accordance with the present disclosure. Such techniques are fully explained in the literature. The present invention is further described in the following examples, which do not limit the scope of the claimed methods and compositions of matter. [Example]

[0112] Example 1 - Adeno-associated viruses (AAV) containing ancestral AAV capsid proteins result in safe and efficient cochlear gene transfer The following methods and materials were used in Example 1. viral vectors AAV2 / 1, 2 / 2, 2 / 6, 2 / 8, 2 / 9, and AAV2 / Anc80 containing a CMV-driven eGFP transgene and a woodchuck hepatitis virus posttranscriptional regulator (WPRE) cassette were prepared at the Massachusetts Eye and Ear Gene Transfer Vector Core (vector.meei.harvard.edu) as previously described. AAV2 / Anc80 plasmid reagents are available from addgene.com.

[0113] Animal models and general methods All experiments were approved by Boston Children's Hospital (Protocol #12-02-2146) and the Institutional Biosafety Committee (Protocol #IBC-P00000447). Wild-type C57BL / 6J and CBA / CaJ mice were obtained from Jackson Laboratory (Bar Harbor, ME), and mice of both sexes were used in experiments at an estimated 50 / 50 ratio. Group size per experiment for in vitro and in vivo transformation assays and subsequent endpoints was determined by specimen access and technical feasibility. The reported observations for Anc80 transformation were qualitatively confirmed in subsequent experiments using various vector lots (except for human vestibular tissue transformation due to the unique and limited accessibility of specimens). Due to limited specimen access and the qualitative nature of the reported findings, statistical analysis between serotype transformation efficiencies was not performed.

[0114] Example 1A - In vivo injection Injections were performed through the round window membrane (RWM) of mouse pups (P0–P2) using a beveled glass microinjection pipette. The pipette was pulled from a glass capillary tube (WPI) on a P-2000 pipette puller (Sutter Instrument, Novato, CA) and beveled using a micropipette beveler (Sutter Instrument, Novato, CA) (tip diameter approximately 20 μm, angle 28°). A sterile swab was used to cover the surgical site (left mastoid), and EMLA cream (lidocaine 2.5% and prilocaine 2.5%) was applied externally for analgesia. Preoperatively, body temperature was maintained at 38°C on a heating pad. Pups were anesthetized and rendered unconscious by rapid hypothermia induction in ice / water for 2–3 minutes, and maintained in this state for 5–10 minutes on a cooling platform during surgery. The surgical site was disinfected by scrubbing with betadine and wiping with 70% ethanol three times. A postauricular incision was made to expose the transparent otic vesicle, and a micropipette was manually passed through the otic vesicle and overlying fascia, with the tip of the micropipette passing through the RWM. Approximately 1 μL of virus was manually injected into the left ear alone of five (AAV1), four (AAV2), two (AAV8), one (AAV6), and three (Anc80) C57BL / 6 mice within 1 minute. To control for factors related to specific vector preparations, such as quality and purity, the Anc80 results were confirmed in subsequent tests using a different vector lot from an independent specimen, corroborating our qualitative findings presented herein (data not shown). Injections were performed blindly within each group. Occasionally, the needle was inserted too deeply, too shallowly, or at an incorrect angle. If there was visible damage to the middle or inner ear structures, the sample was excluded from further analysis. The success rate of injection ranged from approximately 50% to approximately 80%, depending on the experience level of the injector. After injection, the skin incision was closed using 6-0 black monofilament suture (Surgical Specialties, Wyoming, PA). The pups were then placed back on a 38°C heating pad for 5–10 minutes and then returned to their mothers for care.

[0115] Consistent with previous reports, AAV1 transduced IHCs with moderate to high efficiency (Figures 1A and 1B). These experiments show that AAV2, 6, and 8 target a small number of IHCs, with only AAV8 demonstrating roughly equivalent apical and basal transduction (Figure 1B). Also consistent with previous reports, OHC transduction was minimal (<5%) with all conventional AAV serotypes tested. However, Anc80 transduced nearly 100% of IHCs and approximately 90% of OHCs at doses 20-fold (for AAV1) to 3-fold (for AAV2) lower (Figures 2A-2C). As observed by live-cell imaging with epifluorescence microscopy, all serotypes equally transduced 1.36 × 10 12 Transformation at GC doses resulted in substantial IHC and OHC transformation in the case of Anc80, whereas IHC targeting was minimal and absent in OHCs in the cases of AAV1, 2, and 8 (Figures 8C, 8D).

[0116] Anc80-transduced samples were subsequently fixed, stained, and imaged by confocal microscopy, revealing a dose-dependent hair cell transformation (Figure 1E). The unparalleled OHC targeting (Figure 1C, Figure 3) demonstrates a qualitatively different transformation biology for Anc80 compared to other AAVs. Similar levels of Anc80 transformation were found throughout the cochlea, from the base to the apex, of all three Anc80-injected mice (Figure 1A, B, C). A low-magnification view of the cochlear apex (Figure 3A) showed strong eGFP expression far from the injection site. A high-magnification image of the base reveals 100% IHC and 95% OHC transformation (Figure 3B).

[0117] Because some forms of hereditary hearing loss also result in vestibular dysfunction, Anc80 may be a useful vector for gene delivery to the human vestibular organ. To investigate this possibility, human vestibular epithelium was harvested from four adult patients undergoing resection of vestibular schwannomas; the sensory epithelium was cultured as previously described. For AAV-transduced samples, Figure 3C reveals strong eGFP fluorescence in both hair cells and supporting cells throughout the human vestibular epithelium. In Figure 3D, a high-magnification view of the epithelium counterstained with Myo7A revealed that 83% (19 / 23) of the Myo7A-positive hair cells were also eGFP-positive, suggesting that Anc80 can efficiently transduce hair cells in both mice and humans.

[0118] Example 1B - Hair Cell Electrophysiology Cochleae were excised, mounted on coverslips, and viewed under an Axio Examiner.A1 upright microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 63x water-immersion objective and differential interference contrast optics. Electrophysiological recordings were performed at room temperature (22–24°C) in a standard solution (pH 7.4; approximately 310 mOsm / kg) containing (in mM): 137 NaCl, 5.8 KCl, 10 HEPES, 0.7 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, and 5.6 D-glucose, vitamins (1:100), and amino acids (1:50). Recording electrodes (3–4 MΩ) were threaded from R-6 glass (King Precision Glass, Claremont, CA) and filled with intracellular solution (pH 7.4; approximately 280 mOsm / kg) containing (in mM): 140 CsCl, 5 EGTA-KOH, 5 HEPES, 2.5 Na2ATP, 3.5 MgCl2, and 0.1 CaCl2. Using the whole-cell tight-seal technique, mechanotransduction currents were recorded using an Axopatch 200B (Molecular Devices, Sunnyvale, CA). Hair cells were maintained at −84 mV. Currents were filtered at 5 kHz with a low-pass Bessel filter, digitized at ≥20 kHz with a 12-bit acquisition board (Digidata 1440A, Molecular Devices, Sunnyvale, CA), and recorded using pCLAMP10 software (Molecular Devices, Sunnyvale, CA).Hair bundles from IHCs and OHCs were deflected using stiff glass probes attached to PICMA-tip piezo actuators (Physik Instrumente, Karlsruhe, Germany) driven by an LVPZT amplifier (E-500.00, Physik Instrumente, Karlsruhe, Germany). The signal was filtered at 40 kHz by an 8-pole Bessel filter (Model 3384 filter, Krohn-Hite Corporation, Brokton, MA) to remove residual pipette resonance. The stiff glass probes were designed to fit onto the concave side of the row of hair cell stereocilia for whole-bundle recordings (3–4 μm diameter for OHCs and 4–5 μm diameter for IHCs). For whole-cell electrophysiology recordings at >P10, cochlear tissue was dissected at P5–7 and incubated in GlutaMAX™-I medium containing MEM (1x) + 1% FBS at 37°C, 5% CO2 for up to 30 days.

[0119] Representative currents evoked by hair bundle deflection from P7 OHCs and P35 IHCs revealed no differences in amplitude, sensitivity, or kinetics between eGFP-positive and eGFP-negative control cells (Figure 1D). After exposure to Anc80, we recorded from 51 eGFP-positive and 52 eGFP-negative hair cells from all regions of the cochlea at 1 to 5 weeks of age. In all cases, responses were indistinguishable from wild-type responses (Figure 1E), confirming that Anc80 transduction does not have deleterious effects on sensory cell function.

[0120] Example 1E - Hearing Test Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) data were collected as previously described. DPOAE is an assay for correct cochlear amplification and entrainment and a sensitive measure of outer hair cell viability. Stimuli tested in anesthetized mice varied in sound pressure levels from 10 to 90 dB at frequencies of 5.6, 8, 11.3, 16, 22.6, and 32 kHz. Four Anc80-injected ears and four non-injected ears, as well as one negative control ear with injection damage and no eGFP fluorescence, were analyzed at P28–P30.

[0121] The minimum sound threshold required to elicit an ABR was plotted (Figure 1F) and revealed no difference in threshold between injected and uninjected ears. Histological analysis revealed strong eGFP fluorescence in all four injected ears (data not shown). In one case, there were no eGFP-positive cells and the ABR threshold was elevated (Figure 1F), suggesting that the injection had failed and the needle had ruptured the cochlear duct, potentially causing permanent damage. Despite robust outer hair cell transduction with Anc80-eGFP, no difference in DPOAE threshold was observed compared to uninjected control ears (Figure 1G). Thus, the data from ABR and DPOAE indicate that RWM injection, Anc80 transduction, and transgene expression in IHCs and OHCs are all safe for auditory function.

[0122] Example 1F - Rotarod Test Five C57BL / 6 mice were tested for balance behavior on the rotarod apparatus. Mice with impaired vestibular function are known to perform poorly on the apparatus. Previous studies have highlighted the ability of this rotarod test to detect balance dysfunction when only one ear is affected. Three mice were injected at P1 and tested at P36, and two uninjected control mice were tested at P79. All mice were tested using the following rotarod protocol: On day 1, mice were trained to balance on a rod rotating at 4 RPM for 5 min. On day 2, mice were tested for five trials (5 min intervals). For each trial, the rod was accelerated by 1 RPM from a starting speed of 2 RPM. The time (in seconds) until the mouse fell off the apparatus was recorded.

[0123] Because the perilymph of the cochlea is continuous with that of the vestibular labyrinth, we evaluated whether Anc80-eGFP injected through the cochlear RWM transduces vestibular sensory organs. Indeed, whole-tissue preparations of the vestibular epithelium revealed robust eGFP expression in type I and type II hair cells of the utricle (a vestibular organ sensitive to gravity and linear head movements) and in the semicircular canals, which are sensitive to rotational head movements (Figure 2A, 2B). Therefore, to address safety concerns that Anc80 transduction may affect balance, we confirmed that Anc80-eGFP-injected mice performed comparable to uninjected controls on the rotarod test of vestibular function (Figure 4).

[0124] Part 2: Gene therapy restores hearing and vestibular function in a mouse model of Usher syndrome type 1c Example 2 - Mouse model of Usher syndrome The following methods and materials were used in Example 2. tissue specimen For electrophysiological studies, the utricle and organ of Corti from Ush1c c.216G>A heterozygous or homozygous mutant mice were collected between postnatal days 0 and 8 (P0–P8). Postnatal pups were sacrificed by rapid decapitation. The temporal bones were removed and immersed in MEM (Invitrogen, Carlsbad, CA) supplemented with 10 mM HEPES (pH 7.4). The organ of Corti was excised without enzymes as previously described (53). After a 10-minute treatment with 0.1 mg / ml protease (Protease XXIV, Sigma), the utricle was removed. The excised organ was mounted on a round coverslip. A pair of thin glass fibers, previously glued to the coverslip, was placed around the edges of the tissue to stabilize it in a flat position. The tissue was either used immediately or cultured in the presence of 1% fetal bovine serum. For experiments involving in vitro viral vector infection, cultures were maintained for 7–8 days, with medium changes every 2–3 days.

[0125] animal Ush1c c.216G>A knockin mice were obtained from the Louisiana State University Health Science Center. Imported strains on a C57BL6 background were previously bred from the Cdh23 (Ahl) mutation to induce age-related hearing loss (48, 49). All procedures used in this study conformed to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Boston Children's Hospital Institutional Animal Care and Use Committees (protocols #12-02-2146, #14-03-2659R, and #15-01-2878R). Mice were genotyped using toe clips (before P8) or ear punches (after P8), and PCR was performed as previously described (32). Male and female mice were used in approximately equal proportions for all studies. No randomization paradigm was otherwise applied.

[0126] Viral vector generation Total RNA was isolated from the cochlea of ​​c.216AA mutant mice (RNAqueous micro kit, Ambion) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). Trunc-harmonin cDNA was synthesized using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and the primers: Trunc-harmonin.F(KpnI). TIFF0007601916000012.tif3128;Trunc-harmomin.RV(BamHI) The 387-bp PCR product was amplified using TIFF0007601916000013.tif3128. The 387-bp PCR product was cloned using a TA cloning kit (Invitrogen) and confirmed by sequencing. To generate a GFP-fusion construct, the truncated harmonin fragment was subcloned into pEGFP-C1 using KpnI and BamHI. The NheI-XbaI EGFP::trunc-harmonin cDNA was introduced into an AAV shuttle vector. The custom vector was packaged into an AAV1 capsid with AAV2 inverted terminal repeats (ITRs), in which the transgene cassette was driven by a CMV promoter (AAV2 / 1.CMV.EGFP::trunc-harmomin.hGH, 1.92 E14gc / m, BCH).

[0127] Harmonin al and harmonin b1 plasmids were prepared in our laboratory from EGFP-tagged constructs kindly provided by Lily Zheng and James Bartles (52) (Department of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine, Chicago, IL). Harmonin al was originally obtained from mouse kidney, and harmonin b1 was originally obtained from isolated mouse cochlear sensory epithelium. We further modified the harmonin al construct to replace its N-terminal EGFP tag with tdTomato. Fluorescently labeled and unlabeled constructs were packaged into AAV vectors. Viral vectors were generated by the Boston Children's Hospital Viral Core Facility and the Massachusetts Eye and Ear Infirmary Gene Transfer Vector Core. The following vectors were generated: AAV2 / 1.CMV.tdTomato::harmonin-a1 4.33 10^13 gc / ml (BCH); AAV2 / 1.CMV.EGFP::harmonin-b1 2.73 564 10^14 gc / ml (BCH); AAV2 / 1.CMV.EGFP-harmonin-a1: 2.81 10^12 gc / ml (MEEI); AAV2 / 1.CMV.EGFP-trunc-harmonin: 1.92 10^14 gc / ml (BCH); AAV2 / Anc80.CMV.harmonin-a1: 1.93 10^12 gc / ml (MEEI); AAV2 / Anc80.CMV.harmonin-b1: 1.74 10^12 gc / ml (MEEI); AAV2 / Anc80.CMV.trunc-harm.WPRE: 9.02 567 10^12 gc / ml (MEEI). For in vitro experiments, 10 μl of concentrated vector was applied to rapidly dissected tissue in 1 ml of MEM-supplemented medium in the presence of 1% fetal bovine serum for 24 hours. Cultures were then maintained for 10 days.

[0128] Round window membrane (RWM) injection RWM injections were performed as approved by Boston Children's Hospital Institutional Animal Care and Use Committee's Animal Protocol #15-01-2878R. 0.8 μl to 1 μl of AAV vector was injected into neonatal mice at P0-P1 and P10-P12. P0-P1 mice were first anesthetized by hypothermia exposure, and P10-P12 mice were anesthetized with isoflurane. During anesthesia, a postauricular incision was performed to expose the otic vesicle and visualize the cochlea. Injections were performed through the RWM using a glass micropipette controlled by a micromanipulator (Askew et al., 2015). The volume of injected material was controlled at approximately 0.02 μl / min for 10 minutes. Standard postoperative care was applied. Sample size for in vivo experiments was continuously measured to optimize sample size and reduce variance.

[0129] Electrophysiological recordings Recordings were performed in standard artificial perilymph containing (in mM): 144 NaCl, 0.7 NaH2PO4, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 5.6 D-glucose, and 10 HEPES-NaOH, adjusted to pH 7.4 and 320 mOsmol / kg. Vitamins (1:50) and amino acids (1:100) from concentrate (Invitrogen, Carlsbad, CA) were added. Hair cells were observed from the apical surface using an Axioskop FS upright microscope (Zeiss, Oberkochen, Germany) equipped with a 63x water-immersion objective and differential interference contrast optics. Recording pipettes (3–5 MΩ) were pulled from borosilicate glass capillaries (Garner Glass, Claremont, CA) and filled with an intracellular solution containing (in mM): 135 KCl, 5 EGTA-KOH, 10 HEPES, 2.5 KATP, 3.5 MgCl, and 0.1 CaCl at pH 7.4. Currents were recorded under whole-cell voltage clamp at room temperature at a holding potential of −64 mV. Data were acquired using an Axopatch Multiclamp 700A or Axopatch 200A (Molecular Devices, Palo Alto, CA), low-pass Bessel filtered at 10 kHz, and digitized at ≥20 kHz using a 12-bit acquisition board (Digidata 1322) and pClamp 8.2 and 10.5 (Molecular Devices, Palo Alto, CA). Data were analyzed offline using OriginLab software. Unless otherwise stated, values ​​are expressed as mean ± standard deviation.

[0130] statistical analysis To ensure reproducibility, test and control vectors were evaluated in at least three mice per group at each time point. Sample sizes are noted in the figure legends. All mice with successful RWM injections were included in the study analysis. Mice with unsuccessful injections were excluded from the averages but included in the legends for full disclosure. Successful injections were determined by ABR recovery at a threshold >90 dB SPL. Statistical analysis was performed using Origin 2016 (OriginLab Corporation). Data are presented as mean ± standard deviation (SD) or standard error of the mean (SEM), as noted in the text and figure legends. One-way analysis of variance (ANOVA) was used to determine significant differences between means.

[0131] Example 2A - Scanning Electron Microscopy (SEM) SEM was performed along the organ of Corti in control and mutant mice at P7, P18, and approximately P42 (6 weeks). SEM at P18 was performed in collaboration with Dr. Edwin Rubel at the University of Washington. The inner ears were fixed overnight at 4°C in 4% glutaraldehyde in 0.1M sodium phosphate buffer. The following day, specimens were rinsed three times in 0.1M sodium phosphate buffer (PB) and postfixed in 1% osmium tetroxide in 0.1M PB for 30 minutes in an ice bath. The specimens were then rinsed in 0.1M PB and dehydrated through a graded series of ethanol: 35%, 70%, 95%, and 100% (×2). The specimens were critical point dried, mounted on SEM stubs, and sputter-coated with Au / Pd. SEM was performed using a JEOL JSM-840A scanning electron microscope. Similar preparations have been performed by Dr. Geleoc and Dr. Indzhykulian at P8 and P6 weeks. Organ of Corti explants were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (Electron Microscopy Sciences) supplemented with 2 mM CaCl2 for 1 hour at room temperature. Specimens were dehydrated in a graded series of acetone, critical point dried from liquid CO2, sputter-coated with 4-5 nm of platinum (Q150T, Quorum Technologies, United Kingdom), and viewed under a field emission scanning electron microscope (S-4800, Hitachi, Japan).

[0132] Homozygous c.216AA mutant mice are deaf and exhibit circling and head-tossing behaviors characteristic of vestibular dysfunction. A previous study by Lentz et al. (34) described significant inner and outer hair cell degeneration in the cochlear base at P30. Degeneration and hair cell death were also observed in the middle turn, with the apical portion of the organ being better preserved at 1 month of age. Assuming that hair cell degeneration occurs gradually during cochlear development, we performed SEM analysis along the organ of Corti at P8 and P18 to assess hair cell survival at earlier ages. Outer hair cells (OHCs) and inner hair cells (IHCs) in heterozygous c.216GA mice were preserved, and their bundles were correctly oriented at these ages (Figures 5A–5C, 5G, 5I and Figures 12A–12C, 12K). However, at both ages analyzed, disorganized hair bundles were evident along the entire length of the organ of Corti in homozygous c.216AA mice (Figures 12D-12F, 12H, 12J-12L and 19D-19J, 19L). At P8, IHC bundles were mildly disorganized in the basal, middle, and apical regions (Figures 12D-12F, 12J). Numerous IHC bundles showed a wavy pattern and mild disorganization of the stereocilia row (Figure 12J). Although many OHCs in c.216AA mutant mice had well-preserved hair bundles (Figures 12H, 12K), fragmented and disorganized hair bundles were evident scattered along the organ (Figures 12D-12F, 12L). At P18, disorganization was more pronounced, but the majority of hair cells were still present (Figures 12D-12F), as previously reported (35).

[0133] To evaluate hair bundle morphology in mice treated with harmonin b1 gene therapy, temporal bones from 6-week-old untreated (or non-injected) and treated (or injected) mice were prepared for SEM analysis. Untreated c.216AA mice showed severe hair cell loss in the basal and intermediate regions of the organ (Figure 11). In the basal region, OHCs were nearly absent from the first row and scattered in the second and third rows. In the intermediate region of the organ, first-row OHCs were also nearly absent. A milder phenotype was observed in the apical region. High-magnification SEM also revealed severely disorganized hair bundles along the entire length of the organ in c.216AA mutant mice. Notably, no hair bundles retained the typical stepped structure with all three rows of stereocilia in 6-week-old c.216AA mice. Instead, hair cells from c.216AA mice showed disorganized hair bundles, with shrunken stereocilia along the first row, an abnormal second row, and a well-preserved top row. In contrast, reduced hair cell loss and normal hair bundles were observed in c.216AA mice after treatment with harmonin b1. Hair cell number was estimated from the presence or absence of hair bundles in representative fields. Data revealed a remarkable preservation of hair cell number from the base to the apex of the organ in injected mice—40–79% at the base, 68–95% at the middle, and 93–99% at the apex (n = 1824 cells from ears of n = 4 c.216AA mice and n = 792 cells from ears of n = 2 rescued c.216AA mice). Although abnormal hair bundles were also evident in harmonin b1-injected mice, the majority of hair bundles had three rows of stereocilia, exhibiting a morphology nearly indistinguishable from their heterozygous controls (Figure 11).

[0134] Example 2B - FM1-43 Imaging Five micromolar FM1-43 (Invitrogen) was diluted in extracellular recording solution and applied to the tissue for 10 seconds. Excess dye was then removed by three washes in extracellular recording solution to prevent endocytic uptake. After 5 minutes, intracellular FM1-43 was imaged on a Zeiss Axioscope FS plus with 20x, 40x, and 63x water-immersion objectives using an epifluorescence light source, differential interference contrast optics, and an FM1-43 filter set (Chroma Technologies). Images were captured at 16-bit resolution using background fluorescence subtraction using a CCD camera and Argus-20 image processor (Hamamatsu). The same gain and contrast settings were maintained for all image acquisitions, and analysis was performed offline using Adobe Photoshop or Image-J software.

[0135] To assess hair cell function earlier, FM1-43 uptake was analyzed at P4 in rapidly dissected cochleae. Following brief application (<10 s), FM1-43 penetrated hair cells with functional mechanosensitive channels (36, 37, 38). While uniform FM1-43 uptake was observed in hair cells of c.216GA mice (Figure 6A), the level of uptake varied among OHCs of c.216AA mice, suggesting that some, but not all, cells retained functional transduction channels (Figure 6B). Similar observations were made along the entire length of the cochlea. No frequency-specific differences were observed. FM1-43 uptake was also reduced in IHCs of c.216AA mice during the first postnatal week (data not shown). We also assessed FM1-43 uptake in utricle hair cells of mutant mice. Interestingly, in c.216AA mutant mice, at P6, uptake was restricted to the extrastriola region, suggesting that hair cells in the striola region do not have open mechanosensitive channels at rest (Figures 6C, 6D).

[0136] Example 2C - Mechanical Stimulation OHC and IHC: Mechanical stimuli were delivered via a rigid glass probe attached to a one-524-dimensional PICMA tip piezoactuator (Physik Instruments, Waldbronn, Germany) driven by a 400 mA ENV400 Amplifier (Piezosystem Jena, Germany). The tip of the probe was fire-polished (Fire Polisher, H602, World Precision Instruments Inc., Sarasota, FL) to fit the stereocilia bundle (51). Deflection was induced by applying a voltage step filtered at 50 kHz by an 8-pole Bessel filter (Khron-Hite, Brockton, MA) to eliminate residual pipette resonance. Hair bundle deflection was monitored using a C2400 CCD camera (Hamamatsu, Japan). The voltage step was used to calibrate the stimulus probe's movement to ±2 μm from its resting position. To confirm the absence of off-axis motion and to calibrate the probe motion, video images of the probe were recorded (spatial resolution approximately 4 nm). The 10-90% rise time of the probe was approximately 20 μsec.

[0137] VHC: Mechanical stimuli were delivered via a rigid glass probe attached to a piezoelectric bimorph element. Coupling was achieved by gentle suction of the kinecery ciliary tissue into the stimulation pipette. Deflection was induced by applying voltage steps to a piezoelectric device consisting of two bimorphs mounted in series and directly connected to the stimulation probe. The voltage steps were controlled by pClamp 8.0 software and filtered at 1 kHz with an 8-pole Bessel filter (Khron-Hite, Brockton, MA). Hair bundle deflection was monitored using a C2400 CCD camera (Hamamatsu, Japan). Prior to the experiment, the motion of the stimulation probe was calibrated around its resting position (±2 μm).

[0138] During the first postnatal week, the auditory and vestibular epithelia retain mechanosensitive hair cells, including those with relatively normal morphology (Figure 5). In the organ of Corti, recordings were obtained from hair cells with normal-appearing bundles and those with more severely disrupted hair bundles from the middle and apical turns of the cochlea of ​​c.216AA mice from P3 to P6. In c.216AA mutants, OHCs retained mechanosensitivity, but the response amplitude was significantly reduced by approximately 63% to 170 ± 80 pA (n = 24; p < 0.001, Figures 6E, 6F, 6G). A wide range of response amplitudes, from 31 to 292 pA, was observed in OHCs in c.216AA mice. Categorizing the data according to hair bundle morphology revealed a significant difference (p < 0.01): currents evoked in mutant hair cells with severely disrupted bundles were smaller than those evoked in mutant cells with more preserved hair bundles, 120 ± 65 pA (n = 9) and 201 ± 74 pA (n = 15), respectively. Despite the reduced current amplitude, hair cell responses to mechanical displacement retained properties similar to those in heterozygous c.216GA mice. We fitted the stimulus-response [I(X)] curves using the second-order Boltzmann equation (Figure 6F), and used this fit to determine the 10–90% operating range (Figure 13B). No significant differences in operating range were observed between OHCs recorded from c.216GA and c.216AA (p = 0.054). Similarly, hair bundles from IHCs of c.216AA mutant mice appeared mildly disrupted under DIC microscopy, but transduction currents were significantly reduced at P6 (Figures 13E, 13F, and 13G). At a holding potential of -64 mV, maximal transduction currents in IHCs of heterozygous c.216GA mice (P6–P7) averaged 587 ± 96 pA (n = 21), whereas in IHCs of c.216AA mice, they were reduced by 46% to 316 ± 127 pA (n = 19; p < 0.001). A significant (p < 0.01) reduction in the operating range was measured in IHCs of c.216AA mutant mice (Figure 13G).

[0139] Adaptation (defined as a decrease in transduction current in the presence of a constant bundle deflection) was also present in c.216AA mutant mice. Adaptation kinetics was analyzed using a double exponential fit to determine the fast and slow components. Both components were slower in IHCs and OHCs from c.216AA mutant mice, but the difference was significant only for the slow component (p<0.05 in OHCs and p<0.001 in IHCs; Figures 13C, 13D, 13H, 13I). On the other hand, the degree of adaptation measured at Popen=0.5 was significantly lower in OHCs and IHCs from c.216AA hair cells than in c.216GA hair cells (Figures 20E, 20J; p<0.001). Together, these results demonstrate that mechanosensitivity is mildly impaired in inner and outer hair cells of c.216AA mice, and importantly, both cell types survive throughout the first postnatal week (a prerequisite for gene therapy and restoration of cellular function).

[0140] We also observed a reduction in mechanotransduction currents in vestibular hair cells in c.216AA mice. In the extrastriola region, currents in c.216AA mice were significantly reduced (p<0.001) to 109 ± 30 pA (n=9, P5–P7) compared with 231 ± 53 pA in c.216GA mice (n=8, P6–P7) (Figures 6E, 6F, 6H). Consistent with the absence of FM1-43 uptake in the striola region, very little or no current was recorded from hair cells in the striola region (6 ± 13 pA, n=6, P5–P7) (see below; Figures 6C, 6D). By DIC microscopy, utricle hair bundles appeared well preserved overall, but transduction currents were significantly reduced or absent from hair cells in the extrastriola and striola, respectively. Thus, excluding the striola region, these results suggest that the transduction apparatus is correctly assembled and targeted in mutant mice, but the number of functional complexes is reduced in newborn mice.

[0141] Next, we evaluated harmonin function in c.216AA hair cells exposed to an AAV vector driving harmonin expression. To increase the likelihood of functional rescue by exogenous harmonin, we packaged untagged harmonin a1 or harmonin b1 coding sequences driven by a CMV promoter into an AAV capsid known as Anc80 (39). Recently, Anc80 capsids have been shown to transduce 100% of IHCs and 80–90% of OHCs in vivo (40). We hypothesize that harmonin b is required for mechanotransduction in both IHCs and OHCs and for auditory function in both cell types. RWM injections of AAV2 / Anc80.CMV.harmonin-b1 (0.8 μl, 1.9 × 10^12 gc / ml) and a mixture of AAV2 / Anc80.CMV.harmonin-a1 (1.7 × 10^12 gc / ml) and AAV2 / Anc80.CMV.harmonin-b1 (0.5 μl + 0.5 μl) were performed, and mechanotransduction responses were assessed 2 weeks after treatment. Tissue was extracted at P5–P6, before cochlear ossification, and maintained in culture for 10 days. Mature OHCs (>P10) did not survive the ex vivo recording paradigm, but robust electrophysiological recordings were obtained from IHCs at P14–P16. Results are presented in Figure 8. Although IHCs from uninjected mice showed severely reduced transduction currents at P16 (79 ± 43 pA, n = 8), recovery of sensory transduction was evident in AAV-treated mice. Significant recovery (***P < 0.001) was observed in mice injected with harmonin b1 or a combination of b1 and a1 at P1, with mean maximum transduction currents of 388 ± 66 pA (n = 15) and 352 ± 28 pA (n = 7; Figure 8C), respectively. Transduction current amplitudes in IHCs after treatment with harmonin b1 were not significantly different from those in control c.216GA mice. The level of recovery was not significantly altered by co-injection of harmonin b1 and harmonin a1. These results suggest that early delivery of exogenous harmonin b1 via RWM injection can restore mechanotransduction in IHCs.

[0142] Example 2D - Confocal Imaging To prepare tissues for confocal imaging from postnatal mice P0–P8, fixation with 4% paraformaldehyde (PFA) was performed for 15 minutes. Actin filaments were labeled using permeabilization with 0.01% Triton and counterstaining with Alexa Fluor phalloidin (Invitrogen, 1 / 200). Images were obtained on an LSM700 Zeiss confocal microscope. After euthanasia, relatively older mice (4–8 weeks old) were euthanized and their temporal bones were removed and placed in 4% PFA for 1 hour. They were then decalcified with 120 mM EDTA for 24–36 hours. The sensory epithelium was then dissected and injected as described above for immunostaining. Mouse anti-CTBP2 (BD Bioscience #612044, 1 / 200) was applied for 48 hours, followed by counterstaining with Alexa Fluor goat anti-mouse antibody (1 / 200) overnight at 4°C to label ribbon synapses. Images were acquired on a Zeiss LSM 710 laser confocal microscope (IDDRC Imaging Core grant P30 HD18655) and processed with Zeiss LSM Image Viewer 4.2.

[0143] Previous studies have revealed the expression of two alternative splice forms of harmonin in sensory hair cells. To assess the ability of AAV vectors to drive the expression of exogenous harmonin splice forms, the utricle and organ of Corti from neonatal C.216AA and wild-type (C57BL / 6J) mice were exposed to AAV2 / 1 vectors encoding eGFP fused to the N-terminus of harmonin b1 (eGFP::harmonin-b1) or tdTomato fused to the N-terminus of harmonin a1 (tdTomato::harmonin-a1). The vectors were applied in vitro or in vivo by RWM injection (1 μl) at P1. When applied in vitro, P0–P1 tissues were incubated in the presence of the vector for 24 hours and then maintained in culture for one week. Confocal images show successful transduction of hair cells in wild-type c.216GA and C.216AA mice (Figures 7A-7C, 7E). EGFP::harmonin-b1 signal was evident at the tips of stereocilia in VHCs (Figure 7A), IHCs, and OHCs (Figures 7B, 7C). EGFP signal was also detected at P60 in OHCs and IHCs in the basal portion of the cochlea of ​​mice injected at P1 (Figure 14D). tdTomato::harmonin-a1 was detected at the base of auditory hair cells (Figure 14E). Costaining with the ribbon synapse marker CTBP2 revealed frequent colocalization in IHCs at P7 (Figure 14E), but not in the utricle at P7 (data not shown). The localization of the exogenous fusion constructs was consistent with previous studies that localized harmonin b to the distal end of the stereocilia near the sensory thread insertion ( 26 , 27 , 28 ) and harmonin a to the synapse ( 30 , 31 ).

[0144] Example 2E - Auditory Brainstem Response (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) ABRs and DPOAEs were recorded from mice anesthetized with xylazine (5–10 mg / kg intraperitoneally) and ketamine (60–100 mg / kg intraperitoneally). Subdermal needle electrodes were inserted into the skin a) between the two ears dorsally (reference electrode); b) behind the left pinna (recording electrode); and c) dorsally at the rump of the mouse (ground electrode). The ear canal was exposed by trimming the passage at the base of the pinna. For ABR recordings, a 5-ms tone was presented to the ear canal and a hearing device (EPL Acoustic system, MEEI, Boston). Responses were amplified (10,000x), filtered (0.1–3 kHz), and averaged by an analog-to-digital board in a PC-based data acquisition system (EPL, Cochlear function test suite, MEEI, Boston). Sound levels were increased in 5–10 dB steps from 0 to 110 dB sound pressure level (decibels SPL). At each level, 512–1024 responses were averaged (alternating stimulus polarity) after "artifact rejection." Thresholds were determined by visual inspection. Data were analyzed and plotted using Origin-2015 (OriginLab Corporation, MA). Unless otherwise stated, threshold means ± standard deviations are presented. For DPOAEs, f1 and f2 primary tones (f2 / f1 = 1.2) were presented (f2 was varied in half-octave steps between 5.6 and 45.2 kHz, and L1–L2 = 10 dB SPL). At each f2, L2 was varied in 10 dB SPL steps between 10 and 80 dB SPL. DPOAE thresholds were determined from the average spectra as the L2 level eliciting DPOAEs 5 dB SPL above the noise floor. The average noise floor level was below 0 dB SPL at all frequencies. Stimuli were generated by a 24-bit digital IO card (National Instruments PXI-4461) in a PXI-1042Q chassis, amplified by an SA-1 speaker driver (Tucker-Davis Technologies, Inc.), and delivered from two electrostatic drivers (CUI CDMG15008-03A) in our custom sound system.An electret microphone (Knowles FG-23329-P07) on the end of a small probe tube was used to monitor the sound pressure in the ear canal. Most of these experiments were not performed under blinded conditions.

[0145] To determine whether truncated harmonin interferes with normal hearing function, we generated the Anc80.CMV.trunc-harm vector to overexpress the truncated protein. The vector was injected into the inner ear of c.216GA mice via the RWM. Measurement of ABRs and DPOAES at 4, 6, and 12 weeks revealed no difference in thresholds between injected and uninjected c.216GA mice (recordings from a 6-week-old mouse shown in Figures 16C-16D). This data serves as a control for the injection technique, i.e., vector, and importantly, demonstrates that exogenous truncated harmonin does not compete with endogenous full-length harmonin, implying that the endogenous truncated form in c.216AA hair cells is unlikely to interfere with exogenous full-length harmonin expressed via a gene therapy vector.

[0146] To determine whether augmentation of the harmonin gene could rescue hearing and balance function in Ush1c mice, RWM injections of AAV2 / Anc80.CMV.harmonin-a1 (0.8 μl, 1.7 × 10^12 gc / ml) or AAV2 / Anc80.CMV.harmonin-b1 (0.8 μl, 1.9 × 10^12 gc / ml) were performed at P0–P1, and auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), acoustic startle reflex, and open-field and rotarod behavior were assessed. Mice were evaluated at 6 weeks, a time when c.216AA mice suffer profound hearing loss and vestibular dysfunction. Some mice were further examined at 3 and 6 months. Of the 12 mice injected with AAV2 / Anc80.CMV.harmonin-a1, none recovered hearing function at 6 weeks (Figures 9A–9C), suggesting that exogenous expression of harmonin-a1 was insufficient for hearing rescue. However, 19 of the 25 mice injected with AAV2 / Anc80.CMV.harmonin-b1 recovered significant hearing function at 6 weeks. At low frequencies (5.6–16 kHz), the best ABR thresholds in the AAV2 / Anc80.CMV.harmonin-b1-injected ears were 25–30 dB SPL, remarkably similar to those in wild-type mice (Figures 16A–16B). Partial rescue was observed at 22.6 kHz, but little to no rescue was observed at 32 kHz. Consistent with the rescue of function in OHCs, rescue of DPOAE thresholds was also evident (Figure 16C). Eight mice with hearing thresholds <45 dB SPL for 8-11.3 kHz stimuli were subsequently tested to assess the longevity of the rescue. From 6 weeks to 3 months, ABR threshold shifts of approximately 10 dB SPL were observed in the low-frequency range and approximately 30 dB SPL in the high-frequency range (Figure 16D). Similar shifts were observed in DPOAE thresholds (Figure 16E). After this time point, ABR thresholds and DPOAEs remained stable until the final testing time point, 6 months of age (Figures 16D-16E).

[0147] To assess whether both harmonin a1 and harmonin b1 are required for more complete hearing rescue, particularly at the high-frequency end, we co-injected AAV2 / Anc80.CMV.tdTomato::harmonin-a1 (0.5 μl; 238 4.1E^12 gc / ml) and AAV2 / Anc80.CMV.eGFP::harmonin-b1 (0.5 μl; 3.0E^12 gc / ml). Sixty-five percent of hair cells expressed both harmonin a1 and harmonin b1, as evidenced by cells positive for both fluorescent tags (Figure 14). Fluorescently labeled harmonin a1 was occasionally observed in the stereocilia of mice exposed to AAV2 / Anc80.CMV.tdTomato::harmonin-a1, likely due to overexpression. ABR and DPOAE thresholds in mice co-injected with unlabeled harmonin a1 and harmonin b1 vectors (Figure 9) were similar to those in mice injected with harmonin b1 alone and provided no further improvement, suggesting that harmonin a1 is dispensable for auditory function. Importantly, the data demonstrate that harmonin b1 alone is sufficient for significant recovery of hearing thresholds at low frequencies (Figure 9).

[0148] To further evaluate the extent of rescue, ABR waveforms from mice with thresholds ≤45 dB SPL were analyzed and compared between eight control c.216GA mice and five c.216AA mice injected with AAV2 / Anc80.CMV.harmonin-b1. Analysis of responses at 8–11.3 kHz and 16 kHz revealed normal wave 1 amplitudes (non-significant difference, P > 0.2, Student's t-test) and longer peak 1 latencies (P > 0.001) (Figure 15), suggesting possible delays in neurotransmission at the synapse. In many mice, auditory rescue was also observed in the contralateral ear, with ABR thresholds as low as 20 dB SPL at 11.3 kHz (harmonin-b1: mean 59.7 ± 5.3 dB SPL, n = 15 / 25; harmonin-a1+b1: mean 76.2 ± 10.3 dB SPL, n = 4–6). Spread of AAV vectors to the contralateral ear has been observed previously ( 37 ) and likely occurs via the perilymphatic vessels, which remain continuous with the subarachnoid space in neonatal mice.

[0149] We were also interested in whether injection at later developmental stages could result in partial hearing rescue. RWM injections of AAV2 / Anc80.CMV.harmonin-b1 (0.8 μl) were performed at P10–P12, and hearing thresholds were assessed at 6 weeks. None of the mice injected at P10–P12 exhibited detectable DPOAEs, and their ABR thresholds did not differ from those of uninjected c.216AA control mice (n = 10; data not shown), suggesting that the window of opportunity for therapeutic intervention may be limited to early postnatal development, possibly due to low viral transduction efficiency in older tissues or degeneration of the organ of Corti at later developmental stages.

[0150] Example 2F - RT-PCR in the Usher Mouse Model cDNA was prepared from the six auditory organs of wild-type, heterozygous, and homozygous Ush1c c.216G>A mice at P2–P3 using the QuantiTect Revese Transcription Kit (Qiagen). cDNA encoding full-length (450 bp) or truncated harmonin (−35 bp) was amplified with the following primers: forward primer TIFF0007601916000014.tif4128, reverse TIFF0007601916000015.tif4128. These primers are specific for the mouse Ush1c sequence, and because the target sequence is outside the region of the human knock-in portion of the Ush1c c.216A allele, they amplify both endogenous and AAV2-derived Ush1c. DNA and RNA levels were also assessed from mouse tissues collected 6 weeks after treatment. DNA and RNA were isolated from the cochlea using TRIzol Reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. RNA was reverse transcribed using the GoScript Reverse Transcription System (Promega, Madison, WI). Radiolabeled PCR was performed using GoTaq Green Master Mix (Promega, Madison, WI). For viral DNA amplification, primers specific for mouse Ush1c were: TIFF0007601916000016.tif4128 and TIFF0007601916000017.tif4128 was used. Homozygous Ush1 c.216AA mice have the human USH1C c.216A gene knocked in to exons 3 and 4, replacing the mouse sequence (32), so these primers only amplify viral Ush1c DNA. For amplification of full-length (450 bp) and aberrantly spliced / truncated harmonin (415 bp) cDNAs, the same primers as above were used (mUsh1c_Ex2F and mUsh1c_Ex5R). Gapdh primers were TIFF0007601916000018.tif4128 and TIFF0007601916000019.tif4128. Products were separated on a 6% non-denaturing polyacrylamide gel and quantified using a Typhoon 9400 phosphorimager (GE Healthcare).

[0151] Because previous studies have raised the possibility that truncated harmonin may impair function by competing with full-length harmonin for endogenous binding partners (34, 35), we were interested in whether persistent expression of the truncated protein might limit recovery in c.216AA mice injected with a vector expressing exogenous full-length harmonin (Figure 16A). To address this concern, we used an RT-PCR assay to examine the expression of Ush1c transcripts in c.216GA and c.216AA mice. Consistent with previous reports, Ush1c transcripts encoding both full-length and truncated harmonin were detected in c.216GA cochleae, whereas transcripts encoding only truncated harmonin were detected in c.216AA cochleae (Figure 16B).

[0152] To confirm AAV2 / Anc80.CMV.harmonin-b1 expression and investigate the relationship between viral expression levels and ABR thresholds, DNA and RNA were isolated from the injected and contralateral cochleae and quantified by PCR and RT-PCR, respectively. Expression was assessed in 6-week-old c.216GA mice and AAV2 / Anc80.CMV.harmonin-b1-injected (0.8 μl; 1.93 10^12 gc / ml) and uninjected c.216AA mice. Samples included two injected mice with good ABR rescue (threshold ≤ 35 dB SPL at 11.3 kHz) and two mice with poor ABR rescue (threshold ≥ 90 dB SPL at 11.3 kHz). RNA encoding the correct splice form of harmonin (Figure 18A) and AAV2 / Anc80.CMV.harmonin-b1 DNA (Figure 18B) was detected in all injected cochleae and, to a lesser extent, in the contralateral cochleae of all mice tested. Variability in ABR thresholds and the amount of expressed DNA and RNA was observed between mice (Figure 18C). However, a strong correlation was found between AAV2 / Anc80.CMV.harmonin-b1 DNA levels, the amount of RNA encoding the correct splice form of harmonin, and ABR threshold levels, suggesting that the variability in ABR data may be a direct result of AAV expression. To assess long-term hair cell survival in mice with successful ABR threshold restoration, tissues were prepared from five mice at 6 months of age and IHCs and OHCs were counted (Figure 17). Although the number of IHCs did not differ between the two cohorts, the three mice that showed long-term ABR rescue had over 50% remaining OHCs. OHC survival was observed along the entire organ except for the basal turn (Figure 17).

[0153] Example 2G - Acoustic Startle Response Acoustic startle responses (ASRs) were measured using a Startle Monitor (Kinder Scientific). Mice were placed in a small, non-restrictive, cubic Plexiglas recording chamber (27 cm x 10 cm x 652 x 12.5 cm) fixed to a piezoelectric / Plexiglas sensing assembly and allowed to acclimate to 60 dB SPL background white noise for 5 min. Each session consisted of 35 trials, during which a single noise pulse was applied at 10 dB SPL intensities ranging from 60 to 120 dB SPL with an average intertrial interval of 30 seconds (range 25 to 35 seconds). To limit external noise interference, pulses were ordered pseudorandomly over a constant 60 dB SPL background noise. The Startle Monitor system converted responses to each pulse into measurements of the first N, maximum N, and maximum time to response (ms) for calculation of the peak startle response (ASR amplitude) and the time from stimulus to peak startle response (ASR latency). All ASRs were performed blinded.

[0154] To assess whether ABR / DPOAE restoration resulted in behaviorally relevant restoration of auditory function, we measured acoustic startle responses in mice injected with AAV2 / Anc80.CMV.harmonin-a1, mice injected with AAV2 / Anc80.CMV.harmonin-b1, and mice injected with both vectors. Analysis of startle responses to white noise demonstrated partial rescue of responses in 6-week-old mice injected with AAV2 / Anc80.CMV.harmonin-b1 and mice co-injected with both vectors (Figure 10A). Mice receiving harmonin-a1 alone did not recover startle responses, similar to uninjected c.216AA mice.

[0155] Example 2H - Vestibular Assessment Vestibular function was assessed using open-field and rotarod balance tests. The open-field test was conducted in a dimly lit room using a 42-cm diameter circular frame placed within a sound chamber with overhead LED lighting set at 30 lux in the center. Mice were placed one at a time into the circular open field and allowed to explore for 5 minutes. Behavior was recorded and tracked using Ethovision XT, allowing for measurement of distance traveled and speed. All open-field assessments were conducted blinded. The rotarod performance involved placing a mouse on a rod in an enclosed housing that began rotating at 4 rpm and accelerated at a rate of 0.1 rpm s-1. On day 1, mice were placed on the rod for 5 minutes to allow for habituation to the apparatus. The following day, mice were placed on the rod for a total of five trials, with a 5-minute rest period between trials. A timer displayed the length of time the mouse was able to remain on the apparatus before falling to the floor of the housing apparatus, and this was recorded after each trial.

[0156] Because the perilymphatic space is continuous between the cochlea and the vestibular labyrinth, AAV vectors injected through the RWM can also transduce the vestibular sensory organs. To assess vestibular behavior, mice were tested for performance on the rotarod. While c.216AA mice and AAV2 / Anc80.CMV.harmonin-a1-injected c.216AA mice exhibited poor rotarod performance (mean latency to fall <22 seconds), AAV2 / Anc80.CMV.harmonin-b1-injected c.216AA mice and mice co-injected with harmonin a1 and b1 vectors maintained balance function on the rotarod for 60–120 seconds, consistent with control c.216GA mice (Figure 10B).

[0157] Recovery of open field behavior was also observed in c.216AA mice injected with harmonin b1 and in c.216AA mice doubly injected with harmonin a1 and b1. Representative open field exploration traces are plotted in Figure 17C. While c.216GA mice explored the boundaries of the field and exhibited minimal whole-body rotations, c.216AA mice showed more activity throughout the chamber and increased whole-body rotations quantified as rotations / min (Figures 10D-10E). Surprisingly, ABR rescue was not observed in mice injected with AAV2 / Anc80.CMV.harmonin-a1, but open field data demonstrated recovery of vestibular function to the level of control mice. The behavior of c.216GA mice injected with AAV2 / Anc80.CMV.trunc-harmonin did not differ from control c.216GA mice, again indicating no interference between truncated harmonin and wild-type harmonin (Figures 10C-10E).

[0158] Example 3 - Polynucleotide therapy of additional mutations involved in hearing loss Example 3A - In vivo experiments Behavioral assays demonstrated partial vestibular rescue by harmonin a1, as mice injected with harmonin a1 failed the rotarod test, although rotational behavior was avoided. Meanwhile, mice injected with harmonin b1 showed functional recovery in both tests (Figure 10). The absence of transduction and FM1-43 uptake in the striola region indicates that hair cell and possibly type I cell function in the striola region depends on correct harmonin expression (Figure 6).

[0159] Hearing rescue was prominent at low frequencies, but not at high frequencies (Figure 9), and preservation of hair bundle morphology was observed along the entire organ at 6 weeks (Figure 11). The absence of rescue at high frequencies is unlikely to be due to damage caused by the injection. No high-frequency hearing loss was observed in any of the c.216GA mice injected with the AAV vector (Figures 16C-16D). AAV targeting along the entire length of the cochlea argues against a lack of transduction efficiency in the base. One possibility is that other harmonin isoforms, such as short harmonin c, may be required for functional rescue at the basal-high-frequency end of the cochlea. Alternatively, because cochlear development begins at the basal end, it is possible that by P0, hair cells from the basal-high-frequency end have matured beyond the point of repair. If this is the case, embryonic intervention may enable better rescue in the high-frequency region.

[0160] Using a previously described helper virus-free system and double transfection method (Grimm et al., 2003, Mol. Ther., 7:839:50), we generated the Anc80 vector, which carries the coding sequence of mouse TMC1 driven by a modified CMV promoter. To allow visualization of the expressed protein, a triple-flag tag (FLAG) sequence was fused to the C-terminus of the TMC1 coding sequence. The Anc80-CMV-Tmc vector was purified using an iodixanol step gradient followed by ion exchange chromatography. Titers were measured by quantitative PCR using a primer set specific for the human beta globin intron element, reaching 1 x 10. 12 ~1×10 13 The viral aliquots were stored at -80°C and thawed immediately before use.

[0161] Mice (P0–P2) were used for in vivo delivery of viral vectors according to protocols approved by the Boston Children's Hospital Institutional Animal Care and Use Committee (protocols #2659 and #2146) as follows: C57BL / 6J (Jackson Laboratories) or Swiss Webster mouse strain (Taconic) were used as wild-type controls, and mice carrying TMC1 mutant alleles (TMC1Δ / Δ or Tmc1− / −) were on a C57BL / 6J background as previously described (Kawashima et al., 2011, J. Clin. Invest., 121:4796–809).

[0162] To prepare tissue for evaluation, temporal bones were harvested from mouse pups between P0 and P10. Pups were euthanized by rapid decapitation, and the temporal bones were dissected in MEM (Invitrogen) supplemented with 10 mM HEPES, 0.05 mg / ml ampicillin, and 0.01 mg / ml ciprofloxacin, pH 7.40. The membranous labyrinth was isolated under a dissecting scope, the Reissner's membrane was peeled off, and the tectorial membrane and stria vascularis were mechanically removed. The organ of Corti cultures were fixed flat under a pair of thin glass fibers, one end of which was glued to an 18 mm round cover glass with Sylgard. The tissue was used immediately for electrophysiological experiments. For mice older than P10, the temporal bones were harvested after euthanasia by inhaled CO2, and whole-tissue preparations of the cochlea were generated.

[0163] All mean values ​​and error bars shown represent mean ± SD. Comparisons of statistical significance between injected and non-injected ears were performed using a two-tailed paired t-test. P<0.05 was considered significant.

[0164] Example 3B - In vivo injection of viral vectors Injections were performed through the round window membrane (RWM) of mouse pups (P0–P2) using a beveled glass microinjection pipette. The pipette was pulled from a glass capillary tube on a P-2000 pipette puller (Sutter Instruments) and beveled using a micropipette beveler (Sutter Instruments) (tip diameter approximately 20 μm, angle 28°). A sterile swab was used to cover the surgical site (left mastoid), and EMLA cream (lidocaine 2.5% and prilocaine 2.5%) was applied externally for analgesia. Body temperature was maintained on a 37°C heating pad for 30–60 minutes before surgery.

[0165] Mouse pups were anesthetized by rapid hypothermia induction for 2–3 min until they lost consciousness, and this state was maintained for 10–15 min during surgery on a cooling platform. The surgical site was disinfected by scrubbing with betadine and wiping with 70% ethanol three times. A postauricular incision was made to expose the transparent otic vesicle, and a micropipette was inserted through the otic vesicle and overlying fascia using a micromanipulator (MP-30, Sutter Instrument Company), passing the tip of the micropipette through the RWM.

[0166] Using a pneumatic microinjector (WPI Nanoliter 2010), a titer of 10 12 ~10 14 Approximately 1 μl of virus (total number of virus particles: 10 9 ~10 11 ) was infused at 0.1 μl / min into the left ear only. The skin incision was closed using 6-0 monofilament suture (Ethicon). The pups were then returned to the heating pad for recovery.

[0167] Example 3C - Immunofluorescence Immunostaining was performed to determine the distribution of transgene expression delivered by the viral vector. Freshly dissected organs of Corti were immersion-fixed with 4% paraformaldehyde diluted in PBS for 1 hour at room temperature, followed by immunostaining. The tissues were then washed in PBS, permeabilized with 0.01-0.1% Triton X-100 for 30 minutes, and counterstained with AlexaFluor 546 phalloidin (Molecular Probes, 1:200 dilution) for 1 hour to label filamentous actin.

[0168] For localization of exogenously expressed TMC::FLAG fusion proteins, tissues were blocked for 1 hour using 2% BSA and 5% normal goat serum and incubated overnight at 4°C with an antibody against the FLAG motif (BD Biosciences, 1:200 dilution). For hair cell counting, tissues were blocked for 1 hour in normal goat serum, stained overnight at 4°C with rabbit anti-myosin VIIa primary antibody (Proteus Biosciences, 1:1000 dilution), and labeled for 1 hour with goat anti-rabbit antibody conjugated to AlexaFluor 488 (Life Technologies, 1:200 dilution). Samples were mounted on coverslips with Vectashield mounting medium (Vector Laboratories) and imaged at 10x–63x magnification using a Zeiss LSM700 confocal microscope.

[0169] FIG. 13 shows immunofluorescence results demonstrating uniform Anc80 delivery of harmonin to Ush1c mutant mice, and FIG. 28 shows immunofluorescence results demonstrating Anc80 delivery of KCNQ4 to cells in KCNQ4 mutant mice.

[0170] Example 3D - Hair Cell Electrophysiology Organotypic cochlear cultures were immersed in standard artificial perilymph containing 137 mM NaCl, 0.7 mM NaH2PO4, 5.8 mM KCl, 1.3 mM CaCl2, 0.9 mM MgCl2, 10 mM Hepes, and 5.6 mM D-glucose. Vitamins (1:50) and amino acids (1:100) were added to the solution from concentrates (Invitrogen), and the final pH was adjusted to 7.40 (310 mosmol / kg) using NaOH. Recording pipettes (3-5 megohms) were pulled from R6 glass capillaries (King Precision Glass) and filled with an intracellular solution containing 135 mM CsCl, 5 mM Hepes, 5 mM EGTA, 2.5 mM MgCl, 2.5 mM Na-adenosine triphosphate, and 0.1 mM CaCl (final pH adjusted to 7.40 (285 mosmol / kg) using CsOH). Whole-cell, tight-seal voltage-clamp recordings were performed at -84 mV at room temperature (22-24 °C) using an Axopatch 200B amplifier (Molecular Devices). Sensory transduction currents were filtered at 10 kHz with a low-pass Bessel filter and digitized at ≥20 kHz with a 16-bit acquisition board (Digidata 1440A) and pCLAMP10 software (Molecular Devices). Data were saved for offline analysis using OriginPro 8 (OriginLab).

[0171] FIG. 23 shows restoration of potassium currents to near wild-type levels in KCNQ4− / − cells transfected with Anc80-KCNQ4 (FIG. 23A) compared to mutant mice (FIG. 23B).

[0172] Example 3E - Auditory Brainstem Response (ABR) ABR recordings were performed as previously described (Maison et al., 2010, J. Neurosci., 30:6751-62). Briefly, mice between P25 and P30 were anesthetized by intraperitoneal injection of 50 mg ketamine and 5 mg xylazine diluted in 5 ml of 0.9% saline (0.1 ml / 10 g body weight). ABR experiments were performed in a sound-proof chamber at 32°C. To test auditory function, mice were presented with pure-tone stimuli at 5.6 kHz, 8 kHz, 11.3 kHz, 16 kHz, 22.6 kHz, or 32 kHz in 5 dB steps from 10 to 115 dB to determine the threshold intensity eliciting reproducible ABR waveforms (peaks I–IV). Alternating polarity stimuli were used to collect 512–1024 responses, which were averaged for each sound pressure level. Waveforms with amplitudes greater than 15 μV (peak-to-trough) were discarded using the "artifact rejection" function. Before the ABR test began, the skin and cartilage flaps that normally obscure the entrance to the ear canal were trimmed with dissection scissors, and the sound pressure at the entrance to the ear canal was calibrated for each individual test subject at all stimulus frequencies. Acoustic stimuli were applied directly to the test subject's ears through a custom probe-tube speaker / microphone assembly (EPL PXI Systems) consisting of two electrostatic earphones (CUI Miniature Dynamics) to generate the primary tone and a Knowles miniature microphone (Electret Condenser) to record the ear canal sound pressure. Sound stimuli consisted of 5 ms tone bursts (cos 2The ABR signal consisted of a 0.5 ms rise-fall time at the onset, applied at 40 / s. Subdermal needle electrodes inserted into the pinna (active electrode), vertex (reference electrode), and buttocks (ground electrode) were used to collect ABR signals. ABR potentials were amplified (10,000x), pass-filtered (0.3–10 kHz), and digitized using custom data acquisition software (LabVIEW). Sound stimuli and electrode voltages were sampled at 40 μs intervals using a digital IO board (National Instruments) and stored for offline analysis. Thresholds were visually defined as the lowest decibel level at which any wave (I–IV) could be detected and reproduced with increasing sound intensity. ABR thresholds were averaged within each experimental group and used for statistical analysis.

[0173] Figure 20 graphically demonstrates that delivery of an Anc80 viral vector encoding and expressing harmonin can provide near-complete restoration of hearing function, particularly at lower frequencies (e.g., about 5 to about 22 kHz).

[0174] Example 3F - Quantitative RT-PCR analysis Experiments were performed to assess the amount of virus present in the cochlea after in vivo administration. Injections were performed into the left ears of two TMC1- / - mice at P1. Cochleae were excised from both ears and maintained in culture for 3 days at the equivalent of P10. RNA was extracted, quality confirmed using an Agilent Bioanalyzer (Agilent Technologies), and reverse transcribed into cDNA for quantitative RT-PCR analysis using an efficient primer set specific for TMC1 using SYBR GreenER qPCR reagents (Invitrogen) as previously described (Kawashima et al., 2011, J. Clin. Invest., 121:4796-809).

[0175] To amplify a fragment of TMC1, the following primers were used: TIFF0007601916000020.tif17137. Expression levels were The levels of Actb (encoding β-actin) amplified in TIFF0007601916000021.tif10142 were normalized to those of Actb (encoding β-actin). All primers were designed to flank introns and were confirmed using melting curve analysis and negative controls. Data were analyzed using the ΔΔCT method for Actb and the difference between injected and uninjected ears. TMC1 mRNA expression was 12-fold higher in injected ears than in uninjected ears.

[0176] Example 3G - FM1-43 Labeling FM1-43 dye-loading experiments were performed as previously described (Gale et al., 2001, J. Neurosci., 21:7013-25; Meyers et al., 2003, J. Neurosci., 23:4054-65; and Geleoc & Holt, 2003, Nat. Neurosci., 10:1019-20). Coverslips with attached cochlear cultures were placed on a glass-bottom chamber under an upright microscope (Zeiss Axioscope FS Plus). 5 μM FM1-43FX (Invitrogen) diluted in artificial perilymph was applied for 10 seconds, and the tissue was washed three times in artificial perilymph to remove the dye from the outer leaflet of the cell membrane. After 5 minutes, intracellular FM1-43 was imaged using an epifluorescence light source with an FM1-43 filter set and a 63× water-immersion objective. Tissues were fixed and processed for immunofluorescence as described above.

[0177] FIG. 23 is an immunostaining image showing FM1-43 dye uptake by cells exposed to the Anc80 viral vector described herein, and FIG. 28 graphically demonstrates that TMC1 delivered by the Anc80 viral vector described herein restores sensory transduction in Tmcl-deficient hair cells in vivo.

[0178] Example 3H - Distortion Product Otoacoustic Emissions (DPOAEs) DPOAE data were collected under the same conditions and during the same recording session as ABR data. To generate DPOAEs at 2f1-f2, a primary tone was generated with a frequency ratio of 1.2 (f2 / f1) (the f2 level was 10 dB lower than the f1 level for each f2 / f1 pair). The f2 level was swept from 20 to 80 dB in 5 dB steps. Waveform and spectral averaging at each level was used to increase the signal-to-noise ratio of the recorded ear canal sound pressure. The amplitude of DPOAEs at 2f1-f2 was extracted from the averaged spectrum, along with the noise floor at nearby points in the spectrum. Iso-response curves were interpolated from plots of DPOAE amplitude versus sound level. The threshold was defined as the f2 level required to generate DPOAEs at 0 dB.

[0179] Figure 25 graphically demonstrates that TMC1 delivered using the Anc80 viral vector described herein rescues outer hair cell function in TMC1- / - mice, particularly at lower frequencies (e.g., about 5 to about 16 kHz).

[0180] FIG. 26 demonstrates the effect of promoters Pcdh15, Myo6, and KCNQ4 on GFP expression in hair cells.

[0181] Other Aspects While methods and compositions of matter have been described herein with reference to several different aspects, it will be understood that the foregoing descriptions of various aspects are intended to be illustrative of the methods and compositions of matter, and are not intended to limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0182] The recitation of a list of elements in any definition of a variable herein includes definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0183] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

[0184] Disclosed are methods and compositions that can be used for, can be used with, can be used in preparation for, or are products of the disclosed methods and compositions. It is understood that these and other materials are disclosed herein, as are combinations, subsets, interactions, groups, etc., of these methods and compositions. That is, even if specific reference to each of the various individual and collective combinations and permutations of these compositions and methods is not explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and described, and multiple compositions or methods are mentioned, each combination and permutation of those compositions and methods is specifically contemplated unless otherwise indicated. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

[0185] Array List Anc80 capsid protein (SEQ ID NO: 1) TIFF0007601916000022.tif39128X1=K / R;X2=A / S;X3=A / G;X4=R / K;X5=E / Q;X6=T / E;X7=A / T;X8=S / N;X9=Q / E;X 10 =S / A;X 11 =N / D

[0186] Anc80-L0065 capsid protein (SEQ ID NO: 2) TIFF0007601916000023.tif39128

[0187] pAAV-TMC2 (SEQ ID NO: 3) Left inverted terminal repeat (L-ITR): 1~130nt Cytomegalovirus (CMV) promoter: 206 to 799 nt Simian virus 40 (SV40) misc intron: 831-963nt TMC1ex1(Transmembrane channel-like 1):982~3,267nt WPRE(Post-transcriptional regulatory element from Woodchuck hepatitis virus):3,268~3,821nt Bovine growth hormone (bGH) poly(A) signal: 3,822 to 4,086 nt Right inverted terminal repeat (R-ITR): 4,124~4,253nt TIFF0007601916000024.tif30128TIFF0007601916000025.tif183128TIFF0007601916000026.tif146128

[0188] pAAV-TMC1ex2 (SEQ ID NO: 4) L-ITR: 1 to 130 CMV promoter: 206-799 SV40 misc intron: 831-963 TMC1ex2:982~3,255 WPRE: 3,256~3,809 bGH poly(A) signal: 3,810 to 4,074 R-ITR: 4,112 to 4,241 TIFF0007601916000027.tif175128TIFF0007601916000028.tif167128

[0189] pAAV-TMC2 (SEQ ID NO: 5) L-ITR: 1 to 130 CMV promoter: 206-799 SV40 misc intron: 831-963 TMC2:981~3,647 WPRE: 3,655~4,208 bGH poly(A) signal: 4,209 to 4,473 R-ITR: 4,511 to 4,640 TIFF0007601916000029.tif151128TIFF0007601916000030.tif177128TIFF0007601916000031.tif54128

[0190] pAAV-Pmyo6-TMC1ex1(SEQ ID NO: 6) L-ITR: 1 to 141 Myosin 6 (myo6) promoter: 155–1,396 TMC1ex1: 1,425~3,710 bGH poly(A) signal: 3,745-4,225 R-ITR: 4,262~4,402 TIFF0007601916000032.tif101128TIFF0007601916000033.tif184128TIFF0007601916000034.tif76128

[0191] pAAV-Pmyo6-TMC1ex1(SEQ ID NO: 7) L-ITR: 1 to 141 myo6 promoter: 155–1,396 TMC1ex2: 1,425~4,439 hGH poly(A) signal: 4,474-4,954 R-ITR: 4,991 to 5,131 TIFF0007601916000035.tif74128TIFF0007601916000036.tif176128TIFF0007601916000037.tif131128

[0192] pAAV-Pmyo6-TMC2 (SEQ ID NO: 8) L-ITR: 1 to 141 myo6 promoter: 155–1,396 TMC2: 1,425~4,091 hGH poly(A) signal: 4,126 to 4,606 R-ITR: 4,643~4,783 TIFF0007601916000038.tif15128TIFF0007601916000039.tif182128TIFF0007601916000040.tif185128

[0193] Sequence information SEQUENCE LISTING <110> THE CHILDREN'S MEDICAL CENTER CORPORATION <120> MATERIALS AND METHODS FOR DELIVERING NUCLEIC ACIDS TO COCHLEAR AND VESTIBULAR CELLS <150> US 62 / 455,197 <151> 2017-02-06 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 736 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polyp <220> <221> MOD_RES <222> (168)..(168) <223> Lys or Arg <220> <221> MOD_RES <222> (204)..(204) <223> Ala or Ser <220> <221> MOD_RES <222> (266)..(266) <223> Ala or Gly <220> <221> MOD_RES <222> (311)..(311) <223> Arg or Lys <220> <221> MOD_RES <222> (411)..(411) <223> Glu or Gln <220> <221> MOD_RES <222> (460)..(460) <223> Thr or Glu <220> <221> MOD_RES <222> (493)..(493) <223> Ala or Thr <220> <221> MOD_RES <222> (562)..(562) <223> Ser or Asn <220> <221> MOD_RES <222> (576)..(576) <223> Gln or Glu <220> <221> MOD_RES <222> (587)..(587) <223> Ser or Ala <220> <221> MOD_RES <222> (609)..(609) <223> Asn or Asp <400> 1 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Lys Gly Gln Gln Pro Ala Xaa Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Asn Thr Met Xaa Ala Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Xaa Ser Thr Asn Asp Asn Thr 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg Pro Lys Xaa Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr Thr Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Xaa Phe Ser Tyr Thr Phe 405 410 415 Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg 435 440 445 Thr Gln Thr Thr Ser Gly Thr Ala Gly Asn Arg Xaa Leu Gln Phe Ser 450 455 460 Gln Ala Gly Pro Ser Ser Met Ala Asn Gln Ala Lys Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Xaa Asn Gln Asn 485 490 495 Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu Asn 500 505 510 Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Thr His Lys 515 520 525 Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Leu Ile Phe Gly 530 535 540 Lys Gln Gly Ala Gly Asn Ser Asn Val Asp Leu Asp Asn Val Met Ile 545 550 555 560 Thr Xaa Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Xaa 565 570 575 Tyr Gly Thr Val Ala Thr Asn Leu Gln Ser Xaa Asn Thr Ala Pro Ala 580 585 590 Thr Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Xaa Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Asn Lys Ser Thr Asn Val Asp Phe Ala Val Asp Thr Asn Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 2 <211> 736 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Asn Thr Met Ala Ala Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Gly Ser Thr Asn Asp Asn Thr 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg Pro Lys Lys Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr Thr Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe 405 410 415 Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg 435 440 445 Thr Gln Thr Thr Ser Gly Thr Ala Gly Asn Arg Thr Leu Gln Phe Ser 450 455 460 Gln Ala Gly Pro Ser Ser Met Ala Asn Gln Ala Lys Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Thr Asn Gln Asn 485 490 495 Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu Asn 500 505 510 Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Thr His Lys 515 520 525 Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Leu Ile Phe Gly 530 535 540 Lys Gln Gly Ala Gly Asn Ser Asn Val Asp Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Glu 565 570 575 Tyr Gly Thr Val Ala Thr Asn Leu Gln Ser Ala Asn Thr Ala Pro Ala 580 585 590 Thr Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Asn Lys Ser Thr Asn Val Asp Phe Ala Val Asp Thr Asn Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 3 <211> 7064 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 3 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggagaatcg gaattcgccc ttaagctagc tagttattaa tagtaatcaa ttacggggtc 240 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 300 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 360 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 420 cttggcagta catcaagtgt atcatatgcc aagtacgcccc cctattgacg tcaatgacgg 480 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 540 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 600 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 660 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 720 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg 780 tttagtgaac cgtcagatcc tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca 840 aggttacaag acagtttta ggagaccaat agaactggg cttgtcgaga cagagaagac 900 tcttgcgttt ctgataggca cctattggtc ttactgacat ccacttgcc tttctctcca 960 caggtgtcca ggcggccgcg gatgccaccc aaaaaagtgc aaatccaagt ggaggagaaa 1020 gagaggatta cagagagag ctcaagtgaa gagagaag atagctacc cagagagag 1080 agctgagac cccaggaaccg acggaccaga gattcatca atgaggatga cccagaaccg 1140 gaggccgagg agggaac agaaaggca agaaaaag aaggcgag gaggctgcgg 1200 agaggagcgg agagaga agaatttgat gagaggaat tagacggtt aaagcactg 1260 ctcgatgaga atagacaat gatcgctact gtcaatgta aaccttggaa atggagaag 1320 aaaattgaag ttctcagga agcaaagaa ttgtgagtg agaatgaagg cgctcttggg 1380 aaaggaagg gaagaagtg gtttgcattt aagatgatga tggccaagaa atggggcaaa 1440 ttcctccgag attttgagaa cttcaagcg gcttgcgtcc catgggaaaa caaatcaag 1500 gcaattgaaa gtcagtttgg tcctcagtg gcctcgtact tcctgttcct caggtggatg 1560 tacggcgtca acatggttct ctttgtgttg accttcagcc tcatcatgtt accggagtac 1620 ctctggggtt taccgtacgg cagcttacct aggaaaacag tcccaagagc tgaagaagca 1680 tctgcagcca actttggtgt gttgtatgac ttcaatggcc tggcgcagta ctctgtcctc 1740 ttttatggct attacgacaa taaacgcacg atcggatggc tgaatttccg gctacctctt 1800 tcctacttcc tggtggggat tatgtgcatt ggatacagct tcctggttgt cctcaaagcg 1860 atgaccaaaa atattggtga cgatggtggt ggcgatgaca acactttcaa cttcagctgg 1920 1980 aactcttatca cgatgaactt taaggaagcc atcatagaag agagccgc acaggtggag 2040 gagaacatcc acctcatcag atttctgagg tttctcgcta acttcttcgt gttcctcaca 2100 cttggtgcaa gtggatacct catcttttgg gctgtgaagc gatcccagga gttcgcccag 2160 caagatcctg acacccttgg gtggtgggaa aaaaatgaaa tgaacatggt aatgtccctc 2220 ctggggatgt tctgtcccac cctgtttgac ttatttgctg aactgga ttaccatcct 2280 ctcattgctc tgaagtggct cctggggcgc atttttgctc ttcttctagg caacttgtat 2340 gtattcattc tcgccttgat ggatgagatt aacaacaaga ttgaagagga gaagcttgtg 2400 aaggccaata ttaccctgtg ggaagccaac atgattaagg cttacaatga atctctctct 2460 gggctctctg ggaacaccac aggagcaccc tttttcgttc atcctgcaga tgtccctcgc 2520 ggtccctgct gggaaacaat ggtggggcag gaattcgtgc gtctcaccgt ttctgacgtc 2580 ctgaccactt acgtcacgat cctcattggc gacttcctca gagcatgttt cgtgaggttc 2640 tgcaattact gctggtgctg ggacttagaa tatggatatc cttcatacac agaattcgac 2700 atcagtggca acgtcctcgc tctgatcttc aaccaaggca tgatctggat gggctccttc 2760 ttcgctccta gcctcccggg catcaacatc ctccgtctcc acacatccat gtatttccag 2820 tgctgggctg tgatgtgctg caatgttccc gaggccaggg tgttcaaagc ttccagatcc 2880 aacaacttct acctcggcat gctgctactc atcctcttcc tgtccaccat gccggtcctg 2940 tacatgatcg tctccctccc gccatctttt gattgtgggc ccttcagtgg taaaaacagg 3000 atgtttgaag tcatcggtga gaccctggaa catgacttcc caagctggat ggcgaagatc 3060 ctgaggcagc tttctaaccc cggccttgtc attgctgtca ttctggtgat ggttctgacc 3120 atctattatc tcaatgctac tgccaagggc cagaaagcag cgaatctgga cctcaaaaag 3180 aagatgaaac agcaagcttt ggagaacaaa atgcgaaaca agaaaatggc agcggctcga 3240 gcagctgcag ctgctggtgg ccagtaagga tccaatcaac ctctggatta caaaatttgt 3300 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct 3360 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat 3420 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg 3480 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag 3540 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc 3600 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg 3660 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc 3720 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc 3780 ctgctgccgg ctctgcggcc tcttccgcgt cttcgagatc tgcctcgact gtgccttcta 3840 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 3900 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 3960 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 4020 gcaggcatgc tggggactcg agttaagggc gaattcccga taaggatctt cctagagcat 4080 ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg 4140 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc 4200 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat 4260 taacctaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 4320 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 4380 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt 4440 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 4500 agcgccctag cgcccgctcc ttcgctttc ttccctcct tctcgccac gttcgccggc 4560 tttccccgtc aagctctaaa tcggggctc cctttaggt tccgatttag tgctttacgg 4620 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 4680 tagacggttt ttcgccctt gacgttggag tccacgttct ttaatagtgg actcttgttc 4740 caaactggaa caactcaa ccctatctcg gtctattctt ttgatttata agggattg 4800 ccgatttcgg cctattggtt aaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 4860 aaaaaatat taacgtttat aatttcaggt ggcatctttc ggggaatgt gcgcggaacc 4920 cctatttgtt tattttcta atacattca atatgtatc cgctcatgag acaataaccc 4980 tgataaatgc ttcaataata tgaaaaagg aagagtag gtattcaca tttccgtgtc 5040 gcccttattc ccttttttgc ggcattttgc ctcctgttt tgctcaccc agaaacgctg 5100 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tggttacat cgaactggat 5160 ctcaatagtg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 5220 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 5280 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 5340 aagcatctta cggatggcat your hand gaattatgca gtgctgccat aaccatgagt 5400 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 5460 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 5520 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagtaatggt aacaacgttg 5580 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 5640 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 5700 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 5760 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagatca ggcaactatg 5820 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 5880 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 5940 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 6000 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 6060 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 6120 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 6180 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 6240 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 6300 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 6360 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 6420 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 6480 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 6540 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 6600 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 6660 cggttcctgg ccttttgctg cggttttgct cacatgttct ttcctgcgtt atcccctgat 6720 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 6780 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct 6840 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 6900 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 6960 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 7020 acaggaaaca gctatgacca tgattacgcc agatttaatt aagg 7064 <210> 4 <211> 7052 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 4 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgccccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggagaatcg gaattcgccc ttaagctagc tagttattaa tagtaatcaa ttacggggtc 240 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 300 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 360 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 420 cttggcagta catcaagtgt atcatatgcc aagtacgcccc cctattgacg tcaatgacgg 480 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 540 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 600 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 660 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 720 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg 780 tttagtgaac cgtcagatcc tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca 840 aggttacaag acagtttta ggagaccaat agaactggg cttgtcgaga cagagaagac 900 tcttgcgttt ctgataggca cctattggtc ttactgacat ccacttgcc tttctctcca 960 caggtgtcca ggcggccgcg gatgttgcaa atccaagtgg aggagaaaga agaggataca 1020 gaggaaagct caagtgaga agagagat aagctaccca gagagagag cttgagacca 1080 agaggaaac gaccagaga tgtcatcat gaggatgacc cagaaccgga gccggaggat 1140 gagaaaaaaaaagggaaaaaaaaaaaaaaaaaaaaeaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ha agg agghaggs ggctggshagg. gagagaag aattgatga agaggatta gaacggttaa aagcactgct cgatgagaat 1260 agacaatga tcgctactgt aaatgtaaa ccttggaaaa tggagaa attgaagtt 1320 ctcaggaag siaagaatt tgtgagtgag atgaaggcg ctcttgggaa aggaaggga 1380 aagaagtggt ttgcatttaa gatgatgatg gccaagaat gggcaaatt cctccgagat 1440 tttgagaact tcaagcggc ttgcgtcca tgggaaaaaatcaggc aattgaaagt 1500 cagtttggtt cctcagtggc ctcgtacttc ctgttcctca ggtggatgta cggcgtcaac 1560 atggttctct ttgtgttgac cttcagcctc atcatgttac cggagtacct ctggggttta 1620 ccgtacggca gcttacctag gaaaacagtc ccaagagctg aagaagcatc tgcagccaac 1680 tttggtgtgt tgtatgactt caatggcctg gcgcagtact ctgtcctctt ttatggctat 1740 tacgacaata aacgcacgat cggatggctg aatttccggc tacctctttc ctacttcctg 1800 gtggggatta tgtgcattgg atacagcttc ctggttgtcc tcaaagcgat gaccaaaaat 1860 attggtgacg atggtggtgg cgatgacaac actttcaact tcagctggaa ggtgttctgt 1920 agctgggact atctgattgg taaccctgaa acagccgaca acaagtttaa ctctatcacg 1980 atgaacttta aggaagccat catagaagag agagccgcac aggtggagga gaacatccac 2040 ctcatcagat ttctgaggtt tctcgctaac ttcttcgtgt tcctcacact tggtgcaagt 2100 ggatacctca tcttttgggc tgtgaagcga tcccaggagt tcgcccagca agatcctgac 2160 acccttgggt ggtgggaaaa aaatgaaatg aacatggtaa tgtccctcct ggggatgttc 2220 tgtcccaccc tgtttgactt atttgctgaa ctggaagatt accatcctct cattgctctg 2280 aagtggctcc tggggcgcat ttttgctctt cttctaggca acttgtatgt attcattctc 2340 gccttgatgg atgagattaa caacaagatt gaagaggaga agcttgtgaa ggccaatatt 2400 accctgtggg aagccaacat gattaaggct tacaatgaat ctctctctgg gctctctggg 2460 aacaccacag gagcaccctt tttcgttcat cctgcagatg tccctcgcgg tccctgctgg 2520 gaaacaatgg tggggcagga attcgtgcgt ctcaccgttt ctgacgtcct gaccacttac 2580 gtcacgatcc tcattggcga cttcctcaga gcatgtttcg tgaggttctg caattactgc 2640 tggtgctggg acttagaata tggatatcct tcatacacag aattcgacat cagtggcaac 2700 gtcctcgctc tgatcttcaa ccaaggcatg atctggatgg gctccttctt cgctcctagc 2760 ctcccgggca tcaacatcct ccgtctccac acatccatgt atttccagtg ctgggctgtg 2820 atgtgctgca atgttcccga ggccagggtg ttcaaagctt ccagatccaa caacttctac 2880 ctcggcatgc tgctactcat cctcttcctg tccaccatgc cggtcctgta catgatcgtc 2940 tccctcccgc catcttttga ttgtgggccc ttcagtggta aaaacaggat gtttgaagtc 3000 atcggtgaga ccctggaaca tgacttccca agctggatgg cgaagatcct gaggcagctt 3060 tctaaccccg gccttgtcat tgctgtcatt ctggtgatgg ttctgaccat ctattatctc 3120 aatgctactg ccaagggcca gaagcagcg aatctggacc tcaaaaagaa gatgaaacag 3180 caagctttgg agacaaaaat gcgaaacaag aaaatggcag cggctcgagc agctgcagct 3240 gctggtggcc agtaaggatc caatcaacct ctggattaca aaatttgtga aagattgact 3300 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 3360 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 3420 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 3480 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 3540 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 3600 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca 3660 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc 3720 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 3780 ctgcggcctc ttccgcgtct tcgagatctg cctcgactgt gccttctagt tgccagccat 3840 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 3900 tttcctaata aaatgagaa attgcatcgc attgtctgag taggtgtcat tctattctgg 3960 ggggtggggt ggggcaggac agcaaggggg aggattggga agaacaatagc aggcatgctg 4020 gggactcgag ttaagggcga attcccgata aggatcttcc tagagcatgg ctacgtagat 4080 aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga gttggccact 4140 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgccccg 4200 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gccttaatta acctaattca 4260 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 4320 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 4380 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 4440 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 4500 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 4560 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 4620 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 4680 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 4740 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 4800 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 4860 acgtttataa tttcaggtgg catctttcgg ggaaatgtgc gcggaacccc tatttgttta 4920 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 4980 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 5040 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 5100 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caatagtggt 5160 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 5220 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 5280 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 5340 gatggcatga cattaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 5400 5460 atggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 5520 aacgacgagc gtgacaccac gatgcctgta gtaatggtaa caacgttgcg caaactatta 5580 actggcgaac tacttactct agcttccccgg aacaattaa tagactggat ggaggcggat 5640 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 5700 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 5760 5820 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 5880 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 5940 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 6000 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 6060 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 6120 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 6180 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 6240 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 6300 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 6360 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 6420 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 6480 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 6540 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 6600 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 6660 ttttgctgcg gttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 6720 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 6780 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 6840 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 6900 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 6960 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 7020 tatgaccatg attacgccag atttaattaa gg 7052 <210> 5 <211> 7451 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 5 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgccccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggagaatcg gaattcgccc ttaagctagc tagttattaa tagtaatcaa ttacggggtc 240 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 300 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 360 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 420 cttggcagta catcaagtgt atcatatgcc aagtacgcccc cctattgacg tcaatgacgg 480 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 540 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 600 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 660 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 720 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg 780 tttagtgaac cgtcagatcc tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca 840 aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac 900 tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca 960 caggtgtcca ggcggccgcc atgagccccc agtaaagag cttggacgag gaaggtgaca 1020 agtcagcaag aagacccaca aggaaacaaa cctccagagc tgcatgtccc caagacggc 1080 accgagccca atctagccgg areatcctg ctaagggtag cccaagacca gggtcttccc 1140 ggagaaaca gatggaacat ggaagctatc acaaggggtt gcagggacag aaaccacgaa 1200 aggtggagag gtctctacaa gggagaaga aggatcggag aacttccctt aaggagcaga 1260 gagcatctcc aaagaaggag agggaggctc tgagaagg ggcaggcaag cagctgagaa 1320 aacccaggtc cacttcctg ggctccagtg tctctactgg agactccctg tctgaggagg 1380 agctggctca gatcctggaa caggtagaag aaaaaaaagaa gctcatcact accgtgagga 1440 acaaaccctg gcccatggca aagaagctga gggaactcag ggaagcccaa gcctttgtgg 1500 agaagtatga aggagccttg gggaaaggca agggcaaaca cctctacgcc tacaggatga 1560 tgatggctaa gaaatgggtc aagtttaaga gggactttga taatttcaag actcaatgta 1620 ttccctggga aatgaagatc aaggacattg aaagtcactt cggttcttct gtggcatctt 1680 acttcatctt tctccgatgg atgtatggag ttaaccttgt cctttttggc ttaatatttg 1740 gtctagtcat catcccagag gtgctgatgg gcatgcccta tggaagtata cccagaaaga 1800 cggtgcctcg ggctgaggaa gagcgagcca tggacttctc tgtcctttgg gattttgagg 1860 gctacatcaa atattctgct ctcttctatg gctactacaa caaccagcgg accattggat 1920 ggctgaggta caggctgccc atggcttact ttatggtggg ggtcagcgtg tttggctaca 1980 gcttgatgat cgtcattagg tcgatggcca gcaataccca gggtagcacc agtgaggggg 2040 acagtgacag cttcacgttc agcttcaaga tgttcaccag ctgggactac ctcatcggga 2100 attcagagac agcagacaac aaatatgtct ccatcactac cagcttcaag gagtctatag 2160 tggacgaaca agagagtaac aaagaaggga atatccacct gacaagattc ctccgcgtcc 2220 tggccaactt tctcattctc tgctgtctgt gtggaagcgg gtacctcatt tactttgtgg 2280 tgaaacggtc ccaggagttc tccaaaatgc aaaatgtcag ctggtatgaa aggaatgagg 2340 tggagatcgt gatgtctctg ctagggatgt tttgtccccc tctgtttgaa accatcgctg 2400 ccttggagaa ttatcaccca cgaactgggc tgaagtggca gctgggccgc atctttgccc 2460 ttttcctggg aaacctctac acgtttctcc tggccctcat ggacgatgtc caccttaagc 2520 tttctaatga ggaaaaaatc aagaacatca ctcactggac cctgtttaac tattacaatt 2580 cctcaggtgg gaatgagagt gtgccccggc caccaccaca ccctgcagat gtgcccagag 2640 gttcttgctg ggagacagct gtgggcattg agtttatgag gctcaccgtg tctgacatgc 2700 tggtaacata cctcaccatc ttggtcggag atttcctccg agcttgtttt gtccggttca 2760 tgaatcactg ctggtgttgg gacctcgagg ctggttttcc ctcatatgcc gagtttgata 2820 ttagtggaaa tgtgttgggt ttgatcttca accaaggaat gatctggatg ggctccttct 2880 atgctccagg actggtgggc atcaatgtcc tgcgcctgtt gacctccatg tacttccagt 2940 gctgggcagt gatgagcagc aacgttcccc atgagcgtgt gttaaagcc tcccgatcca 3000 acaacttcta catgggcctg ctgctgttgg tgctcttcct cagcctcctg cctgtggcct 3060 acactgtcat gtctctccca ccctcgtttg actgtggccc cttcagtggg aaaaacagaa 3120 tgtacgatgt cctccatgag accatcgaga acgatttccc taagttcctg ggcaagatct 3180 ttgcgttcct tgccaaccca ggcctgatca ttccagccat cctgctaatg tttctggcca 3240 tttactacct gaactcagtt tcaaaaagtc tttccagagc taatgcccag ctgcgaaaga 3300 agatccaagc gctccgtgaa gttgagaaga accataaatc catcaaggga aaagccatag 3360 tcacatattc agaggacaca atcaagaaca gctccaaaaa tgccacccag atacatctta 3420 ctaaagaaga gcccacatct cactcttcca gccaaatcca gaccctggac aagaaagcgc 3480 agggccccca cacctccagt actgagggtg gggcctcgcc gtctacctcc tggcaccatg 3540 ttgggtctca accaccgaga ggcagacgag attctggcca accccagtct cagacttata 3600 caggcaggtc accttctgga aagagaaccc agaggcctca caactgataa gcttggatcc 3660 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 3720 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 3780 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 3840 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 3900 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 3960 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 4020 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 4080 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 4140 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 4200 cgagatctgc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 4260 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 4320 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 4380 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggactcgagt taagggcgaa 4440 ttcccgataa ggatcttcct agagcatggc tacgtagata agtagcatgg cgggttaatc 4500 attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 4560 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 4620 gtgagcgagc gagcgcgcag ccttaattaa cctaattcac tggccgtcgt tttacaacgt 4680 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 4740 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 4800 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 4860 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 4920 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 4980 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 5040 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 5100 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 5160 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 5220 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttataat ttcaggtggc 5280 atctttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 5340 5400 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 5460 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 5520 gcacgagtgg gttacatcga actggatctc aatagtggta agatccttga gagttttcgc 5580 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 5640 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 5700 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 5760 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 5820 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 5880 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 5940 atgcctgtag taatggtaac aacgttgcgc aaactattaa ctggcgaact acttactcta 6000 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 6060 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 6120 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 6180 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 6240 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 6300 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 6360 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 6420 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 6480 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 6540 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 6600 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 6660 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 6720 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6780 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 6840 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 6900 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6960 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 7020 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctgcgg ttttgctcac 7080 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 7140 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 7200 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 7260 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 7320 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 7380 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaga 7440 tttaattaag g 7451 <210> 6 <211> 6999 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 6 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120 actccatcac taggggttcc tgcggccgca cgcgtgcaag aaccctcact ggctgaacta 180 tcttgccagc cccttatttt gttttcatat taacctcttt tttctagtaa aggagatgtt 240 tgctctcaaa tttgcatagg aatgtaatat ttaatttaaa aagatgaccc acatatgacc 300 ttataaggac agtaaaatta aacaaccgga aagataaagc gggccagttg gctcagttct 360 ataaaaccag cccacaagga ttgtcactat tcttaggctt gcgcgggcta catgatgagt 420 tccaggactg cctggttaca gaccgagact ctctcaagag tccagataaa caacaacaaa 480 gggggcgagg tggaaataca ggggctgtaa gaagtaaata tgatatctgc atgggaggct 540 agccagagaa gaaaaaattt tcttccgtgg ttcaatcctc caagggctga acaggaagtt 600 gacgcaggca ggtgaggagc acgagcctag atgggctgcg gtgccaccct taatccccac 660 aagcgagttc ctccgcaatt cgcctgtccc actctcaact tttcttcaac tgactctttg 720 ctgtggtccc tcgctgtggc agtggaaaca actaccactg cgaggtaggg aatgtcatga 780 ggggctacct gcagcccttg gcttgcaggg atgcagggat gcggtcggaa cctgaggccc 840 cgcccttc ttgccccacg ccattaggcc acgcccctac ccagcactcc ttcaaccacc 900 cccttccccg gcgcctcatg aggtcccgcc cctctcaacc ctagctcttg aggcctcccc 960 ttcacagccg ccccggcgtt ccttgacttg agccacgtc cctctgctcc ttcattccca 1020 agaccctacg ctttgcgagt cctccctgtc ctgctgccta ggaccccgcc cctctcagcc 1080 cttctgcccc agaccccgc cccttaggct gttcccccccc actggccaat gagacccgc 1140 cctttcttta gccgccccgc cccggtcccca caaatcccg cctccggccc cgccctcccgc 1200 ccccttgggc gctccgtagc agtgacgtgc gcaggctggg cactctgcag gctctctgg 1260 ccggcgggtg gagaccgatc cgggatctgt cccagcagga agcgtaccc ggccgccgtc 1320 gtgctgtcgt ctccggtgct cgctctcggc cgcggtgtcg cgcttgccct tcgcgcccgc 1380 agcccggcag cctctcgagc tcaagctcg aattcgtcga caggatgcca cccaaaaag 1440 tgcaatcca agtggaggag aagaagagg atacagagga aagctcaagt gagagaag 1500 aagataagct accagaga gagagcttga gaccaagag gaaacgacc agagatgtca 1560 tcaatgagga tgacccagaa ccggagccgg aggatgaag aacaagaag gcaagagaa 1620 agaaaggcg gaggaggctg cggaggagg cggaagg agagaaatt gaggagggg 1680 aattagaacg gttaaaagca ctgctcgatg agaatagaca aatgatcgct actgtcaaat 1740 gtaaaccttg gaaaatggag aagaaaattg aagttctcaa ggaagcaaag aaatttgtga 1800 gtgagaatga aggcgctctt gggaaaggaa agggaaagaa gtggtttgca tttaagatga 1860 tgatggccaa gaaatgggca aaattcctcc gagattttga gaacttcaaa gcggcttgcg 1920 tcccatggga aaacaaaatc aaggcaattg aaagtcagtt tggttcctca gtggcctcgt 1980 acttcctgtt cctcaggtgg atgtacggcg tcaacatggt tctctttgtg ttgaccttca 2040 gcctcatcat gttaccggag tacctctggg gtttaccgta cggcagctta cctaggaaaa 2100 cagtcccaag agctgaagaa gcatctgcag ccaactttgg tgtgttgtat gacttcaatg 2160 gcctggcgca gtactctgtc ctcttttatg gctattacga caataaacgc acgatcggat 2220 ggctgaattt ccggctacct ctttcctact tcctggtggg gattatgtgc attggataca 2280 gcttcctggt tgtcctcaaa gcgatgacca aaaatattgg tgacgatggt ggtggcgatg 2340 acaacacttt caacttcagc tggaaggtgt tctgtagctg ggactatctg attggtaacc 2400 ctgaaacagc cgacaacaag tttaactcta tcacgatgaa ctttaaggaa gccatcatag 2460 aagagagagc cgcacaggtg gaggagaaca tccacctcat cagatttctg aggtttctcg 2520 ctaacttctt cgtgttcctc acacttggtg caagtggata cctcatcttt tgggctgtga 2580 agcgatccca ggagttcgcc cagcaagatc ctgacaccct tgggtggtgg gaaaaaaatg 2640 aaatgaacat ggtaatgtcc ctcctgggga tgttctgtcc caccctgttt gacttatttg 2700 ctgaactgga agattaccat cctctcattg ctctgaagtg gctcctgggg cgcatttttg 2760 ctcttcttct aggcaacttg tatgtattca ttctcgcctt gatggatgag attaacaaca 2820 agattgaaga ggagaagctt gtgaaggcca atattaccct gtgggaagcc aacatgatta 2880 aggcttacaa tgaatctctc tctgggctct ctgggaacac cacaggagca ccctttttcg 2940 ttcatcctgc agatgtccct cgcggtccct gctgggaaac aatggtgggg caggaattcg 3000 tgcgtctcac cgtttctgac gtcctgacca cttacgtcac gatcctcatt ggcgacttcc 3060 tcagagcatg tttcgtgagg ttctgcaatt actgctggtg ctgggactta gaatatggat 3120 atccttcata cacagaattc gacatcagtg gcaacgtcct cgctctgatc ttcaaccaag 3180 gcatgatctg gatgggctcc ttcttcgctc ctagcctccc gggcatcaac atcctccgtc 3240 tccacacatc catgtatttc cagtgctggg ctgtgatgtg ctgcaatgtt cccgaggcca 3300 gggtgttcaa agcttccaga tccaacaact tctacctcgg catgctgcta ctcatcctct 3360 tcctgtccac catgccggtc ctgtacatga tcgtctccct cccgccatct tttgattgtg 3420 ggcccttcag tggtaaaaac aggatgtttg aagtcatcgg tgagaccctg gaacatgact 3480 tcccaagctg gatggcgaag atcctgaggc agctttctaa ccccggcctt gtcattgctg 3540 tcattctggt gatggttctg accatctatt atctcaatgc tactgccaag ggccagaaag 3600 cagcgaatct ggacctcaaa aagaagatga aacagcaagc tttggagaac aaaatgcgaa 3660 acaagaaaat ggcagcggct cgagcagctg cagctgctgg tggccagtaa gcggccgctc 3720 gagcctaagc ttctagaaga tctacgggtg gcatccctgt gacccctccc cagtgcctct 3780 cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg 3840 catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg 3900 gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag 3960 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat 4020 tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta 4080 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc 4140 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac 4200 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg 4260 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4320 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 4380 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc 4440 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa 4500 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 4560 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 4620 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 4680 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 4740 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 4800 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct 4860 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 4920 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 4980 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 5040 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 5100 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 5160 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 5220 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 5280 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 5340 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 5400 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 5460 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 5520 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 5580 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 5640 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 5700 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 5760 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 5820 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 5880 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 5940 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 6000 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg tagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca fathercttt agttgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 6480 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 6600 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgac 6720. tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 6780 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 6840 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 6900 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 6960 tacggttcct ggccttttgc tggccttttg ctcacatgt 6999 <210> 7 <211> 7728 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 7 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120 actccatcac taggggttcc tgcggccgca cgcgtgcaag aaccctcact ggctgaacta 180 tcttgccagc cccttatttt gttttcatat taacctcttt tttctagtaa aggagatgtt 240 tgctctcaaa tttgcatagg aatgtaatat ttaatttaaa aagatgaccc acatatgacc 300 ttataaggac agtaaaatta aacaaccgga aagataaagc gggccagttg gctcagttct 360 ataaaaccag cccacaagga ttgtcactat tcttaggctt gcgcgggcta catgatgagt 420 tccaggactg cctggttaca gaccgagact ctctcaagag tccagataaa caacaacaaa 480 gggggcgagg tggaaataca ggggctgtaa gaagtaaata tgatatctgc atgggaggct 540 agccagagaa gaaaaaattt tcttccgtgg ttcaatcctc caagggctga acaggaagtt 600 gacgcaggca ggtgaggagc acgagcctag atgggctgcg gtgccaccct taatccccac 660 aagcgagttc ctccgcaatt cgcctgtccc actctcaact tttcttcaac tgactctttg 720 ctgtggtccc tcgctgtggc agtggaaaca actaccactg cgaggtaggg aatgtcatga 780 ggggctacct gcagcccttg gcttgcaggg atgcagggat gcggtcggaa cctgaggccc 840 cgcccttc ttgccccacg ccattaggcc acgcccctac ccagcactcc ttcaaccacc 900 cccttccccg gcgcctcatg aggtcccgcc cctctcaacc ctagctcttg aggcctcccc 960 ttcacagccg ccccggcgtt cttgacttg aggccacgtc cctctgctcc ttcattccca 1020 agaccctacg ctttgcgagt cctccctgtc ctgctgccta ggaccccgcc cctctcagcc 1080 cttctgcccc aagaccccgc cccttaggct gttcccgccc actggccaat gaagacccgc 1140 cctttcttta gccgccccg cccggtccca caaaatcccg cctccggccc cgcctcccgc 1200 ccccttgggc gctccgtagc agtgacgtgc gcaggctggg cactctgcag ggctctctgg 1260 ccggcgggtg gagaccgatc cgggatctgt cccagcagga agcgtatccc ggccgccgtc 1320 gtgctgtcgt ctcggtgct cgctctcggc cgcggtgcg cgcttgccct tcgcgcgc 1380 agcccggcag cctctcgagc tcaagcttcg aattcgtcga caggatgttg caaatccaag 1440 tggaggagaa agaagaggat acagaggaaa gctcaagtga agaagaagaa gataagctac 1500 ccagaagaga gagcttgaga ccaaagagga aacggaccag agatgtcatc aatgaggatg 1560 acccagaacc ggagccggag gatgaagaaa caagaaaggc aagagaaaaa gaaaggcgga 1620 ggaggctgcg gagaggagcg gaagaagaag aagaaattga tgaagaggaa ttagaacggt 1680 taaaagcact gctcgatgag atagacaaa tgatcgctac tgtcaaatgt aaaccttgga 1740 aaatggagaa gaaaattgaa gttctcaagg aagcaaagaa atttgtgagt gagaatgaag 1800 gcgctcttgg gaaaggaaag ggaaagaagt ggtttgcatt taagatgatg atggccaaga 1860 aatgggcaaa attcctccga gattttgaga acttcaaagc ggcttgcgtc ccatgggaaa 1920 acaaaatcaa ggcaattgaa agtcagtttg gttcctcagt ggcctcgtac ttcctgttcc 1980 tcaggtggat gtacggcgtc aacatggttc tctttgtgtt gaccttcagc ctcatcatgt 2040 taccggagta cctctggggt ttaccgtacg gcagcttacc taggaaaaca gtcccaagag 2100 ctgaagaagc atctgcagcc aactttggtg tgttgtatga cttcaatggc ctggcgcagt 2160 actctgtcct cttttatggc tattacgaca ataaacgcac gatcggatgg ctgaatttcc 2220 ggctacctct ttcctacttc ctggtgggga tttgtgcat tggatacagc ttcctggttg 2280 tcctcaaagc gatgaccaaa aatattggtg acgatggtgg tggcgatgac aacactttca 2340 acttcagctg gaaggtgttc tgtagctggg actatctgat tggtaaccct gaaacagccg 2400 acaacaagtt taactctatc acgatgaact ttaaggaagc catcatagaa gagagagccg 2460 cacaggtgga ggagaacatc cacctcatca gatttctgag gtttctcgct aacttcttcg 2520 tgttcctcac acttggtgca agtggatacc tcatcttttg ggctgtgaag cgatcccagg 2580 agttcgccca gcaagatcct gacacccttg ggtggtggga aaaaaatgaa atgaacatgg 2640 taatgtccct cctggggatg ttctgtccca ccctgtttga cttatttgct gaactggaag 2700 attaccatcc tctcattgct ctgaagtggc tcctggggcg catttttgct cttcttctag 2760 gcaacttgta tgtattcatt ctcgccttga tggatgagat taacaacaag attgaagagg 2820 agaagcttgt gaaggccaat attaccctgt gggaagccaa catgattaag gcttacaatg 2880 aatctctctc tgggctctct gggaacacca caggagcacc ctttttcgtt catcctgcag 2940 atgtccctcg cggtccctgc tgggaaacaa tggtggggca ggaattcgtg cgtctcaccg 3000 tttctgacgt cctgaccact tacgtcacga tcctcattgg cgacttcctc agagcatgtt 3060 tcgtgaggtt ctgcaattac tgctggtgct gggacttaga atatggatat ccttcataca 3120 cagaattcga catcagtggc aacgtcctcg ctctgatctt caaccaaggc atgatctgga 3180 tgggctcctt cttcgctcct agcctcccgg gcatcaacat cctccgtctc cacacatcca 3240 tgtatttcca gtgctgggct gtgatgtgct gcaatgttcc cgaggccagg gtgttcaaag 3300 cttccagatc caacaacttc tacctcggca tgctgctact catcctcttc ctgtccacca 3360 tgccggtcct gtacatgatc gtctccctcc cgccatcttt tgattgtggg cccttcagtg 3420 gtaaaaacag gatgtttgaa gtcatcggtg agaccctgga acatgacttc ccaagctgga 3480 tggcgaagat cctgaggcag ctttctaacc ccggccttgt cattgctgtc attctggtga 3540 tggttctgac catctattat ctcaatgcta ctgccaaggg ccagaaagca gcgaatctgg 3600 acctcaaaaa gaagatgaaa cagcaagctt tggagaacaa aatgcgaaac aagaaaatgg 3660 cagcggctcg agcagctgca gctgctggtg gccagtggat ccaccggccg gtcgccacca 3720 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3780 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 3840 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3900 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 3960 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 4020 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 4080 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 4140 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 4200 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 4260 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 4320 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 4380 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag 4440 cggccgctcg agcctaagct tctagaagat ctacgggtgg catccctgtg acccctcccc 4500 agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa 4560 attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg 4620 ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg 4680 ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt 4740 tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag 4800 gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt 4860 ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca 4920 ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc 4980 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5040 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5100 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5160 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 5220 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 5280 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 5340 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 5400 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 5460 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 5520 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 5580 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 5640 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 5700 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 5760 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 5820 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 5880 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 5940 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6000 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6060 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6120 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6180 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 6240 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 6300 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 6360 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 6420 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 6480 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 6540 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 6600 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 6660 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 6720 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 6780 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagagtc 6840 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 6900 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 6960 tttaatttaa aaggaatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7020 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7080 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7140 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7200 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 7260 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 7320 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 7380 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 7440 acaccgaact gagataccta cagcgtgagc tatgagaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggac aggagagcgc acgagggagc 7560 ttccaggggg aaacgcctgg tatcttata gtcctgtcgg gtttcgccac ctctgacttg 7620 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 7680 cggcctttttt acggttcctg gccttttgct ggccttttgc tcacatgt 7728 <210> 8 <211> 7380 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 8 60. cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccggggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120 actccatcac taggggttcc tgcggccgca cgcgtgcaag aaccctcact ggctgaacta tcttgccagc cccttatttt gttttcatat taacctcttt tttctagtaa aggagatgtt tgctctcaaa tttgcatagg aatgtaatat ttaatttaaa aagatgaccc acatatgacc 300 ttataaggac agtaaaatta aacaaccgga aagataaagc gggccagttg gctcagttct 360 ataaaaccag cccacaagga ttgtcactat tcttaggctt gcgcgggcta catgatgagt 420 tccaggactg cctggttaca gaccgagact ctctcaagag tccagataaa caacaacaaa 480 gggggcgagg tggaaataca ggggctgtaa gaagtaaata tgatatctgc atgggaggct 540 agccagagaa gaaaaaattt tcttccgtgg ttcaatcctc caagggctga acaggaagtt 600 gacgcaggca ggtgaggagc acgagcctag atgggctgcg gtgccaccct taatccccac 660 aagcgagttc ctccgcaatt cgcctgtccc actctcaact tttcttcaac tgactctttg 720 ctgtggtccc tcgctgtggc agtggaaaca actaccactg cgaggtaggg aatgtcatga 780 ggggctacct gcagcccttg gcttgcaggg atgcagggat gcggtcggaa cctgaggccc 840 cgcccttc ttgccccacg ccattaggcc acgcccctac ccagcactcc ttcaaccacc 900 cccttccccg gcgcctcatg aggtcccgcc cctctcaacc ctagctcttg aggcctcccc 960 ttcacagccg ccccggcgtt ccttgacttg aggccacgtc cctctgctcc ttcattccca 1020 agaccctacg ctttgcgagt cctccctgtc ctgctgccta ggaccccgcc cctctcagcc 1080 cttctgcccc aagaccccgc cccttaggct gttcccgccc actggccaat gaagacccgc 1140 cctttcttta gccgccccgc cccggtccca caaaatcccg cctccggccc cgcctcccgc 1200 ccccttgggc gctccgtagc agtgacgtgc gcaggctggg cactctgcag ggctctctgg 1260 ccggcgggtg gagaccgatc cgggatctgt cccagcagga agcgtatccc ggccgccgtc 1320 gtgctgtcgt ctccggtgct cgctctcggc cgcggtgtcg cgcttgccct tcgcgcccgc 1380 agcccggcag cctctcgagc tcaagcttcg aattcgtcga caggatgagc ccccagttaa 1440 agagcttgga cgaggaaggt gacaagtcag caagaagacc cacaaggaaa caaacctcca 1500 gagctgcatg tccccaagac gggcaccgag cccaatctag ccggaaggat cctgctaagg 1560 gtagcccaag accagggtct tcccggaaga aacagatgga acatggaagc tatcacaagg 1620 ggttgcaggg acagaaacca cgaaaggtgg agaggtctct acaagggagg aagaaggatc 1680 ggagaacttc ccttaaggag cagagagcat ctccaaagaa ggagagggag gctctgagga 1740 aggaggcagg caagcagctg agaaaaccca ggtccacttc cttgggctcc agtgtctcta 1800 ctggagactc cctgtctgag gaggagctgg ctcagatcct ggaacaggta gaagaaaaaa 1860 agaagctcat cactaccgtg aggaacaaac cctggcccat ggcaaagaag ctgagggaac 1920 tcagggaagc ccaagccttt gtggagaagt atgaaggagc cttggggaaa ggcaagggca 1980 aacacccta cgcctacagg atgatgatgg ctaagaaatg ggtcaagtttt aagaggggact 2040 ttgataattt caagactcaa tgtattccct gggaaatgaa gatcaaggac attgaaagtc 2100 acttcggttc ttctgtggca tcttacttca tctttctccg atggatgtat ggagttaacc 2160 ttgtcctttt tggcttaata tttggtctag tcatcatccc agaggtgctg atgggcatgc 2220 cctatggaag tatacccaga aagacggtgc ctcgggctga ggaagagcga gccatggact 2280 tctctgtcct ttgggatttt gagggctaca tcaaatattc tgctctcttc tatggctact 2340 acaacaacca gcggaccatt ggatggctga ggtacaggct gcccatggct tactttatgg 2400 tggggtcag cgtgtttggc tacagcttga tgatcgtcat taggtcgatg gccagcaata 2460 cccaggggtag caccagtgag ggggacagtg acagcttcac gttcagcttc aagatgttca 2520 ccagctggga ctacctcatc gggaattcag agacagcaca caaaatat gtctccatca 2580 ctaccagctt caaggagtct atagtggacg aacaagagag taacaaaaga gggaatatcc 2640 acctgacaag attcctccgc gtcctggcca actttctcat tctctgctgt ctgtgtggaa 2700 gcgggtacct catttacttt gtggtgaaac ggtcccagga gttctccaaa atgcaaaatg 2760 tcagctggta tgaaaggaat gaggtggaga tcgtgatgtc tctgctaggg atgttttgtc 2820 cccctctgtt tgaaaccatc gctgccttgg agaattatca cccacgaact gggctgaagt 2880 ggcagctggg ccgcatcttt gcccttttcc tgggaaacct ctacacgttt ctcctggccc 2940 tcatggacga tgtccacctt aagctttcta atgaggaaaa aatcaagaac atcactcact 3000 ggaccctgtt taactattac aattcctcag gtgggaatga gagtgtgccc cggccaccac 3060 cacaccctgc agatgtgccc agaggttctt gctgggagac agctgtgggc attgagttta 3120 tgaggctcac cgtgtctgac atgctggtaa catacctcac catcttggtc ggagatttcc 3180 tccgagcttg ttttgtccgg ttcatgaatc actgctggtg ttgggacctc gaggctggtt 3240 ttccctcata tgccgagttt gatattagtg gaaatgtgtt gggtttgatc ttcaaccaag 3300 gaatgatctg gatgggctcc ttctatgctc caggactggt gggcatcaat gtcctgcgcc 3360 tgttgacctc catgtacttc cagtgctggg cagtgatgag cagcaacgtt ccccatgagc 3420 gtgtgtttaa agcctcccga tccaacaact tctacatggg cctgctgctg ttggtgctct 3480 tcctcagcct cctgcctgtg gcctacactg tcatgtctct cccaccctcg tttgactgtg 3540 gccccttcag tgggaaaaac agaatgtacg atgtcctcca tgagaccatc gagaacgatt 3600 tccctaagtt cctgggcaag atctttgcgt tccttgccaa cccaggcctg atcattccag 3660 ccatcctgct aatgtttctg gccatttact acctgaactc agtttcaaaa agtctttcca 3720 gagctaatgc ccagctgcga aagaagatcc aagcgctccg tgaagttgag aagaaccata 3780 aatccatcaa gggaaaagcc atagtcacat attcagagga cacaatcaag aacagctcca 3840 aaaatgccac ccagatacat cttactaaag aagagcccac atctcactct tccagccaaa 3900 tccagaccct ggacaagaaa gcgcagggcc cccacacctc cagtactgag ggtggggcct 3960 cgccgtctac ctcctggcac catgttgggt ctcaaccacc gagaggcaga cgagattctg 4020 gccaacccca gtctcagact tatacaggca ggtcaccttc tggaaagaga acccagaggc 4080 ctcacaactg agcggccgct cgagcctaag cttctagaag atctacgggt ggcatccctg 4140 tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct 4200 tgtcctaata aaattaagtt gcatcattt gtctgactag gtgtccttct ataatattat 4260 ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg 4320 cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc 4380 cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc 4440 atgcatgacc aggctcagct aatttttgtt ttttggtag agacggggtt tcaccatatt 4500 ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt 4560 gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac 4620 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg 4680 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc 4740 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat 4800 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg 4860 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 4920 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 4980 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 5040 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat 5100 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 5160 tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag 5220 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 5280 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct 5340 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 5400 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 5460 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 5520 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 5580 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 5640 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 5700 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 5760 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 5820 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 5880 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 5940 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 6000 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 6060 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 6120 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 6180 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 6240 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 6300 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 6360 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 6420 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 6480 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 6540 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 6600 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 6660 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 6720 aaggatcttc ttgagatcct ttttttgc gcgtaatctg ctgcttgcaa acaaaaaaac 6780 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg 6840 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 6900 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 6960 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 7020 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 7080 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 7140 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 7200 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 7260 acctctgact tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 7320 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 7380 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 catctgcagc caactttggt gtgt 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 agaggtagcc ggaaattcag ccat 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 11 tgagcgcaag tactctgtgt ggat 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 12 actcatcgta ctcctgcttg ctga 24 <210> 13 <211> 734 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Asn Thr Met Ala Ala Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Ala Leu Pro Thr Tyr Asn Asn His Leu Tyr 245 250 255 Lys Gln Ile Ser Ser Gln Ser Gly Gly Ser Thr Asn Asp Asn Thr Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Lys Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Thr Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Gln Thr Thr Ser Gly Thr Ala Gly Asn Arg Thr Leu Gln Phe Ser Gln 450 455 460 Ala Gly Pro Ser Ser Met Ala Asn Gln Ala Lys Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Thr Asn Gln Asn Asn 485 490 495 Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Thr His Lys Asp 515 520 525 Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ala Gly Asn Ser Asn Val Asp Leu Asp Asn Val Met Ile Thr 545 550 555 560 Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Glu Tyr 565 570 575 Gly Thr Val Ala Thr Asn Leu Gln Ser Ala Asn Thr Ala Pro Ala Thr 580 585 590 Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp Gln Asp 595 600 605 Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615 620 Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 625 630 635 640 His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 645 650 655 Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670 Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Glu Leu Gln Lys Glu 675 680 685 Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn 690 695 700 Lys Ser Thr Asn Val Asp Phe Ala Val Asp Thr Asn Gly Val Tyr Ser 705 710 715 720 Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 <210> 14 <211> 760 <212> PRT <213> Homo sapiens <400> 14 Met Ser Pro Lys Lys Val Gln Ile Lys Val Glu Glu Lys Glu Asp Glu 1 5 10 15 Thr Glu Glu Ser Ser Ser Glu Glu Glu Glu Glu Val Glu Asp Lys Leu 20 25 30 Pro Arg Arg Glu Ser Leu Arg Pro Lys Arg Lys Arg Thr Arg Asp Val 35 40 45 Ile Asn Glu Asp Asp Pro Glu Pro Glu Pro Glu Asp Glu Glu Thr Arg 50 55 60 Lys Ala Arg Glu Lys Glu Arg Arg Arg Arg Leu Lys Arg Gly Ala Glu 65 70 75 80 Glu Glu Glu Ile Asp Glu Glu Glu Leu Glu Arg Leu Lys Ala Glu Leu 85 90 95 Asp Glu Lys Arg Gln Ile Ile Ala Thr Val Lys Cys Lys Pro Trp Lys 100 105 110 Met Glu Lys Lys Ile Glu Val Leu Lys Glu Ala Lys Lys Phe Val Ser 115 120 125 Glu Asn Glu Gly Ala Leu Gly Lys Gly Lys Gly Lys Arg Trp Phe Ala 130 135 140 Phe Lys Met Met Met Ala Lys Lys Trp Ala Lys Phe Leu Arg Asp Phe 145 150 155 160 Glu Asn Phe Lys Ala Ala Cys Val Pro Trp Glu Asn Lys Ile Lys Ala 165 170 175 Ile Glu Ser Gln Phe Gly Ser Val Ser Ala Ser Phe Leu Phe Leu 180 185 190 Arg Trp Met Tyr Gly Val Asn Met Val Leu Phe Ile Leu Thr Phe Ser 195 200 205 Leu And Met Leu Pro Glu Tyr Trp Gly Leu Pro Tyr Gly Ser Leu 210 215 220 Pro Arg Lys Thr Val Pro Arg Ala Glu Glu Ala Ser Ala Ala Asn Phe 225 230 235 240 Gly Val Leu Tyr Asp Phe Asn Gly Leu Ala Gln Tyr Ser Val Leu Phe 245 250 255 Tyr Gly Tyr Tyr Asp Asn Lys Arg Thr Ile Gly Trp Met Asn Phe Arg 260 265 270 Leu Pro Leu Ser Tyr Phe Leu Val Gly Ile Met Cys Ile Gly Tyr Ser 275 280 285 Phe Leu Val Val Leu Lys Ala Met Thr Lys Asn Ile Gly Asp Asp Gly 290,295,300 Gly Gly Asp Asp Asn Thr Phe Asn Phe Ser Trp Lys Val Phe Thr Ser 305 310 315 320 Trp Asp Tyr Leu Ile Gly Asn Pro Glu Thr Ala Asp Asn Lys Phe Asn 325 330 335 Ser Ile Thr Met Asn Phe Lys Glu Ala Ile Thr Glu Glu Lys Ala Ala 340 345 350 Gln Val Glu Glu Asn Val His Leu Ile Arg Phe Leu Arg Phe Leu Ala 355 360 365 Asn Phe Phe Val Phe Leu Thr Leu Gly Gly Ser Gly Tyr Leu Ile Phe 370 375 380 Trp Ala Val Lys Arg Ser Gln Glu Phe Ala Gln Gln Asp Pro Asp Thr 385 390 395 400 Leu Gly Trp Trp Glu Lys Asn Glu Met Asn Met Val Met Ser Leu Leu 405 410 415 Gly Met Phe Cys Pro Thr Leu Phe Asp Leu Phe Ala Glu Leu Glu Asp 420 425 430 Tyr His Pro Leu Ile Ala Leu Lys Trp Leu Leu Gly Arg Ile Phe Ala 435 440 445 Leu Leu Leu Gly Asn Leu Tyr Val Phe Ile Leu Ala Leu Met Asp Glu 450 455 460 Ile Asn Asn Lys Ile Glu Glu Glu Lys Leu Val Lys Ala Asn Ile Thr 465 470 475 480 Leu Trp Glu Ala Asn Met Ile Lys Ala Tyr Asn Ala Ser Phe Ser Glu 485 490 495 Asn Ser Thr Gly Pro Pro Phe Phe Val His Pro Ala Asp Val Pro Arg 500 505 510 Gly Pro Cys Trp Glu Thr Met Val Gly Gln Glu Phe Val Arg Leu Thr 515 520 525 Val Ser Asp Val Leu Thr Thr Tyr Val Thr Ile Leu Ile Gly Asp Phe 530 535 540 Leu Arg Ala Cys Phe Val Arg Phe Cys Asn Tyr Cys Trp Cys Trp Asp 545 550 555 560 Leu Glu Tyr Gly Tyr Pro Ser Tyr Thr Glu Phe Asp Ile Ser Gly Asn 565 570 575 Val Leu Ala Leu Ile Phe Asn Gln Gly Met Ile Trp Met Gly Ser Phe 580 585 590 Phe Ala Pro Ser Leu Pro Gly Ile Asn Ile Leu Arg Leu His Thr Ser 595 600 605 Met Tyr Phe Gln Cys Trp Ala Val Met Cys Cys Asn Val Pro Glu Ala 610 615 620 Arg Val Phe Lys Ala Ser Arg Ser Asn Asn Phe Tyr Leu Gly Met Leu 625 630 635 640 Leu Leu Ile Leu Phe Leu Ser Thr Met Pro Val Leu Tyr Met Ile Val 645 650 655 Ser Leu Pro Pro Ser Phe Asp Cys Gly Pro Phe Ser Gly Lys Asn Arg 660 665 670 Met Phe Glu Val Ile Gly Glu Thr Leu Glu His Asp Phe Pro Ser Trp 675 680 685 Met Ala Lys Ile Leu Arg Gln Leu Ser Asn Pro Gly Leu Val Ile Ala 690 695 700 Val Ile Leu Val Met Val Leu Ala Ile Tyr Tyr Leu Asn Ala Thr Ala 705 710 715 720 Lys Gly Gln Lys Ala Ala Asn Leu Asp Leu Lys Lys Lys Met Lys Met 725 730 735 Gln Ala Leu Glu Asn Lys Met Arg Asn Lys Lys Met Ala Ala Ala Arg 740 745 750 Ala Ala Ala Ala Ala Gly Arg Gln 755 760 <210> 15 <211> 3201 <212> Ms <213> Homo sapiens <400> 15 cagaaactat gagggcagaa cccagcaatc tgtgcttct ttcacaagcc ctccaggagt 60 tgctgaaatt taggaatcat tgccccaaa agtggccctc ataatgatgc cagatgggat 120 cttactctgt tgcccaggct ggtgcagt ggtgcgatct cggctctg CAcctccgc 180 ctcccaggtt caagtgatc tcctgcctcg gcctcctgag tagctggt ttcaggccat 240 gaaagatcac tgtttagtc tgcgtgtgc agtggaacag atagacctcg gtttgaatct 300 cagctctact gtttactaga catgaaatgg ggaaatctaa atgagatgc cagaagccctc 360 aaaaatggaa aaccccctgt gctcacatc tgaaatctc tgctggggc agcaactttg 420 agcctgtggg gaaggaactg tccacgtgga gtggtctggt gatgcttaa ggagctgcag 480 aagggaagtc cctctccaaa ctagccagcc actgagacct tctgacagga caccccagg 540 atgtcacca aaaaagtaca atcaagtg gaggaaaag aagacgac tgaggaagc 600 tcaagtgaag aggagagga ggtggagat aagctacctc gagagagag cttgagacca 660 agaggaaac ggaccagaga tgttatcaat gaggatgacc cagaacctga accagaggat 720 gagaaaaaaaaaaaaaaaaaaaaaaaggggaaaaaagg ggctaagaaaaagg 780 gagaaaa ttgatgaaga ggattgaaa agattgaagg cagagttaga tgagaaaaga 840 caaatattg ctactgtca atgcaacca tggagatgg agaagaaat tgaagttctc 900 aaggaggcaaaaaatttgt gagtgaaat gaggggctc ttgggaagg aaaaaaaaaaaaaaaaaaaaaaaaaaaaaalling 960 cggtggtttg cattttagat gatgatggcc aagaatggg caaatttcct ccgtgatttt 1020 gagaacttca aagctgcgtg tgtcccatgg gaaataaaa tcaggctat tgaagtcag 1080 tttggctcct cagtggccctc atactcctc ttcttgagat ggatgtatgg agtcaatg 1140 gttctcttta tcctgacatt tagcctcatc atgttgccag agtaccctg gggtttgcca 1200 tatggcagtt tacctaggaaaccgttccc agagccgaag agcatcggc agcaacttt 1260 ggtgtgttgt acgactca tggtttgca caatattccg ttctcttta tggctattat 1320 gabaataac gaacaatttgg atggatgaat ttcaggttgc cgctctccta ttttctagtg 1380 gggattatgt gcattggata cagctttctg gttgtcctca aagcaatgac caaaaacatt 1440 ggtgatgatg gaggtggaga tgacaacact ttcaatttca gctggaaggt cttaccagc 1500 tgggactacc tgatcggcaa tcctgaaaca gcagacaaca aatttaattc tatcacaatg 1560 aactttaagg aagctatcac agaagaaaaa gcagcccaag tagaagaaaa cgtccacttg 1620 atcagattcc tgaggtttct ggctaacttc ttcgtgtttc taacacttgg agggagtgga 1680 tacctcatct tttgggctgt gaagcgatcc caggaatttg cacagcaaga tcctgacacc 1740 cttgggtggt gggaaaaaaa tgaaatgaac atggttatgt ccctcctagg gatgttctgt 1800 ccaacattgt ttgacttatt tgctgaatta gaagactacc atcctctcat cgctttgaaa 1860 tggctactgg gacgcatttt tgctcttctt ttaggcaatt tatacgtatt tattcttgca 1920 ttaatggatg agattaacaa caagattgaa gaggagaagc tagtaaaggc caatattacc 1980 ctttgggaag ccaatatgat caaggcctac aatgcatcat tctctgaaaa tagcactgga 2040 ccacccttt ttgttcaccc tgcagatgta cctcgaggac cttgctggga aacaatggtg 2100 ggacaggagt ttgtgaggct gacagtctct gatgttctga ccacctacgt cacaatcctc 2160 attggggact ttctaagggc atgttttgtg aggttttgca attattgctg gtgctgggac 2220 ttggagtatg gatatccttc atacaccgaa ttcgacatca gtggcaacgt cctcgctctg 2280 atcttcaacc aaggcatgat ctggatgggc tccttctttg ctcccagcct cccaggcatc 2340 aatatccttc gactccatac atccatgtac ttccagtgct gggccgttat gtgctgcaat 2400 gttcctgagg ccagggtctt caaagcttcc agatcaaata acttctacct gggcatgcta 2460 ctgctcatcc tcttcctgtc cacaatgcct gtcttgtaca tgatcgtgtc cctcccacca 2520 tcttttgatt gtggtccatt cagtggcaaa aatagaatgt ttgaagtcat tggagagacc 2580 ctggagcacg atttcccaag ctggatggcg aagatcttga gacagctttc aaaccctggg 2640 ctggtcattg ctgtcatttt ggtgatggtt ttggccatct attatctcaa tgctactgcc 2700 aagggccaga aggcagcgaa tctggatctc aaaaagaaga tgaaaatgca agctttggag 2760 aacaaaatgc gaaacaagaa aatggcagct gcacgagcag ctgcagctgc tggtcgccag 2820 tataagtat cctgagagcc cagaaaaggt acactttgcc ttgctgttta aaagtaatgc 2880 aatatgtgaa cgcccagaga acaagcactg tggaactgct atttcctgt tctacccttg 2940 atggattttc aaggtcatgc tggccaatta aggcatcatc agtcctacct gagcaacaag 3000 aatctaaact ttattccaag tcagaaactg tttctgcaga gccactctct cccctgctcc 3060 atttcgtgac tttttttttt ttttaacaa attgagttta gaagtgagtg taatccagca 3120 atacagttta ctggtttagt tggtgggtta attaaaaaaa atttgctcat atgaactttc 3180 atttatatg tttctttgc c 3201 <210> 16 <211> 906 <212> PRT <213> Homo sapiens <400> 16 Met Ser His Gln Val Lys Gly Leu Lys Glu Glu Ala Arg Gly Gly Val 1 5 10 15 Lys Gly Arg Val Lys Ser Gly Ser Pro His Thr Gly Asp Arg Leu Gly 20 25 30 Arg Arg Ser Ser Ser Lys Arg Ala Leu Lys Ala Glu Gly Thr Pro Gly 35 40 45 Arg Arg Gly Ala Gln Arg Ser Gln Lys Glu Arg Ala Gly Gly Ser Pro 50 55 60 Ser Pro Gly Ser Pro Arg Arg Lys Gln Thr Gly Arg Arg Arg Arg His Arg 65 70 75 80 Glu Glu Leu Gly Glu Gln Glu Arg Gly Glu Ala Glu Arg Thr Cys Glu 85 90 95 Gly Arg Arg Lys Arg Asp Glu Arg Ala Ser Phe Gln Glu Arg Thr Ala 100 105 110 Ala Pro Lys Arg Glu Lys Glu Ile Pro Arg Arg Glu Glu Lys Ser Lys 115 120 125 Arg Gln Lys Lys Pro Arg Ser Ser Ser Leu Ala Ser Ser Ala Ser Gly 130 135 140 Gly Glu Ser Leu Ser Glu Glu Glu Leu Ala Gln Ile Leu Glu Gln Val 145 150 155 160 Glu Glu Lys Lys Lys Leu Ile Ala Thr Met Arg Ser Lys Pro Trp Pro 165 170 175 Met Ala Lys Lys Leu Thr Glu Leu Arg Glu Ala Gln Glu Phe Val Glu 180 185 190 Lys Tyr Glu Gly Ala Leu Gly Lys Gly Lys Gly Lys Gln Leu Tyr Ala 195 200 205 Tyr Lys Met Leu Met Ala Lys Lys Trp Val Lys Phe Lys Arg Asp Phe 210 215 220 Asp Asn Phe Lys Thr Gln Cys Ile Pro Trp Glu Met Lys Ile Lys Asp 225 230 235 240 Ile Glu Ser His Phe Gly Ser Ser Val Ala Ser Tyr Phe Ile Phe Leu 245 250 255 Arg Trp Met Tyr Gly Val Asn Leu Val Leu Phe Gly Leu Ile Phe Gly 260 265 270 Leu Val Ile Ile Pro Glu Val Leu Met Gly Met Pro Tyr Gly Ser Ile 275 280 285 Pro Arg Lys Thr Val Pro Arg Ala Glu Glu Glu Lys Ala Met Asp Phe 290 295 300 Ser Val Leu Trp Asp Phe Glu Gly Tyr Ile Lys Tyr Ser Ala Leu Phe 305 310 315 320 Tyr Gly Tyr Tyr Asn Asn Gln Arg Thr Ile Gly Trp Leu Arg Tyr Arg 325 330 335 Leu Pro Met Ala Tyr Phe Met Val Gly Val Ser Val Phe Gly Tyr Ser 340 345 350 Leu Ile Ile Val Ile Arg Ser Met Ala Ser Asn Thr Gln Gly Ser Thr 355 360 365 Gly Glu Gly Glu Ser Asp Asn Phe Thr Phe Ser Phe Lys Met Phe Thr 370 375 380 Ser Trp Asp Tyr Leu Ile Gly Asn Ser Glu Thr Ala Asp Asn Lys Tyr 385 390 395 400 Ala Ser Ile Thr Thr Ser Phe Lys Glu Ser Ile Val Asp Glu Gln Glu 405 410 415 Ser Asn Lys Glu Glu Asn Ile His Leu Thr Arg Phe Leu Arg Val Leu 420 425 430 Ala Asn Phe Leu Ile Ile Cys Cys Leu Cys Gly Ser Gly Tyr Leu Ile 435 440 445 Tyr Phe Val Val Lys Arg Ser Gln Gln Phe Ser Lys Met Gln Asn Val 450 455 460 Ser Trp Tyr Glu Arg Asn Glu Val Glu Ile Val Met Ser Leu Leu Gly 465 470 475 480 Met Phe Cys Pro Pro Leu Phe Glu Thr Ile Ala Ala Leu Glu Asn Tyr 485 490 495 His Pro Arg Thr Gly Leu Lys Trp Gln Leu Gly Arg Ile Phe Ala Leu 500 505 510 Phe Leu Gly Asn Leu Tyr Thr Phe Leu Leu Ala Leu Met Asp Asp Val 515 520 525 His Leu Lys Leu Ala Asn Glu Glu Thr Ile Lys Asn Ile Thr His Trp 530 535 540 Thr Leu Phe Asn Tyr Tyr Asn Ser Ser Gly Trp Asn Glu Ser Val Pro 545 550 555 560 Arg Pro Pro Leu His Pro Ala Asp Val Pro Arg Gly Ser Cys Trp Glu 565 570 575 Thr Ala Val Gly Ile Glu Phe Met Arg Leu Thr Val Ser Asp Met Leu 580 585 590 Val Thr Tyr Ile Thr Ile Leu Leu Gly Asp Phe Leu Arg Ala Cys Phe 595 600 605 Val Arg Phe Met Asn Tyr Cys Trp Cys Trp Asp Leu Glu Ala Gly Phe 610 615 620 Pro Ser Tyr Ala Glu Phe Asp Ile Ser Gly Asn Val Leu Gly Leu Ile 625 630 635 640 Phe Asn Gln Gly Met Ile Trp Met Gly Ser Phe Tyr Ala Pro Gly Leu 645 650 655 Val Gly Ile Asn Val Leu Arg Leu Leu Thr Ser Met Tyr Phe Gln Cys 660 665 670 Trp Ala Val Met Ser Ser Asn Val Pro His Glu Arg Val Phe Lys Ala 675 680 685 Ser Arg Ser Asn Asn Phe Tyr Met Gly Leu Leu Leu Leu Val Leu Phe 690 695 700 Leu Ser Leu Leu Pro Val Ala Tyr Thr Ile Met Ser Leu Pro Pro Ser 705 710 715 720 Phe Asp Cys Gly Pro Phe Ser Gly Lys Asn Arg Met Tyr Asp Val Leu 725 730 735 Gln Glu Thr Ile Glu Asn Asp Phe Pro Thr Phe Leu Gly Lys Ile Phe 740 745 750 Ala Phe Leu Ala Asn Pro Gly Leu Ile Ile Pro Ala Ile Leu Leu Met 755 760 765 Phe Leu Ala Ile Tyr Tyr Leu Asn Ser Val Ser Lys Ser Leu Ser Arg 770 775 780 Ala Asn Ala Gln Leu Arg Lys Lys Ile Gln Val Leu Arg Glu Val Glu 785 790 795 800 Lys Ser His Lys Ser Val Lys Gly Lys Ala Thr Ala Arg Asp Ser Glu 805 810 815 Asp Thr Pro Lys Ser Ser Ser Lys Asn Ala Thr Gln Leu Gln Leu Thr 820 825 830 Lys Glu Glu Thr Thr Pro Pro Ser Ala Ser Gln Ser Gln Ala Met Asp 835 840 845 Lys Lys Ala Gln Gly Pro Gly Thr Ser Asn Ser Ala Ser Arg Thr Thr 850 855 860 Leu Pro Ala Ser Gly His Leu Pro Ile Ser Arg Pro Pro Gly Ile Gly 865 870 875 880 Pro Asp Ser Gly His Ala Pro Ser Gln Thr His Pro Trp Arg Ser Ala 885 890 895 Ser Gly Lys Ser Ala Gln Arg Pro Pro His 900 905 <210> 17 <211> 552 <212> PRT <213> Homo sapiens <400> 17 Met Asp Arg Lys Val Ala Arg Glu Phe Arg His Lys Val Asp Phe Leu 1 5 10 15 Ile Glu Asn Asp Ala Glu Lys Asp Tyr Leu Tyr Asp Val Leu Arg Met 20 25 30 Tyr His Gln Thr Met Asp Val Ala Val Leu Val Gly Asp Leu Lys Leu 35 40 45 Val Ile Asn Glu Pro Ser Arg Leu Pro Leu Phe Asp Ala Ile Arg Pro 50 55 60 Leu Ile Pro Leu Lys His Gln Val Glu Tyr Asp Gln Leu Thr Pro Arg 65 70 75 80 Arg Ser Arg Lys Leu Lys Glu Val Arg Leu Asp Arg Leu His Pro Glu 85 90 95 Gly Leu Gly Leu Ser Val Arg Gly Gly Leu Glu Phe Gly Cys Gly Leu 100 105 110 Phe Ile Ser His Leu Ile Lys Gly Gly Gln Ala Asp Ser Val Gly Leu 115 120 125 Gln Val Gly Asp Glu Ile Val Arg Ile Asn Gly Tyr Ser Ile Ser Ser 130 135 140 Cys Thr His Glu Glu Val Ile Asn Leu Ile Arg Thr Lys Lys Thr Val 145 150 155 160 Ser Ile Lys Val Arg His Ile Gly Leu Ile Pro Val Lys Ser Ser Pro 165 170 175 Asp Glu Pro Leu Thr Trp Gln Tyr Val Asp Gln Phe Val Ser Glu Ser 180 185 190 Gly Gly Val Arg Gly Ser Leu Gly Ser Pro Gly Asn Arg Glu Asn Lys 195 200 205 Glu Lys Lys Val Phe Ile Ser Leu Val Gly Ser Arg Gly Leu Gly Cys 210 215 220 Ser Ile Ser Ser Gly Pro Ile Gln Lys Pro Gly Ile Phe Ile Ser His 225 230 235 240 Val Lys Pro Gly Ser Leu Ser Ala Glu Val Gly Leu Glu Ile Gly Asp 245 250 255 Gln Ile Val Glu Val Asn Gly Val Asp Phe Ser Asn Leu Asp His Lys 260 265 270 Glu Ala Val Asn Val Leu Lys Ser Ser Arg Ser Leu Thr Ile Ser Ile 275 280 285 Val Ala Ala Ala Gly Arg Glu Leu Phe Met Thr Asp Arg Glu Arg Leu 290 295 300 Ala Glu Ala Arg Gln Arg Glu Leu Gln Arg Gln Glu Leu Leu Met Gln 305 310 315 320 Lys Arg Leu Ala Met Glu Ser Asn Lys Ile Leu Gln Glu Gln Gln Glu 325 330 335 Met Glu Arg Gln Arg Arg Lys Glu Ile Ala Gln Lys Ala Ala Glu Glu 340 345 350 Asn Glu Arg Tyr Arg Lys Glu Met Glu Gln Ile Val Glu Glu Glu Glu 355 360 365 Lys Phe Lys Lys Gln Trp Glu Glu Asp Trp Gly Ser Lys Glu Gln Leu 370 375 380 Leu Leu Pro Lys Thr Ile Thr Ala Glu Val His Pro Val Pro Leu Arg 385 390 395 400 Lys Pro Lys Tyr Asp Gln Gly Val Glu Pro Glu Leu Glu Pro Ala Asp 405 410 415 Asp Leu Asp Gly Gly Thr Glu Glu Gln Gly Glu Gln Asp Phe Arg Lys 420 425 430 Tyr Glu Glu Gly Phe Asp Pro Tyr Ser Met Phe Thr Pro Glu Gln Ile 435 440 445 Met Gly Lys Asp Val Arg Leu Leu Arg Ile Lys Lys Glu Gly Ser Leu 450 455 460 Asp Leu Ala Leu Glu Gly Gly Val Asp Ser Pro Ile Gly Lys Val Val 465 470 475 480 Val Ser Ala Val Tyr Glu Arg Gly Ala Ala Glu Arg His Gly Gly Ile 485 490 495 Val Lys Gly Asp Glu Ile Met Ala Ile Asn Gly Lys Ile Val Thr Asp 500 505 510 Tyr Thr Leu Ala Glu Ala Glu Ala Ala Leu Gln Lys Ala Trp Asn Gln 515 520 525 Gly Gly Asp Trp Ile Asp Leu Val Val Ala Val Cys Pro Pro Lys Glu 530 535 540 Tyr Asp Asp Glu Leu Thr Phe Phe 545 550 <210> 18 <211> 2237 <212> DNA <213> Homo sapiens <400> 18 agctccgagg gcggctggcc cggtcgcggt cgcggctctt tccagctcct ggcagccggg 60 cacccgaagg aacgggtcgt gcaacgacgc agctggacct ggcccagcca tggaccgaaa 120 agtggcccga gaattccggc ataaggtgga ttttctgatt gaaaatgatg cagagaagga 180 ctatctctat gatgtgctgc gaatgtacca ccagaccatg gacgtggccg tgctcgtggg 240 agacctgaag ctggtcatca atgaacccag ccgtctgcct ctgtttgatg ccattcggcc 300 gctgatccca ctgaagcacc aggtggaata tgatcagctg accccccggc gctccaggaa 360 gctgaaggag gtgcgtctgg accgtctgca ccccgaaggc ctcggcctga gtgtgcgtgg 420 tggcctggag tttggctgtg ggctcttcat ctcccacctc atcaaaggcg gtcaggcaga 480 cagcgtcggg ctccaggtag gggacgagat cgtccggatc aatggatatt ccatctcctc 540 ctgtacccat gaggaggtca tcacctcat tcgaccaag aaaactgtgt ccatchaagt 600 gagacacatc ggcctgatcc ccgtgaaaag ctctcctgat gagcccctca cttggcagta 660 tgtggatcag ttgtgtcgg aatctggggg cgtgcgaggc agcctgggct cccctggaaa 720 tcgggaaac aaggagaaga aggtctcat cagcctggta ggctcccgag gccttggctg 780 cagcatttcc agcggcccca tccagaagcc tggcatcttt atcagccatg tgaaacctgg 840 ctccctgtct gctgaggtgg gattggagat agggaccag attgtcgaag tcaatggcgt 900 cgacttctct aacctggatc acaaggaggc tgtaatgtg ctgaagagta gccgcagcct 960 gaccatctcc attgtagctg cagctggccg ggagctgttc atgacagacc gggagcggct 1020 ggcagaggcg cggcagcgtg agctgcagcg gcaggagctt ctcatgcaga agcggctggc 1080 gatggagtcc aaaagatcc tccaggagca gcaggagatg gagcggcaaa ggagaaaag 1140 aattgcccag aaggcagcag aggaaatga gagataccgg aaggagatgg aacagattgt 1200 agaggagaa gagagatttta agagagaatg gagagac tggggctca aggaacagct 1260 actcttgcct aaaaccatca ctgctgaggt acaccagta ccccttcgca agccaaagta tgatcaggga gtggaacctg agctcgagcc cgcagatgac ctggatggag gcacggagga 1380 gcagggagag caggatttcc ggaaatatga ggaaggcttt gacccctact ctatgttcac cccagagcag atcatgggga aggatgtccg gctcctacgc atcaagaagg aggatcctt agacctggcc ctggaaggcg gtgtggactc ccccattggg aaggtggtcg tttctgctgt 1560 gtatgagcgg ggagctgctg agcggcatgg tggcattgtg aaaggggacg agatcatggc 1620 aatcaacggc aagattgtga cagactacac cctggctgag gctgaggctg ccctgcagaa ggcctggaat cagggcgggg actggatcga ccttgtggtt gccgtctgcc ccccaaagga 1740. gtatgacgat gagctgacct tcttctgaag tccaaaaggg gaaaccaaat tcaccgttag gaaacagtga gctccggccc cacctcgtga acacaaagcc tcggatcagc cttgagagag gccacactac acacaccaga tggcatcctt gggacctgaa tctatcaccc aggaatctca aactcccttt ggccctgac cagggccaga tagcag ctcgggccac tcttctgag gccaacgtgg aggaaaggga gcagccagcc atttgggaga agatctcaag gatccagact 2040 ctcattcctt tcctctggcc cagtgaattt ggtctctccc agctctgggg gactccttcc 2100 ttgaacccta ataagacccc actggagtct ctctctctcc atccctctcc tctgccctct 2160 gctctaattg ctgccaggat tgtcactcca aaccttactc tgagctcatt aataaaatag 2220 atttattttc cagctta 2237 <210> 19 <211> 2385 <212> DNA <213> Homo sapiens <400> 19 agctccgagg gcggctggcc cggtcgcggt cgcggctctt tccagctcct ggcagccggg 60 cacccgaagg aacgggtcgt gcaacgacgc agctggacct ggcccagcca tggaccgaaa 120 agtggcccga gaattccggc ataaggtgga ttttctgatt gaaaatgatg cagagaagga 180 ctatctctat gatgtgctgc gaatgtacca ccagaccatg gacgtggccg tgctcgtggg 240 agacctgaag ctggtcatca atgaacccag ccgtctgcct ctgtttgatg ccattcggcc 300 gctgatccca ctgaagcacc aggtggaata tgatcagctg accccccggc gctccaggaa 360 gctgaaggag gtgcgtctgg accgtctgca ccccgaaggc ctcggcctga gtgtgcgtgg 420 tggcctggag tttggctgtg ggctcttcat ctcccacctc atcaaaggcg gtcaggcaga 480 cagcgtcggg ctccaggtag gggacgagat cgtccggatc aatggatatt ccatctcctc 540 ctgtacccat gaggaggtca tcaacctcat tcgaaccaag aaaactgtgt ccatcaaagt 600 gagacacatc ggcctgatcc ccgtgaaaag ctctcctgat gagcccctca cttggcagta 660 tgtggatcag tttgtgtcgg aatctgggg cgtgcgaggc agcctgggct cccctggaaa 720 tcgggaaaac your area aggtcttcat cagcctggta ggctcccgag gccttggctg 780 cagcatttcc agcggcccca tccagaagcc tggcatcttt atcagccatg tgaaacctgg 840 ctccctgtct gctgaggtgg gattggagat agggaccag attgtcgaag tcaatggcgt 900 960 gaccatctcc attgtagctg cagctggccg ggagctgttc atgacagacc gggagcggct 1020 ggcagaggcg cggcagcgtg agctgcagcg gcaggagctt ctcatgcaga agcggctggc 1080 gatggagtcc aaaagatcc tccaggagca gcaggagatg gagcggcaaa ggagaaaag 1140 aattgcccag aaggcagcag aggaaatga gagataccgg aaggagatgg aacagattgt 1200 agaggagaa gagagatttta agagagaatg gagagac tggggctca aggaacagct 1260 actctgcct aaaaccatca ctgctgaggt accaccagta ccccttcgca agccaaagta 1320 tgatcaggga gtgaacctg agctcgagcc cgcagatgac ctggatggag gcacggagga 1380 gcagggagag gagaagaa gaagccaag tattggcagcc tgcaggactt 1440 gagaagaat agaagac tggatttga gcaaagctt agaagaaaaaaah 1500 gctggagaag gaaagcagc windowcatcaccggctggcc caggatt tccggaata 1560 tgaggaggc ttgaccct actctatgtt caccccagag cagatcatgg ggaaggatgt 1620 ccggctccta cgcatcaaga aggaggtc cttagacctg gccctggaag gcggtgtgga 1680 ctcccccatt gggaaggtgg tcgtttctgc tgtgtatgag cggggagctg ctgagcggca 1740 tggtggcatt gtgaaagggg multi-cat ggcaatcac ggcaattg tgacagacta 1800 caccctggct gaggctgagg ctgccctgca gaaggcctgg aatcagggcg gggactggat 1860 cgaccttgtg gttgccgtct gccccccaaa ggagtatgac gatgagctga ccttcttctg 1920 aagtccaaaa ggggaaacca aattcaccgt taggaaacag tgagctccgg ccccacctcg 1980 tgaacacaaa gcctcggatc agccttgaga gaggccacac tacacacacc agatggcatc 2040 cttgggacct gaatctatca cccaggaatc tcaaactccc tttggccctg aaccagggcc 2100 agataaggaa cagctcgggc cactcttctg aaggccaacg tggaggaaag ggagcagcca 2160 gccatttggg agaagatctc aaggatccag actctcattc ctttcctctg gcccagtgaa 2220 tttggtctct cccagctctg ggggactcct tccttgaacc ctaataagac cccactggag 2280 tctctctc tccatccctc tctctgccc tctgctctaa ttgctgccag gattgtcact 2340 ccaaacctta ctctgagctc attaataaaa tagatttatt ttcca 2385 <210> 20 <211> 899 <212> PRT <213> Homo sapiens <400> 20 Met Asp Arg Lys Val Ala Arg Glu Phe Arg His Lys Val Asp Phe Leu 1 5 10 15 Ile Glu Asn Asp Ala Glu Lys Asp Tyr Leu Tyr Asp Val Leu Arg Met 20 25 30 Tyr His Gln Thr Met Asp Val Ala Val Leu Val Gly Asp Leu Lys Leu 35 40 45 Val Ile Asn Glu Pro Ser Arg Leu Pro Leu Phe Asp Ala Ile Arg Pro 50 55 60 Leu Ile Pro Leu Lys His Gln Val Glu Tyr Asp Gln Leu Thr Pro Arg 65 70 75 80 Arg Ser Arg Lys Leu Lys Glu Val Arg Leu Asp Arg Leu His Pro Glu 85 90 95 Gly Leu Gly Leu Ser Val Arg Gly Gly Leu Glu Phe Gly Cys Gly Leu 100 105 110 Phe Ile Ser His Leu Ile Lys Gly Gly Gln Ala Asp Ser Val Gly Leu 115 120 125 Gln Val Gly Asp Glu Ile Val Arg Ile Asn Gly Tyr Ser Ile Ser Ser 130 135 140 Cys Thr His Glu Glu Val Ile Asn Leu Ile Arg Thr Lys Lys Thr Val 145 150 155 160 Ser Ile Lys Val Arg His Ile Gly Leu Ile Pro Val Lys Ser Ser Pro 165 170 175 Asp Glu Pro Leu Thr Trp Gln Tyr Val Asp Gln Phe Val Ser Glu Ser 180 185 190 Gly Gly Val Arg Gly Ser Leu Gly Ser Pro Gly Asn Arg Glu Asn Lys 195 200 205 Glu Lys Lys Val Phe Ile Ser Leu Val Gly Ser Arg Gly Leu Gly Cys 210 215 220 Ser Ile Ser Ser Gly Pro Ile Gln Lys Pro Gly Ile Phe Ile Ser His 225 230 235 240 Val Lys Pro Gly Ser Leu Ser Ala Glu Val Gly Leu Glu Ile Gly Asp 245 250 255 Gln Ile Val Glu Val Asn Gly Val Asp Phe Ser Asn Leu Asp His Lys 260 265 270 Glu Ala Val Asn Val Leu Lys Ser Ser Arg Ser Leu Thr Ile Ser Ile 275 280 285 Val Ala Ala Ala Gly Arg Glu Leu Phe Met Thr Asp Arg Glu Arg Leu 290 295 300 Ala Glu Ala Arg Gln Arg Glu Leu Gln Arg Gln Glu Leu Leu Met Gln 305 310 315 320 Lys Arg Leu Ala Met Glu Ser Asn Lys Ile Leu Gln Glu Gln Gln Glu 325 330 335 Met Glu Arg Gln Arg Arg Lys Glu Ile Ala Gln Lys Ala Ala Glu Glu 340 345 350 Asn Glu Arg Tyr Arg Lys Glu Met Glu Gln Ile Val Glu Glu Glu Glu 355 360 365 Lys Phe Lys Lys Gln Trp Glu Glu Asp Trp Gly Ser Lys Glu Gln Leu 370 375 380 Leu Leu Pro Lys Thr Ile Thr Ala Glu Val His Pro Val Pro Leu Arg 385 390 395 400 Lys Pro Lys Ser Phe Gly Trp Phe Tyr Arg Tyr Asp Gly Lys Phe Pro 405 410 415 Thr Ile Arg Lys Lys Gly Lys Asp Lys Lys Ala Lys Tyr Gly Ser 420 425 430 Leu Gln Asp Leu Arg Lys Asn Lys Lys Glu Leu Glu Phe Glu Gln Lys 435 440 445 Leu Tyr Lys Glu Lys Glu Glu Met Leu Glu Lys Glu Lys Gln Leu Lys 450 455 460 Ile Asn Arg Leu Ala Gln Glu Val Ser Glu Thr Glu Arg Glu Asp Leu 465 470 475 480 Glu Glu Ser Glu Lys Ile Gln Tyr Trp Val Glu Arg Leu Cys Gln Thr 485 490 495 Arg Leu Glu Gln Ile Ser Ser Ala Asp Asn Glu Ile Ser Glu Met Thr 500 505 510 Thr Gly Pro Pro Pro Pro Pro Pro Ser Val Ser Pro Leu Ala Pro Pro 515 520 525 Leu Arg Arg Phe Ala Gly Gly Leu His Leu His Thr Thr Asp Leu Asp 530 535 540 Asp Ile Pro Leu Asp Met Phe Tyr Tyr Pro Pro Lys Thr Pro Ser Ala 545 550 555 560 Leo Pro Val Met Pro His Pro Pro Pro Ser Asn Pro Pro His Lys Val 565 570 575 Pro Ala Pro Pro Val Leu Pro Leu Ser Gly His Val Ser Ala Ser Ser 580 585 590 Ser Pro Trp Val Gln Arg Thr Pro Pro Pro Ile Pro Ile Pro Pro Pro 595 600 605 Pro Ser Val Pro Thr Gln Asp Leu Thr Pro Thr Arg Pro Leu Pro Ser 610 615 620 Ala Leu Glu Glu Ala Leu Ser Asn His Pro Phe Arg Thr Gly Asp Thr 625 630 635 640 Gly Asn Pro Val Glu Asp Trp Glu Ala Lys Asn His Ser Gly Lys Pro 645 650 655 Thr Asn Ser Pro Val Pro Glu Gln Ser Phe Pro Pro Thr Pro Lys Thr 660 665 670 Phe Cys Pro Ser Pro Gln Pro Pro Arg Gly Pro Gly Val Ser Thr Ile 675 680 685 Ser Lys Pro Val Met Val His Gln Glu Pro Asn Phe Ile Tyr Arg Pro 690 695 700 Ala Val Lys Ser Glu Val Leu Pro Gln Glu Met Leu Lys Arg Met Val 705 710 715 720 Val Tyr Gln Thr Ala Phe Arg Gln Asp Phe Arg Lys Tyr Glu Glu Gly 725 730 735 Phe Asp Pro Tyr Ser Met Phe Thr Pro Glu Gln Ile Met Gly Lys Asp 740 745 750 Val Arg Leu Leu Arg Ile Lys Lys Glu Gly Ser Leu Asp Leu Ala Leu 755 760 765 Glu Gly Gly Val Asp Ser Pro Ile Gly Lys Val Val Val Ser Ala Val 770 775 780 Tyr Glu Arg Gly Ala Ala Glu Arg His Gly Gly Ile Val Lys Gly Asp 785 790 795 800 Glu Ile Met Ala Ile Asn Gly Lys Ile Val Thr Asp Tyr Thr Leu Ala 805 810 815 Glu Ala Glu Ala Ala Leu Gln Lys Ala Trp Asn Gln Gly Gly Asp Trp 820 825 830 Ile Asp Leu Val Val Ala Val Cys Pro Pro Lys Glu Tyr Asp Asp Glu 835 840 845 Leu Ala Ser Leu Pro Ser Ser Val Ala Glu Ser Pro Gln Pro Val Arg 850 855 860 Lys Leu Leu Glu Asp Arg Ala Ala Val His Arg His Gly Phe Leu Leu 865 870 875 880 Gln Leu Glu Pro Thr Asp Leu Leu Leu Lys Ser Lys Arg Gly Asn Gln 885 890 895 Ile His Arg <210> 21 <211> 2180 <212> DNA <213> Homo sapiens <400> 21 agctccgagg gcggctggcc cggtcgcggt cgcggctctt tccagctcct ggcagccggg 60 cacccgaagg aacgggtcgt gcaacgacgc agctggacct ggcccagcca tggaccgaaa 120 180 ctatctctat gatgtgctgc gaatgtacca ccagaccatg gacgtggccg tgctcgtggg 240 agacctgaag ctggtcatca atgaacccag ccgtctgcct ctgtttgatg ccattcggcc 300 gctgatccca ctgaagcacc aggtggataa tgatcagctg accccccggc gctccaggaa 360 gctgaaggag gtgcgtctgg accgtctgca ccccgaaggc ctcggcctga gtgtgcgtgg 420 tggcctggag tttggctgtg ggctcttcat ctcccacctc atcaaaggcg gtcaggcaga 480 cagcgtcggg ctccaggtag gggacgagat cgtccggatc aatggatatt ccatctcctc 540 ctgtacccat gaggaggtca tcaacctcat tcgaaccaag aaaactgtgt ccatcaaagt 600 gagacacatc ggcctgatcc ccgtgaaaag ctctcctgat gagcccctca cttggcagta 660 tgtggatcag tttgtgtcgg aatctgggg cgtgcgaggc agcctgggct cccctggaaa 720 tcgggaaaac your area aggtcttcat cagcctggta ggctcccgag gccttggctg 780 cagcatttcc agcggcccca tccagaagcc tggcatcttt atcagccatg tgaaacctgg 840 ctccctgtct gctgaggtgg gattggagat aggggaccag attgtcgaag tcaatggcgt 900. cgacttctct aacctggatc acaaggaggg ccggggagctg ttcatgacag accggggagcg gctggcagag gcgcggcagc gtgagctgca gcggcaggag cttctcatgc agaagcggct ggcgatggag tccaacaaga tcctccagga gcagcaggag atggagcggc aaaggagaaa agaaattgcc cagaaggcag cagaggaaaa tgagagatac cggaaggaga tggaacagat tgtagaggag gagagagagt ttagagagca atgggaga gactggggct caaaggaca gctactcttg cctaaacca tcactgctga ggtacaccca gtaccccttc gcaagccaaa gtatgatcag ggagtggaac ctgagctcga gcccgcagat gacctggatg gaggcacgga ggagcaggga ggaggatt tccggaata tggaggaggc tttgacccct actctatgtt caccccagag cagatcatgg ggaaggatgt ccggctccta cgcatcaaga aggagggatc cttagacctg gccctggaag gcggtgtgga ctcccccatt gggaaggtgg tcgtttctgc tgtgtatgag cggggagctg ctgagcggca tggtggcatt gtgaaagggg acgagatcat 1560 ggcaatcaac ggcaagattg tgacagacta caccctggct gaggctgagg ctgccctgca 1620 gaaggcctgg aatcagggcg gggactggat cgaccttgtg gttgccgtct gccccccaaa 1680 ggagtatgac gatgagctga ccttcttctg aagtccaaaa ggggaaacca aattcaccgt 1740 taggaaacag tgagctccgg ccccacctcg tgaacacaaa gcctcggatc agccttgaga 1800 gaggccacac tacacacacc agatggcatc cttgggacct gaatctatca cccaggaatc 1860 tcaaactccc tttggccctg aaccagggcc agataaggaa cagctcgggc cactcttctg 1920 aaggccaacg tggaggaaag ggagcagcca gccatttggg agaagatctc aaggatccag 1980 actctcattc ctttcctctg gcccagtgaa tttggtctct cccagctctg ggggactcct 2040 tccttgaacc ctaataagac cccactggag tctctctctc tccatccctc tcctctgccc 2100 tctgctctaa ttgctgccag gattgtcact ccaaacctta ctctgagctc attaataaaa 2160 tagattatt ttccagctta 2180 <210> 22 <211> 533 <212> PRT <213> Homo sapiens <400> 22 Met Asp Arg Lys Val Ala Arg Glu Phe Arg His Lys Val Asp Phe Leu 1 5 10 15 Ile Glu Asn Asp Ala Glu Lys Asp Tyr Leu Tyr Asp Val Leu Arg Met 20 25 30 Tyr His Gln Thr Met Asp Val Ala Val Leu Val Gly Asp Leu Lys Leu 35 40 45 Val Ile Asn Glu Pro Ser Arg Leu Pro Leu Phe Asp Ala Ile Arg Pro 50 55 60 Leu Ile Pro Leu Lys His Gln Val Glu Tyr Asp Gln Leu Thr Pro Arg 65 70 75 80 Arg Ser Arg Lys Leu Lys Glu Val Arg Leu Asp Arg Leu His Pro Glu 85 90 95 Gly Leu Gly Leu Ser Val Arg Gly Gly Leu Glu Phe Gly Cys Gly Leu 100 105 110 Phe Ile Ser His Leu Ile Lys Gly Gly Gln Ala Asp Ser Val Gly Leu 115 120 125 Gln Val Gly Asp Glu Ile Val Arg Ile Asn Gly Tyr Ser Ile Ser Ser 130 135 140 Cys Thr His Glu Glu Val Ile Asn Leu Ile Arg Thr Lys Lys Thr Val 145 150 155 160 Ser Ile Lys Val Arg His Ile Gly Leu Ile Pro Val Lys Ser Ser Pro 165 170 175 Asp Glu Pro Leu Thr Trp Gln Tyr Val Asp Gln Phe Val Ser Glu Ser 180 185 190 Gly Gly Val Arg Gly Ser Leu Gly Ser Pro Gly Asn Arg Glu Asn Lys 195 200 205 Glu Lys Lys Val Phe Ile Ser Leu Val Gly Ser Arg Gly Leu Gly Cys 210 215 220 Ser Ile Ser Ser Gly Pro Ile Gln Lys Pro Gly Ile Phe Ile Ser His 225 230 235 240 Val Lys Pro Gly Ser Leu Ser Ala Glu Val Gly Leu Glu Ile Gly Asp 245 250 255 Gln Ile Val Glu Val Asn Gly Val Asp Phe Ser Asn Leu Asp His Lys 260 265 270 Glu Gly Arg Glu Leu Phe Met Thr Asp Arg Glu Arg Leu Ala Glu Ala 275 280 285 Arg Gln Arg Glu Leu Gln Arg Gln Glu Leu Leu Met Gln Lys Arg Leu 290 295 300 Ala Met Glu Ser Asn Lys Ile Leu Gln Glu Gln Gln Glu Met Glu Arg 305 310 315 320 Gln Arg Arg Lys Glu Ile Ala Gln Lys Ala Ala Glu Glu Asn Glu Arg 325 330 335 Tyr Arg Lys Glu Met Glu Gln Ile Ser Glu Glu Glu Glu Lys Phe Lys 340 345 350 Lys Gln Trp Glu Glu Asp Trp Gly Ser Lys Glu Gln Leu Ile Leu Pro 355 360 365 Lys Thr Ile Thr Ala Glu Val His Pro Val Pro Leu Arg Lys Pro Lys 370 375 380 Tyr Asp Gln Gly Val Glu Pro Glu Leu Glu Pro Ala Asp Asp Leu Asp 385 390 395 400 Gly Gly Thr Glu Glu Gln Gly Glu Gln Asp Phe Arg Lys Tyr Glu Glu 405 410 415 Gly Phe Asp Pro Tyr Ser Met Phe Thr Pro Glu Gln Ile Met Gly Lys 420 425 430 Asp Val Arg Leu Leu Arg Ile Lys Lys Glu Gly Ser Leu Asp Leu Ala 435 440 445 Leu Glu Gly Gly Val Asp Ser Pro Ile Gly Lys Val Val Val Ser Ala 450 455 460 Val Tyr Glu Arg Gly Ala Ala Glu Arg His Gly Gly Ile Val Lys Gly 465 470 475 480 Asp Glu Ile Met Ala Ile Asn Gly Lys Ile Val Thr Asp Tyr Thr Leu 485 490 495 Ala Glu Ala Glu Ala Ala Leu Gln Lys Ala Trp Asn Gln Gly Gly Asp 500 505 510 Trp Ile Asp Leu Val Val Ala Val Cys Pro Pro Lys Glu Tyr Asp Asp 515 520 525 Glu Leu Thr Phe Phe 530 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 23 gaggtaccat ggaccggaag gtggcccgag 30 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 24 caggatccgg acaatttcat cccctac 27 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 25 ctcattgaaa atgacgcaga gaagg 25 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 26 tctcactttg atggacacgg tctt 24 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 27 gaacccaacc gcctgccg 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 28 tgcagacggt ccaagcgt 18 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 29 gtgaggccgg tgctgagtat g 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 30 gccaaagttg tcatggatga c 21 <210> 31 <211> 909 <212> PRT <213> Mus sp. <400> 31 Met Asp Arg Lys Val Ala Arg Glu Phe Arg His Lys Val Asp Phe Leu 1 5 10 15 Ile Glu Asn Asp Ala Glu Lys Asp Tyr Leu Tyr Asp Val Leu Arg Met 20 25 30 Tyr His Gln Thr Met Asp Val Ala Val Leu Val Gly Asp Leu Lys Leu 35 40 45 Val Ile Asn Glu Pro Asn Arg Leu Pro Leu Phe Asp Ala Ile Arg Pro 50 55 60 Leu Ile Pro Leu Lys His Gln Glu Tyr Asp Gln Leu Thr Pro Arg Arg 65 70 75 80 Ser Arg Lys Leu Lys Glu Val Arg Leu Asp Arg Leu His Pro Glu Gly 85 90 95 Leu Gly Leu Ser Val Arg Gly Gly Leu Glu Phe Gly Cys Gly Leu Phe 100 105 110 Ile Ser His Leu Ile Lys Gly Gly Gln Ala Asp Ser Val Gly Leu Gln 115 120 125 Val Gly Asp Glu Ile Val Arg Ile Asn Gly Tyr Ser Ile Ser Ser Cys 130 135 140 Thr His Glu Glu Val Ile Asn Leu Ile Arg Thr Lys Lys Thr Val Ser 145 150 155 160 Ile Lys Val Arg His Ile Gly Leu Ile Pro Val Lys Ser Ser Pro Glu 165 170 175 Glu Ser Leu Lys Trp Gln Tyr Val Asp Gln Phe Val Ser Glu Ser Gly 180 185 190 Gly Val Arg Gly Gly Leu Gly Ser Pro Gly Asn Arg Thr Thr Lys Glu 195 200 205 Lys Lys Val Phe Ile Ser Leu Val Gly Ser Arg Gly Leu Gly Cys Ser 210 215 220 Ile Ser Ser Gly Pro Ile Gln Lys Pro Gly Ile Phe Val Ser His Val 225 230 235 240 Lys Pro Gly Ser Leu Ser Ala Glu Val Gly Leu Glu Thr Gly Asp Gln 245 250 255 Ile Val Glu Val Asn Gly Ile Asp Phe Thr Asn Leu Asp His Lys Glu 260 265 270 Ala Val Asn Val Leu Lys Ser Ser Arg Ser Leu Thr Ile Ser Ile Val 275 280 285 Ala Gly Ala Gly Arg Glu Leu Phe Met Thr Asp Arg Glu Arg Leu Glu 290 295 300 Glu Ala Arg Gln Arg Glu Leu Gln Arg Gln Glu Leu Leu Met Gln Lys 305 310 315 320 Arg Leu Ala Met Glu Ser Asn Lys Ile Leu Gln Glu Gln Gln Glu Met 325 330 335 Glu Arg Gln Arg Arg Lys Glu Ile Ala Gln Lys Ala Ala Glu Glu Asn 340 345 350 Glu Arg Tyr Arg Lys Glu Met Glu Gln Ile Ser Glu Glu Glu Glu Lys 355 360 365 Phe Lys Lys Gln Trp Glu Glu Asp Trp Gly Ser Lys Glu Gln Leu Ile 370 375 380 Leu Pro Lys Thr Ile Thr Ala Glu Val His Pro Val Pro Leu Arg Lys 385 390 395 400 Pro Lys Ser Phe Gly Trp Phe Tyr Arg Tyr Asp Gly Lys Phe Pro Thr 405 410 415 Ile Arg Lys Lys Ala Lys Glu Lys Lys Lys Ala Lys Tyr Asp Ser Leu 420 425 430 Gln Asp Leu Arg Lys Asn Lys Lys Glu Leu Glu Phe Glu Gln Lys Leu 435 440 445 Tyr Lys Glu Lys Glu Glu Met Leu Glu Lys Glu Lys Gln Leu Lys Ile 450 455 460 Asn Arg Leu Ala Gln Glu Val Ser Glu Thr Glu Arg Glu Asp Leu Glu 465 470 475 480 Glu Ser Glu Lys Thr Gln Tyr Trp Val Glu Arg Leu Cys Gln Thr Arg 485 490 495 Leu Glu Gln Ile Ser Ser Ala Glu Asn Glu Ile Pro Glu Met Thr Thr 500 505 510 Gly Pro Pro Pro Pro Pro Pro Ser Val Ser Pro Leu Ala Pro Pro Leu 515 520 525 Arg Arg Phe Ala Gly Gly Ile His Leu His Thr Thr Asp Leu Asp Asp 530 535 540 Ile Pro Leu Asp Met Phe Tyr Tyr Pro Pro Lys Thr Pro Ser Ala Leu 545 550 555 560 Pro Val Met Pro His Pro Pro Ser Val Asn Ser Pro Ser Lys Val Pro 565 570 575 Ala Pro Pro Val Leu Pro Ser Ser Gly His Val Ser Ser Ser Ser Ser 580 585 590 Pro Trp Val Gln Arg Thr Pro Pro Pro Ile Pro Ile Pro Pro Pro Pro 595 600 605 Ser Ile Pro Thr Gln Asp Leu Thr Pro Thr Arg Pro Leu Pro Ser Ala 610 615 620 Leu Glu Glu Ala Leu Gly Asn His Pro Phe Arg Thr Gly Asp Pro Gly 625 630 635 640 His Pro Ala Asp Asp Trp Glu Ala Asn Thr His Ser Gly Lys Pro Ser 645 650 655 Ser Ser Pro Thr Thr Glu Arg Ser Phe Pro Pro Ala Pro Lys Thr Phe 660 665 670 Cys Pro Ser Pro Gln Pro Pro Arg Gly Pro Gly Val Ser Thr Ile Ser 675 680 685 Lys Pro Val Met Val His Gln Glu His Asn Phe Val Tyr Arg Pro Ala 690 695 700 Val Lys Ser Glu Val Leu Pro Gln Glu Met Leu Lys Arg Met Val Val 705 710 715 720 Tyr Gln Thr Ala Phe Arg Gln Asp Phe Arg Lys Tyr Glu Glu Gly Phe 725 730 735 Asp Pro Tyr Ser Met Phe Ser Pro Glu Gln Ile Ala Gly Lys Asp Val 740 745 750 Arg Leu Leu Arg Ile Lys Lys Glu Gly Ser Leu Asp Leu Ala Leu Glu 755 760 765 Gly Gly Val Asp Ser Pro Val Gly Lys Val Val Val Ser Ala Val Tyr 770 775 780 Glu Gly Gly Ala Ala Glu Arg His Gly Gly Val Val Lys Gly Asp Glu 785 790 795 800 Ile Met Ala Ile Asn Gly Lys Ile Val Thr Asp Tyr Thr Leu Ala Glu 805 810 815 Ala Glu Ala Ala Leu Gln Lys Ala Trp Asn Gln Gly Gly Asp Trp Ile 820 825 830 Asp Leu Val Val Ala Val Cys Pro Pro Lys Glu Tyr Asp Asp Glu Leu 835 840 845 Ser Ser Leu Pro Ser Ser Ala Ala Glu Ser Pro Gln Leu Ala Arg Lys 850 855 860 Gln Leu Glu Ala Tyr Glu Pro Val Cys Arg His Gly Phe Phe Leu Gln 865 870 875 880 Leu Glu Pro Thr Asn Leu Leu Leu Lys Ser Arg Glu Arg Asn Gln Thr 885 890 895 Asp Pro Ser Trp Arg Pro Ala Ser Ser Ala Pro Ser Pro 900 905 <210> 32 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 Met Asp Arg Lys Val Ala Arg Glu Phe Arg His Lys Val Asp Phe Leu 1 5 10 15 Ile Glu Asn Asp Ala Glu Lys Asp Tyr Leu Tyr Asp Val Leu Arg Met 20 25 30 Tyr His Gln Thr Met Asp Val Ala Val Leu Val Gly Asp Leu Lys Leu 35 40 45 Val Ile Asn Glu Pro Asn Arg Leu Pro Leu Phe Asp Ala Ile Arg Pro 50 55 60 Leu Ile Pro Leu Lys His Gln Glu Thr Glu Gly Gly Thr Leu Gly Pro 65 70 75 80 Ser Ala Pro Arg Arg Ser Arg Pro Gln Arg Ala Trp Arg Pro Gly Ile 85 90 95 Trp Leu Trp Thr Leu Tyr Leu Pro Pro His Gln Arg Trp Pro Gly Arg 100 105 110 Gln Arg Trp Ala Ser Gly Arg Gly 115 120

Claims

1. A method for producing a nucleic acid sequence encoding the Anc80 capsid protein shown in SEQ ID NO: 1 or SEQ ID NO: 2, A promoter selected from the group consisting of Espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter; and comprising a polynucleotide selected from TMC1 or TMC2, 1. An inner ear hair cell targeting synthetic adeno-associated virus (AAV) vector comprising: the promoter directs expression of the polynucleotide; the vector transduces inner and outer hair cells with an efficiency of at least about 70% or higher; vector.

2. The inner ear hair cell targeting synthetic adeno-associated virus (AAV) vector of claim 1, wherein the polynucleotide is TMC1.

3. The inner ear hair cell targeting synthetic adeno-associated virus (AAV) vector of claim 1, wherein the polynucleotide is TMC2.

4. A cell comprising the inner ear hair cell targeting synthetic adeno-associated virus (AAV) vector described in claim 1.

5. The cell described in claim 4, which is an outer hair cell or an inner hair cell.

6. A pharmaceutical composition for treating a subject having a genetic abnormality in hearing and / or vestibular mechanosensation, comprising the inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector described in claim 1.

7. A pharmaceutical composition for introducing a wild-type form of a defective gene into a subject having a genetic abnormality in hearing and / or vestibular mechanosensation, comprising the inner ear hair cell-targeting synthetic adeno-associated virus (AAV) vector described in claim 1.

8. The pharmaceutical composition of claim 6, the administration of which improves hearing loss.

9. The pharmaceutical composition of claim 7, the administration of which improves hearing loss.

10. The pharmaceutical composition of claim 8, wherein the hearing loss is partial hearing loss or complete hearing loss.

11. The pharmaceutical composition of claim 9, wherein the hearing loss is partial hearing loss or complete hearing loss.

12. The pharmaceutical composition of claim 8, wherein recovery of hearing function is associated with preservation of hair bundle morphology and / or restoration of mechanotransduction.

13. The pharmaceutical composition of claim 9, wherein recovery of hearing function is associated with preservation of hair bundle morphology and / or restoration of mechanotransduction.

14. The pharmaceutical composition of claim 8, wherein the genetic abnormality is selected from the group consisting of DFNB7, DFNB11, and DFNA36.

15. The pharmaceutical composition of claim 9, wherein the genetic abnormality is selected from the group consisting of DFNB7, DFNB11, and DFNA36.

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